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Natriuretic Peptides As A Humoral Link BetweenThe Heart And The Gastrointetsinal SystemAnteneh AddisuUniversity of South Florida

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Natriuretic Peptides As A Humoral Link Between The Heart And The

Gastrointetsinal System

by

Anteneh Addisu

A dissertation submitted in partial fulfillment of the requirements for the degree of

Doctor of Medicine Department of Molecular Pharmacology and Physiology

College of Medicine University of South Florida

Co-Major Professor: John R. Dietz, Ph.D. Co-Major Professor: William R. Gower. Jr., Ph.D.

Kendall F. Morris, Ph.D. Stanley J. Nazian, Ph.D.

Ravi Sankar, Ph.D.

Date of Approval: March 18, 2008

Keywords: BNP, volume regulation, heart failure, interorgan communication, non-muscle myosins

© Copyright 2008 , Anteneh Addisu

DEDICATION

I dedicate this thesis to my loving family, my wife Sirgut Tirusew and

my children Leeyu and Yonaas, my parents Addisu Merdassa and my

mom Tsedale Sahlemariam. Your, dedication, sacrifices, prayers and

love has made this possible and for that I am eternally thankful.

ACKNOWLEDGMENTS

I owe a huge debt of gratitude to my mentor Dr. John R. Dietz.

Your exemplary guidance has enriched my training in so many ways;

but more importantly in encouraging my keenness to become an

independent researcher. Your unreserved commitment to my success

will always be cherished. Thank you.

My co-major professor, Dr. William R. Gower Jr. has been most

generous with his valuable time and resources to assure my success,

and I would like to take this opportunity to thank him.

I thank my committee members, Drs. Kendall F. Morris, Stanley

J. Nazian and Ravi Sankar for their support and encouragement. Each

one of you has been very supportive whenever I needed your guidance

or assistance. Thank you for everything.

Our department Chair Dr. Bruce Lindsey had been a source of

continued encouragement and resourceful advice. It truly was an

honor and privilege to have been a member of your department.

A heart felt thanks also goes to Dr. Kersti K. Linask, she not only

kindled my interest in non-muscle myosins, but also kindly hosted me

in her lab where I learned the techniques of immuno staining. Without

her help the work on non-muscle myosins would not have been

possible. Similarly, the electron microscopy images would not have

been possible without the help of Dr. Truitt Sutton; and I would like to

express my thanks to him.

I also owe a debt of gratitude to Carol Landon, who has ‘held my

hand’ during those early days of mouse surgery. Your superb surgical

and technical skills will always inspire me and I thank you very much

for all you have done. I also thank Barbara Nicholson for her kindness

and patience in addressing the seemingly endless questions. Bridget

and Joyce have always been most helpful in handling the numerous

paper work that inevitably come up in the course of a graduate study.

I would also like to thank the staff at the office of graduate and post

doctoral affairs, Kathy Zahn, Susan Chapman and Francjesca Jackson

for their continued assistance through the myriad of formalities that

arose at every juncture.

Once again, I would like to acknowledge my immediate and

extended family including my brothers and sisters, uncles and aunts

and grandparents who have kept my spirits up when at times I felt it

may not be worth it. Thanks for all the support. I will always treasure

your love and will forever be grateful.

i

TABLE OF CONTENTS

LIST OF TABLES iv LIST OF FIGURES v ABSTRACT viii CHAPTER ONE – INTRODUCTION AND BACKGROUND Body Fluid Volume Sensing and Regulation 1 The Natriuretic Peptides 5 Natriuretic Peptide Receptors 11 Tissue Distribution of Natriuretic Peptides and their Receptors 15 Physiological Effects of the Natriuretic Peptides 15 Gastrointetsinal Effects of Natriuretic Peptides 17 CHAPTER TWO – MATERIALS AND METHODS Measurement of Gastric Contractility and Intragastric pressure 22 Experimental Animals 22 Surgical Preparation 23 Positive and Negative Controls 25 Blood Pressure Measurement 28 Measurement of Gastric Emptying 28 Measurement of Absorption 29 Induction of Myocardial Ischemia (Myocardial Injury) 31

ii

Plasma BNP measurement by Radioimmunoassay 32 Immunostaining for Non-Muscle Myosin 33 Statistical Methods 36 CHAPTER THREE- THE EFFECT OF NATRIURETIC PPETIDES ON INTRAGASTRIC PRESSURE, GASTRIC EMPTYING AND ABSORPTION Introduction 37 Results 39 Discussion 56 CHAPTER FOUR – THE ROLE OF BNP IN THE GASTROINTETSINAL MANIFESTAION OF MYOCARDIAL INJURY Introduction 62 Results 65 Discussion 72 CHAPTER FIVE – BNP AND NON MUSCLE MYOSINS IN THE GASTROINTETSINAL TRACT Introduction 77 Results 85 Discussion 92 CHAPTER SIX – SUMMARY AND CONCLUSIONS 98 PERSPECTIVE 100 REFERENCES CITED 102

iii

BIBLIOGRAPHY 128 APPENDICES 129 Appendix A - Abbreviations Used 130 ABOUT THE AUTHOR End Page

iv

LIST OF TABLES Table 1 Composition of modified Krebs’s solution 26 Table 2 Molar concentration of modified Krebs solution 27 Table 3 Peptides used in this study 30

v

LIST OF FIGURES Figure 1 Linear peptide structure of the natriuretic peptides 6 Figure 2 Schematic representation of the synthesis of natriuretic peptides ANP, BNP and CNP 8 Figure 3 Schematic representations of the natriuretic peptide receptors 12 Figure 4 A model for natriuretic peptide receptor activation 13 Figure 5 Typical pressure wave pattern of a basal gastric

contraction 39 Figure 6 Simultaneous recording of blood pressure and intragastric pressure 40 Figure 7 The effect of intravenous administration of Gherelin

on intragastric pressure 41 Figure 8 Three individual experiments showing intragastric pressure after a 10 ng/g iv bolus of ANP, BNP and CNP 42 Figure 9 Typical recoding of post peptide injection gastric contraction wave pattern 43 Figure 10 The effect of C-ANP4-23 on gastric pressure 44 Figure 11 BNP did not change gastric pressure in

NPR-A knock out mice 45

vi

Figure 12 Determination of baseline and peak intragastric pressure before and after peptide injection 46 Figure 13 Mean reduction in intragastric pressure following

intravenous BNP injection 48 Figure 14 Mean reduction in intragastric pressure following

intravenous ANP injection 49 Figure 15 Mean reduction in intragastric pressure following

intravenous CNP injection 50 Figure 16 The Effect of BNP on gastric emptying 51 Figure 17 Dose dependent reduction of gastric emptying 52 Figure 18 The Effect of BNP on absorption 53 Figure 19 Comparison of plasma fluorescence following intravenous FITC-dextran injection; BNP vs. Vehicle 54 Figure 20 Representative histological appearance of the myocardium 14 days after cryoinfarction 66 Figure 21 Comparison of percent gastric emptying in Sham vs. MI wild Type mice, 1 week post MI 67 Figure 22 Comparison of percent gastric emptying in Sham vs. MI Wild Type mice 68 Figure 23 Comparison of percent gastric emptying in Sham vs. MI NPR-A knockout mice 69 Figure 24 Comparison of absorption measured in relative plasma fluorescence units. Sham vs. MI, WT mice 70 Figure 25 Comparison of absorption measured in relative plasma fluorescence units. Sham vs. MI, NPR-A KO 71

vii

Figure 26 Schematic diagram of non muscle myosin-II 78 Figure 27 Schematic depiction and electron micrograph of

the structures of intestinal villi 79 Figure 28 Cytoskeletal structures in the intestinal microvillus 80 Figure 29 Ultra structures of the microvilli cytoskeleton 81 Figure 30 Transmission Electron micrographs of intestinal villi in various state of contraction 82 Figure 31 Schematic depiction of tight junctions 83 Figure 32 Toluidine blue stained 3µm sections of a mouse

intestinal tissues 85 Figure 33 Higher magnification view of jejunal villi 86 Figure 34 Electron micrographs of the intestinal microvilli

Control vs. BNP treated 87 Figure 35 Jejunal villi immunostained for non muscle

myosin type IIB – Control 88 Figure 36 Comparative images of control vs. BNP treated

jejunal villi 89 Figure 37 Jejunal villi and smooth muscle bundles

immunostained for non muscle myosin 90 Figure 38 A 3 µm sections of a mouse jejunum immunostained for non muscle myosin type IIB. Control vs. BNP 91

viii

NATRIURETIC PEPTIDES AS A HUMORAL LINK BETWEEN THE

HEART AND THE GASTROINTETSINAL SYSTEM

Anteneh Addisu ABSTRACT

Natriuretic peptides are a family of hormones released by several

different tissues and exert various physiological functions by coupling

with cell surface receptors and increasing intracellular cyclic gyanylyl

monophosphate (cGMP). Atrial Natriuretic Peptide (ANP) and B-type

Natriuretic Peptide (BNP) are released in response to mechanical

stretch of the atrial or ventricular myocardium, respectively and their

plasma level is markedly elevated during myocardial infarction and

heart failure. Heart failure in turn is associated with symptoms

suggestive of perturbed gastrointestinal function such as nausea,

indigestion and malabsorption.

Intragastric pressure was monitored using a balloon catheter in

anesthetized mice. The pressure before and after treatment with a 10

ng/g intravenous dose of ANP, BNP, CNP or vehicle was compared and

analyzed. All the natriuretic peptides significantly decreased

intragastric pressure compared to vehicle. These effects were

ix

attenuated or absent in natriuretic peptide receptor type-A (NPR-A)

knockout mice. Furthermore, the effect of BNP on gastric emptying

and intestinal absorption was examined using a meal consisting of

fluorescence labeled dextran gavage fed to awake mice. BNP

significantly decreased gastric emptying and absorption as compared

to vehicle control. Using a cryoinfarction acute myocardial injury

model, our investigation showed that mice with acute cryoinfarction

had a significantly lower gastric emptying and absorption of a gavage

fed meal compared to sham. Circulating BNP levels were significantly

higher in the infarcted mice compared to controls. Immunostaining

showed amplified distribution of the non-muscle myosin type-II (MCH-

II) in BNP treated mice. MCH-II is involved in movement of intestinal

villi.

In summary, natriuretic peptides in general and BNP in

particular, have gastrointestinal effects including reduced gastric

contractility, emptying and absorption. In addition to their effect on

smooth muscle relaxation mediated by cGMP, natriuretic peptides

appear to have an effect on distribution of MHC-II in cells of the

intestinal villi.

We postulate that these effects are aimed at mediating a

‘communication’ between the cardiovascular and gastrointestinal

systems. Further characterization of such a link will not only add a

x

dimension to the understanding of the pathophysiology of heart failure

but also enhances the search for further therapeutic targets.

1

CHAPTER ONE

INTRODUCTION AND BACKGROUND

Since maintaining body fluid volume is one of the most tightly

regulated physiological functions, animals have evolved with

increasingly sophisticated mechanisms for rapidly detecting and

responding to changes in fluid volume. In vertebrates, the vascular low

pressure volume sensors are embedded within the walls of the

myocardium and large pulmonary blood vessels. These volume sensors

detect changes in pressure (or volume) induced stretch of the vessel

and myocardial wall. Signals from these receptors travel via afferent

fibers of the vagus nerve to the solitary tract nucleus of the medulla

oblongata in the brain stem (Donald & Shepherd, 1979; Thorén et al.,

1976). Feedback activation or deactivation of neural signals back to

the target organs then modulate mechanisms that result in more or

less diuresis, natriuresis or vascular tone depending on the body’s

requirement. However even as early as the first half of the last century

there were studies that suggested there may be humoral (non

neuronal) system of receptors that responded to the change in

2

pressure or volume stretching the atrial myocardium. For instance,

expansion of plasma volume by blood transfusion to healthy dogs was

shown to increase urine flow (Metcalf, 1944). Similarly infusion of

isooncotic solution of albumin was shown to produce marked diuresis

in human volunteers (Welt & Orloff, 1951). Strauss et al reported that

infusion of isotonic (0.9%) saline to healthy volunteers increased

“water diuresis’’ during recumbency; and they noted that the body

position influenced how the infused volume is distributed and sensed

by the atrium (Strauss et al., 1951). The role of atrial stretch in such

volume induced diuresis was already experimentally established in the

mid 1950s (Henry & Pearce, 1956). Henry and colleagues used a

balloon to distend the atrium in dogs and showed that urine output

was increased corresponding to the degree of atrial distension. In their

paper entitled “The possible role of cardiac atrial stretch receptors in

the induction of changes in urine flow”, Henry and Pearce noted that

vagotomy did not abolish the diuretic response to isotonic infusions;

and this led to their conclusion in 1956 that “the body may be

provided with other receptors or pathways by which it receives

information which makes possible the regulation of blood volume by

the control of urine flow”.

Nevertheless, until only a little over two decades ago; the

outcome of the activation of atrial and venous stretch receptors was

3

believed to almost entirely depend on the modulation of autonomic

nerve activity or varying levels of secretion of antidiuretic hormone

(ADH) from the neurohypohysis.

In 1980 Adolfo de Bold reported his seminal discovery that

intravenous injection of extract from the rat atrium produced a marked

diuresis (de Bold et al., 1981).

Thus it became apparent that the atrium produced a diuretic and

natriuretic substance that was released into the circulation and worked

through a receptor system that is distinct from the neurally mediated

feedback inhibition. This natriuretic ‘substance’ was initially termed

atrial natriuretic factor (ANF). With this discovery, it also became

apparent that the heart has an endocrine function and directly

‘communicates’ with the kidney by virtue of its own cardiac hormone

systems.

The presence of granular structures in the atrial myocytes of

several different species was one of the earliest findings of the advent

of electron microscopy (Jamieson & Palade, 1964; Kisch, 1956; Palade,

1961). Moreover, the possible relationship between atrial granules and

the degree of sensitivity of the atrium to volume or salt loading was

suggested as early as 1976 (Marie et al., 1976). However it was with

de Bold’s seminal experiments that the content of these atrial granules

was clearly hinted to be a potent natriuretic substance that directly

4

influenced renal handling of salt and water (de Bold et al., 1981; de

Bold., 1985). The atrial natriuretic factor was later determined to be a

peptide produced by the atrial myocardium (Currie et al., 1984; Flynn

TG, 1983; Misono et al., 1984) and it is currently more commonly

referred to as Atrial Natriuretic Peptide (ANP). The ANP gene was

subsequently sequenced revealing the remarkable similarity and

genetic conservation of the natriuretic peptides across many species

(Seidman et al., 1984).

5

The Natriuretic Peptides

The identification of ANP in 1985 was soon followed by the

discovery of a similar peptide in porcine brain (Sudoh et al., 1988)

hence termed as brain natriuretic peptide (BNP). However it was later

discovered that BNP was another cardiac hormone and is actually

absent in brains of some species (Ogawa et al., 1991; Ogawa et al.,

1990). In 1990, C-type natriuretic peptide (CNP) was isolated from the

brain of pigs, bullfrogs and two species of teleost fishes (Sudoh et al.,

1990; Suzuki et al., 1991; Yoshihara et al., 1990). The three

natriuretic peptides share a common structural feature, a conserved

17 amino acid ring with variable N- and C-terminal sequences (figure

1). The number of amino acid residues extending from the C terminal

is usually 5 for ANP, 6 for BNP and 0 for CNP with few exceptions.

Thus the C-terminal sequence appears to be a major determinant of

biological activity of the natriuretic peptides. CNP is the most

conserved of the three peptides across species and it is believed to

have evolved earlier in the phylogenetic tree, ANP and BNP being

derived from it at a later point in evolution. In recent years a fourth

natriuretic peptide, a 38 amino acid peptide known as deandropsis

natriuretic peptide (DNP) has been isolated from the venom of the

green mamba snake (Munagala et al., 2004). Immunoreactivity to DNP

has been shown in human and rodent plasma (Johns et al., 2007;

Schirger et al., 1999). However, a gene coding for it has not yet been

isolated and the physiological significance of DNP in humans (if any)

remains poorly understood.

Figure 1. Linear peptide structure of the natriuretic peptides

Adapted from (Cea, 2005)

In humans, the ANP and BNP genes are localized in tandem on

chromosome 1. Transcription of the ANP gene yields an mRNA that

encodes a 151 amino acid metabolic precursor known as preproANP.

6

7

PreproANP is rapidly converted to a 126 amino acid peptide proANP.

ProANP is the predominant storage form of ANP and the major

constituent of atrial granules. Atrial distension is the major signal for

the release of ANP (Anderson et al., 1986; Dietz, 1984; Dietz et al.,

1991; Kinnunen et al., 1992; Sato et al., 1986). When such a signal is

sensed; proANP is cleaved by a cardiac serine protease (corin) into an

amino fragment pro-ANP (amino acids 1-98) and ANP, the biologically

active fragment (amino acids 99-126) (Bloch et al., 1986; Vuolteenaho

et al., 1985). Further cleavage of proANP (1-98) results in more

peptide fragments (1-30) and (31-67); these fragments also have

biological activity and neutralization of proANP 1-30 was shown to

exacerbate hypertension in spontaneously hypertensive rats (Dietz et

al., 2001; Dietz et al., 2003; Dietz et al., 1995; Dietz & Villarreal,

1995; Vesely et al., 1999). The amino acid sequences of the main

biologically active hormone ANP 99-126 are identical in all mammalian

species except at residue 110, which is methionine in humans

(Kangawa et al., 1984; Lewicki et al., 1986; Vlasuk et al., 1986) but

isoleucine in rats, mice and rabbits (Oikawa et al., 1985; Seidman et

al., 1984; Yamanaka et al., 1984). Alternate processing of the prepro

ANP in the kidney produces a 32 amino acid peptide known as

urodilatin. Urodilatin is secreted into the lumen of the distal nephron

(medullary collecting duct) where it is believed to be involved in

regulation of sodium and water absorption in the kidney (Forssmann et

al., 1998; Kuhn, 2005). Physiologically plasma ANP is markedly

increased in response to pressure or volume overload or in

pathological states such as heart failure or ventricular hypertrophy.

However the plasma half life is rather short, averaging less than 3

minutes which indicates that the release of ANP serves to counteract

the effect of acute pressure or volume overload (Lang et al., 1985;

Ruskoaho, 1992).

Figure 2. Synthesis of natriuretic peptides and prohormones

Adapted from Koller and Goeddel 1992

8

9

Human BNP is produced as a 132 amino acid residue preproBNP

that is subsequently cleaved to a 108 amino acid prohormone.

Additional cleavage yields the 32 amino acid active hormone and an

inactive 76 amino acid amino terminal (NT) fragment sometimes

known as NT-proBNP (Saito et al., 1989; Seilhamer et al., 1989;

Sudoh et al., 1988). Ventricular BNP is not stored in granules in the

myocytes, instead BNP production is regulated at the transcription

level by various stimuli, the main stimulus for BNP synthesis is stretch

of the ventricular wall by volume and/or pressure overload (Grépin et

al., 1994; Thuerauf et al., 1994). Even though the BNP gene is located

in tandem with the ANP gene; BNP expression doesn’t always parallel

ANP gene expression. The response of the BNP gene is quicker than

that of ANP suggesting a more acute and sustained role for BNP in

response to a volume overload. Increase in BNP mRNA is detected

within one hour of increased ventricular wall tension induced by

increased venous volume or acute myocardial infarction (Hama et al.,

1995; Nakagawa et al., 1995). BNP has a much longer half-life (than

ANP) of about 20 minutes (Espiner et al., 1995) and with sustained

cardiac stress; as in the case of heart failure, BNP mRNA levels have

been shown to remain increased (Tokola et al., 2001). BNP secretion

takes two forms, constitutive, where the BNP is secreted as fast as it is

being formed and a regulated pathway where the BNP is stored in

10

granules prior to being secreted (Kelly, 1985). Ventricular myocytes

shift between the two pathways depending on the stimulus, the

constitutive secretion being called upon when there is acute need for

BNP secretion and the regulated pathways operating when there is a

sustained myocardial stress such as chronic heart failure (Bloch et al.,

1986; Kelly, 1985).

CNP is mainly expressed in the brain, chondrocytes and

endothelial tissue. Myocardial tissue has a much smaller amount of

CNP than ANP or BNP and CNP is not stored in granules (Yandle,

1994). In humans, proCNP contains 103 amino acid residues and it is

cleaved to a 53 amino acid CNP in the brain, the heart and endothelial

tissue. Further cleavage yields a 22 amino acid peptide which is the

predominantly circulating form of CNP (Stingo et al., 1992; Totsune et

al., 1994; Wu et al., 2003). While CNP 53 is believed to be

predominantly a neurotransmitter, it is also involved in bone and

cartilage growth; CNP 22 is mainly involved in autocrine and paracrine

regulation of vascular tone (D'Souza et al., 2004). CNP has less effect

on diuresis and natriuresis than ANP or BNP and a more potent effect

on smooth muscle relaxation, it is thus believed to be mainly involved

in the regulation of coronary vascular tone in the heart (Clavell et al.,

1993; Komatsu et al., 1992; Sudoh et al., 1990).

11

Natriuretic Peptide Receptors

The natriuretic peptide receptors are members of the

transmembrane guanylyl cyclase family of enzymes that are widely

distributed in human and animal tissues. There are at least seven

different guanylyl cyclase enzymes identified so far (Anand-Srivastava

& Trachte, 1993; Garbers et al., 2006). Natriuretic peptide receptor

(NPR) types A and B (NPR-A and NPR-B) are structurally similar and

exist as homodimers or homotetramers in intact mammalian cells

(Chinkers & Wilson, 1992; Iwata et al., 1991; Katafuchi et al., 1994).

The receptor consists of an extra cellular ligand-binding domain, a

membrane spanning domain and intracellular kinase-like and guanylyl

cyclase domains (Nagase et al., 1997; Potter, 2005). Both ANP and

BNP bind to NPR-A although ANP is known to bind with at least 10

times more affinity to NPR-A (Kambayashi et al., 1990; Nakao et al.,

1991). NPR-B has similar structure to NPR-A, but selectively binds to

CNP. Natriuretic peptide receptor type-C (NPR-C) has a short

intracellular sequence that doesn’t have a guanylyl cyclase domain.

Figure 3. Schematic representation of the natriuretic

peptide receptors

Adapted from Potter et al., 2006

Ligand binding to the receptor leads to the activation of the

guanylyl cyclase and results in a conformational change. The C-

terminal guanylyl cyclase then comes into a tight association that

leads to conversion of guanosine triphospate (GTP) to 3´5´- cyclic

guanosine monophosphate (cGMP) (Chinkers & Wilson, 1992; Foster et

al., 1999). The ATP binding site in the kinase homology domain (KHD)

is believed to be essential for ligand-induced signal transduction and

12

deletion of the KHD depresses the guanylyl cyclase catalytic region

(Chinkers & Garbers, 1989). Phosphorylation of amino acid residues in

the KHD of the receptor is essential for normal function and

dephosphorylation appears to be one method of the natriuretic peptide

receptor desensitization (Potter & Garbers, 1992; Potter & Hunter,

1998).

Figure. 4. A model for natriuretic peptide receptor activation

Adapted from Silberbach and Roberts 2001

13

14

The end result of these mechanisms of receptor activation is

increased concentration of intracellular cGMP; cGMP in turn exerts its

physiological effects by binding to one of three cGMP binding proteins.

cGMP dependent protein kinases (PKG), cGMP binding

phosphodiesterases (PDE) and cyclic nucleotide gated ion channels

(Pfeifer et al., 1996; Rybalkin et al., 2003; Smolenski et al., 1998).

As mentioned earlier, natriuretic peptide receptor type-C (NPR-

C) has a short intracellular sequence that doesn’t have a guanylyl

cyclase domain. The major role of NPR-C was initially believed to be as

a clearance receptor and regulation of the plasma concentration of ANP

and BNP through receptor mediated internalization and degradation

(Matsukawa et al., 1999). However recent evidence suggests that

NPR-C is involved in signaling that leads to reduction of adenylyl

cyclase activity through activation of inhibitory G protein (Gi) (Anand-

Srivastava & Trachte, 1993; Rose & Giles, 2007).

The natriuretic peptides are cleared from the circulation by three

mechanisms. Endocystosis and degradation by coupling with NPR-C,

enzymatic cleavage by neutral endopetidases and by glomerular

filtration and excretion in the urine (Boerrigter & Burnett, 2004; Freda

& Francis, 2006).

15

Tissue Distribution of Natriuretic Peptides and their Receptors

The atrial and ventricular cardiomyocytes are the main sites of

production and storage of ANP and BNP, respectively. This was

confirmed through seminal experiments that showed that atrial

appendectomy resulted in marked reduction of both the plasma levels

of ANP and the diuretic and natriuretic effect seen in response to a

volume load with isotonic saline (Veress & Sonnenberg, 1984;

Villarreal et al., 1986). Nevertheless, ANP and BNP mRNA have been

detected in several non-cardiac tissues including the adrenal glands,

the kidneys, lung, the gonads, lymphoid tissue and the gut (Gerbes et

al., 1994; Gower et al., 1994; Gower et al., 2003; Li et al., 2006;

Nguyen et al., 1990; Sharkey et al., 1991; Vollmar, 1990). Natriuretic

peptide receptors have also been detected in the gastrointestinal (GI)

tract of both non-mammalian and mammalian species including

humans (Gower & Skvorak, 1997; Li & Goy, 1993; Lowe et al., 1989;

Ohyama et al., 1992; Schulz et al., 1998).

Physiological Effects of the Natriuretic Peptides

Vascular relaxation is one of the most important physiological

actions of the natriuretic peptides. Binding of cGMP to PKG is known to

regulate ion channels in vascular smooth muscle cells with a cascade

of molecular events that culminate in a lower concentration of

16

intracellular calcium. These effects include reduction of calcium influx,

increase of calcium efflux and promotion of calcium sequestration in

sarcoplasmic reticulum (Tamaoki et al., 1997). Reduced vascular tone

and vasodilatation in turn results in lower total peripheral vascular

resistance which has important physiological benefit on the

cardiovascular system; especially in the face of cardiac ischemia or

volume overload. Such vascular relaxation and vasodilatation will also

result in reduced pressure in the renal afferent arterioles thereby

increasing glomerular filtration, promoting diuresis and natriuresis. In

addition to these effects on vascular smooth muscle cells, the direct

effect of ANP and BNP on renal tubules and mesangial cells as well as

inhibition of the renin angiotensin aldosterone (RAAS) system results

in decreased sodium absorption from the renal tubules (Lohmeier et

al., 1995; Zeidel, 1993).

ANP and BNP are also known to decrease sympathetic outflow

and catecholamine release from peripheral sympathetic neurons which

benefits the cardiovascular system since it leads to lower blood

pressure, decreased heart rate and natriuresis (Levin et al., 1988).

Further support for the importance of the natriuretic peptides in blood

pressure and blood volume regulation comes from data showing that

NPR-A transgenic mice show chronic hypertension and ventricular

hypertrophy (John et al., 1995). NPR-A transgenic mice were recently

17

shown to have an upregulation of the angiotensin converting enzyme

(ACE) and angiotensin II type 1a receptor (AT1) mRNA by up to four-

fold signifying the importance of the natriuretic peptides in

counteracting the effect of the renin angiotensin aldosterone (RAS)

system (Vellaichamy et al., 2007). It is also shown that NPR-A

knockout mice exhibit dysregulation of matrix metalloproteinases and

tissue inhibitors of metalloproteinases; enzymes involved in the

regulation of collagen synthesis and organization of myocardial fibrils

(Li et al., 2000; Spinale, 2002). As a result NPR-A knockout mice show

increased tendency towards myocardial fibrosis, hypertrophy and

eventually heart failure that leads to premature death as compared to

the wild type mice.

Gastrointestinal Effects of Natriuretic Peptides

Natriuretic peptides are known to have several effects on

contractile and absorptive functions in the GI tract. ANP has been

shown to inhibit intestinal sodium and water absorption in teleost and

mammalian intestines (Barros et al., 1990; Matsushita et al., 1991;

O'Grady et al., 1985). A decrease in jejunal water absorption in

response to intravascular volume overload was also shown in rats.

Such a decrease in transjejunal water absorption was absent in rats

that underwent right atrial appendectomy and this effect returned

18

when ANP was exogenously administered (Pettersson & Johnsson,

1989). Intravenous administration of BNP and CNP has also been

shown to decrease jejunal electrolyte and water absorption in dogs

(Morita et al., 1992). Earlier investigations have also shown that

injection of either ANP or BNP into the cerebral ventricles inhibits thirst

induced by water deprivation or angiotensin (Antunes-Rodrigues et al.,

1985; Itoh et al., 1988; Zhu & Herbert, 1996). Furthermore, ANP and

BNP have been shown to have an indirect effect on uptake and

excretion of sodium and water through inhibition of vasopressin and

aldoseterone secretion (Januszewicz et al., 1986; Nguyen et al.,

1989).

The effect of natriuretic peptides on contractility of GI smooth

muscle has been documented beginning with the early discovery of

these peptides and their receptors in the GI tract (Scott & Maric,

1991). Subsequent studies have confirmed these findings showing that

ANP, BNP and CNP all inhibit contractility of isolated gastric and

intestinal smooth muscle cells from different species including

humans(GuoCui et al., 2003; GuoJin et al., 2003; Yasuda et al.,

2000).

Apart from the traditional ‘cardiac’ natriuretic peptides ANP, BNP,

and CNP; the GI tract is also known to be a source of two peptides

with structures very similar to the natriuretic peptides. Guanylin and

19

uroguanylin were first isolated from rat intestine and opossum urine

and later found to be widely distributed in non-mammalian and

mammalian species including humans (Beltowski, 2001; Date et al.,

1998). In humans, uroguanylin is mainly expressed in the

enterochromaaffin (EC) cells of the duodenum (where ANP is also

expressed), whereas guanylin is expressed in the jejunum and the

colon (Beltowski, 2001; Li et al., 2006). Uroguanylin is secreted in

response to oral salt load and the circulating hormone is known to

mediate sodium balance in the post-prandial state by increasing renal

excretion of sodium and potassium while the luminally secreted

hormone leads to increased chloride and bicarbonate secretion in to

the lumen of the intestine (Beltowski, 2001; Forte et al., 1996).

Uroguanylin and guanylin mRNA are detectable in the atrial and

ventricular myocardium and interestingly, plasma levels of guanylin

and uroguanylin are increased during heart failure and renal failure

(Beltowski, 2001; Forte et al., 1996). Both guanylin and uroguanylin

receptors are membrane bound guanylyl cyclase enzymes and

activation of these receptors leads to increased intracellular cGMP

(Carrithers et al., 1999; Forte et al., 1996; Forte et al., 1999). Both

synergistic and antagonistic interaction between ANP and BNP on one

hand and uroguanylin and guanylin on the other have been shown and

there has been some suggestion that this evidence points to a

20

probable regulatory link between the kidney and the GI tract in the

process of sodium and water balance (Santos-Neto et al., 2006).

Taken together, the remarkable genetic conservation of the

natriuretic peptides, the stimuli for their release and their effect on

sodium and water uptake is strong evidence that they serve crucial

physiological functions that conferred survival benefit earlier in

evolutionary times and have since evolved to be important conveyers

of signals among the various organ systems involved in cardiovascular

homeostasis.

While the cardio-renal link is now sufficiently well established,

there have been very few studies conducted to assess the functional

significance of the effect of natriuretic peptides on the GI tract and

how this all fits in the bigger scheme of volume regulation. The early

observations on the effect of natriuretic peptides as well as recent

evidence showing the presence and role of a possible ‘intestinal’

natriuretic peptide system raise several intriguing questions. If the

heart sends humoral signals to the kidney to decrease sodium

absorption, shouldn’t it also use the same signals to limit sodium and

water absorption from the GI tract? GI symptoms such as nausea,

indigestion and malabsorption are frequent clinical findings in the face

of acute heart attack or chronic heart failure, states where BNP levels

are elevated. Could BNP be responsible for some of these symptoms?

21

If so, could there be potential targets of therapy for heart failure in the

GI tract?

Our studies will provide evidence that the effect of natriuretic

peptides on gastric and intestinal smooth muscle cell contractility has a

functional dimension. By directly testing the effect of ANP, BNP and

CNP on gastric contractility, gastric emptying and absorption we show

that these peptides do decrease gastric emptying and absorption.

Moreover BNP, a peptide with increasing clinical utility, is shown here

to cause decreased gastric emptying and intestinal absorption

following acute myocardial injury using a whole animal cryo induced

acute MI model. While our data suggests that these effects may be

mediated by NPR-A, we also show data that suggests a more direct

effect on absorptive structures at the level of intestinal microvilli.

Further characterization of the signals involved in these functions not

only adds a new dimension to cardiovascular research but could also

lead to identification of new therapeutic targets for the treatment of

heart failure.

22

CHAPTER TWO

MATERIALS AND METHODS

Measurement of Gastric Contractility and Intragastric Pressure

These protocols were approved by the University of South Florida

Institutional Animal Care and Use Committee.

Experimental Animals

NPR-A Knockout (KO) mice were obtained from our resident

colony that was founded with pathogen-free breeding pairs and were

genetically monitored by PCR of tail-snip DNA. The generation of NPR-

A knockout mice has previously been described in detail (Lopez et al.,

1995). Wild type (C57BL/6) mice were purchased from commercial

sources. The wild type (WT) mice were all males with ages ranging

from 8 to 12 weeks and weight ranging from 18-26 grams at the time

of the experiment. The NPR-A KO mice ranged from 10 to 32 weeks in

age and 24-38 grams in weight at the time of the experiment, with

equal number of male and female KO mice in the experiment and

vehicle group. The parental strain of the knockout mice was C57BL/6.

23

Surgical Preparation

In order to objectively determine the effect of natriuretic

peptides on gastric contractility, a method of measuring intragastric

pressure before and after peptide injection was developed as follows.

The mice were anesthetized with sodium pentobarbital (0.9 mg/10 gm

body weight) given intraperitoneally (ip) and supplemental doses of

0.5 mg given ip as needed. The mice were then placed on a

temperature controlled surgical table and a tracheotomy was

performed using a 20 ga. Luer-stub adapter. The right jugular vein

was catheterized with polyethylene (PE)10 tubing for injections and

infusions and the right carotid was catheterized with a 5 cm piece of

PE tubing (o.d. 0.012”, i.d. 0.006”: Braintree Scientific, Inc.) attached

to a 12-18” segment of PE 50 tubing for arterial blood pressure

measurements. A 10 mm left sub-costal skin incision was made to

access the stomach. A 3 mm vertical incision of the stomach fundus

was made with cautery carefully choosing a site that has minimal or no

visible blood vessels. A 2-3 mm latex balloon fitted with PE tubing and

primed with saline was inserted into the stomach. The balloon was

then minimally distended by adding 20-25 µl of saline and attached to

a standard pressure transducer (Gould/Statham DB25). The

intragastric catheter was held in place by the minimal distension of the

balloon and suturing the stomach incision was not necessary. The skin

24

incision was closed with 1-2 interrupted surgical sutures. Following

these surgical preparations, the mice received a 100 µl bolus of 0.9%

saline intravenously (iv) via the jugular catheter and then allowed a

one hour equilibration period while being infused with 0.9 % saline iv

at 5 µl/minute. The saline bolus and infusion were administered to

compensate for blood loss associated with the surgical procedure and

maintenance fluid requirement. Arterial blood pressure, heart rate and

intragastric pressure were monitored continuously via the carotid and

intragastric catheters and recorded on a data acquisition system

(DATAQ Instruments, Akron, OH).

The change in intragastric pressure was measured as the

difference between the peak and baseline pressure. The

measurements were taken for three 30 minute periods. The basal

gastric pressure was measured from 30 to 0 minutes before injection.

Post injection period was from 10 to 40 minutes after injection to

correspond with the peak plasma level of the peptides and the

recovery period was from 90 to 120 minutes after injection

corresponding to the period later than 5 peptide half-lives (12). The

changes in intragastric pressure during each of the three periods were

averaged for each mouse and the differences in intragastric pressure

between the experimental and vehicle groups during the three periods

were compared using a one way ANOVA with Fisher’s least significant

25

difference test (LSD) used as a post hoc test. A “p” value of <0.05 was

considered the criteria for statistical significance.

Positive and Negative Controls

Gherelin, a hormone with known prokinetic gastrointestinal

effect in rodents, was used as a positive control to ascertain that the

waves of contraction we observed and recorded were indeed changes

in gastric motility (Depoortere et al., 2005). Gherelin (Rat, Phoenix

pharmaceuticals, C# 031-31, Lot # 423341) was administered at a

dose of 50 µg/kg body weight in 100 µl of vehicle iv, a dose previously

established to increase gastric motility in rodents.

The vehicle was used as a negative control. The vehicle

consisted of modified Krebs-Hensledt bicarbonate buffer equilibrated

final PH = 7.4, dissolved in the order shown in table 1.

26

Table 1. Composition of modified Kreb’s solution (g/l)

CaCl2 0.220g

MgSO4 0.144g

KCl 0.224g

KH2PO4 0.163g

NaCl 6.72g

NaHCO3 2.1g

Glucose 1g

Albumin 1g

27

Table 2. Millimolar concentration of modified Kreb’s solution

Na+ 140

K+ 4.2

Ca2+ 1.5

Cl- 123

Mg2+ 1.2

HCO3 25

H2PO4 1.2

SO4 1.2

Glucose 2.5

Albumin

(BSA fraction V)

0.1%

28

Blood Pressure Measurement

Mean arterial pressure was continuously monitored using an

intra-carotid catheter and recorded. Average blood pressure was

measured for the same three periods as for the gastric pressure

measurement. Pre vs. post peptide injection blood pressures were

compared to ascertain that our findings were not confounded by

differences in blood pressure.

Measurement of Gastric Emptying

Conscious WT and NPR-A KO mice (n = 5 in each group) were

given BNP at dose of 10 ng/g through the tail vein dissolved in 100 µl

of vehicle (modified Krebs solution) or the vehicle alone and

immediately gavaged with 0.1 ml of 0.5 Mmol 70 kDa fluorescein-

isothiocyanate (FITC)-dextran. The 70 kDa FITC-dextran is known to

be non-diffusible across intestinal membrane thus suitable for

measurement of emptying (Thorball, 1981). Thirty minutes after the

gavage meal, the animals were euthanized and the stomach was

separated and the intestine divided into 8 equal segments, each

flushed with 3 ml of PBS and centrifuged for 10 minutes. Fluorescence

of the supernatant fluid was measured and the percent gastric

emptying rate was compared in BNP treated vs. control for both WT

and NPR-A KO mice. This method of evaluating gastric emptying has

been previously established (Aube et al., 2006).

29

Measurement of Absorption

Conscious WT & NPR-A KO mice (n = 5 in each group) were

given 10 ng/g of BNP through the tail vein dissolved in 100 µl of

vehicle (modified Krebs solution) or the vehicle alone. The mice were

then gavaged with 0.01 ml/g of a solution containing 22 mg/ml of 4

kDa FITC-dextran. Blood was collected via cardiac puncture under

pentobarbital anesthesia. Fluorescence was quantified using relative

fluorescence units in the plasma. The plasma fluorescence measured 1

hour after gavage feeding in WT and NPR-A KO mice was compared for

BNP treated vs. control. Similar comparisons were made for the

subsequent experiments between sham vs. Mi in both WT and NPR-A

KO mice. The group differences were analyzed using a t-test with

p<0.05 considered the significant level for statistical difference. This

method of evaluating gastric absorption has been previously

established (Aube et al., 2006).

We also compared the fluorescence of a 50 µl plasma sample

taken 1 hour after iv administration of 100 µl of 0.5 mmol 4kDa FITC-

dextran in BNP treated vs. vehicle WT mice. The purpose of this test

was to rule out the possibility that the changes in plasma fluorescence

were produced by other actions of BNP such as increased excretion,

redistribution or metabolism of the dextran as this effect of BNP has

been previously established (Huxley et al., 1987).

30

The concentration of fluorescein was determined using a

fluorimeter (FLUOstar Galaxy, BMG Labtechnologies) with an excitation

wavelength at 485 nm and an emission wavelength of 520 nm using

serially diluted samples of the marker as standard.

Table 3. Peptides used in this study

ANP Rat ANP, Sigma, P # A8208

BNP Rat BNP-32, Phoenix Pharmaceuticals, 011-14,

Lot # 421752

CNP Rat 32-53, Bachem, P # H-1296, Lot # B00656

c-ANF 4-23 Rat, Phoenix Pharmaceuticals

Gherelin Rat, Phoenix Pharmaceuticals, C # 031-31, Lot # 423341

In the initial experiments, the peptides were administered

intravenously at 10 ng/g body weight. This dose was chosen to raise

and sustain the plasma levels above 500 pg/ml; a level that is

consistent with a greater than 90% likelihood of heart failure (for BNP)

in humans (Maisel & Mehra, 2005). For subsequent dose response

experiments we administered BNP at 1, 5, 10 and 100 ng/g body

weight iv.

31

Induction of Myocardial Ischemia (Myocardial Injury)

Wild-type and NPR-A knockout mice were anesthetized with 2-

3% isoflurane-oxygen flow at 500ml/minute. A 2 mm incision was

made through the skin, just distal to the 4th and 5th intercostal space.

Blunt dissection through all thoracic musculature, using a fine-tipped

instrument was performed to accommodate passage of a probe

induction catheter (PIC). Using digital pressure, the distal tip of the

PIC (catheter cap in place) was flattened as much as possible (to aid in

atraumatic insertion), and the PIC was then passed through the

muscle wall. Prior to removing the cap, the operator pinched the

proximal tip of the PIC shut (to aid in the prevention of

pneumothorax). The oxygen flow was then turned up to 2 L/min. The

liquid-nitrogen-cooled probe was then quickly threaded through the

PIC, to the full length of the PIC. If needed, the PIC was then gently

repositioned so the bevel and the cooled probe were in direct contact

with the left ventricle of the heart. Correct placement of the probe,

with subsequent cardiac thermal injury, was confirmed as the super

cooled probe "grabbed" the warmer tissue of the heart, and remained

affixed until the probe had warmed enough for release. After passive

release, the probe and PIC, as one unit, were quickly withdrawn and

the ribs were then immediately brought into apposition. A pre-loaded

1/2 cc syringe of tissue glue (VetBond™) was then used for closure.

32

The experiment to study emptying and absorption were performed at 1

and 2 weeks after this surgical procedure (cryo induced myocardial

injury/infarction).

Plasma BNP Measurement by Radioimmunoassay

Plasma BNP levels were measured by radioimmunoassay (RIA).

(Peninsula Laboratories, 5-2104 RIAS 9085). The assay is based upon

the competition of 125I-labeled peptide and unlabeled peptide

(unknown sample) binding to the limited quantity of antibodies specific

for peptide in each reaction mixture. As the quantity of peptide in the

unknown sample in the reaction increases, the amount of 125I-bound

peptide able to bind to the antibody is decreased. By measuring the

amount of 125I- bound peptide as a function of the concentration of the

peptide in standard reaction mixtures, a “standard curve” is

constructed from which the concentration of peptide in the unknown

sample can be determined.

The summary of the assay protocol is as follows:

1- A 100 µl of unknown sample is pipetted in to duplicates glass

test tubes 2- 100 µl primary antibody is added to unknown samples,

vortexed, and incubated overnight at 4°C.

33

3- 100 µl of the I 125 labeled peptide is added and incubated

overnight at 4°C. 4- Goat rabbit antiserum and normal rabbit serum is added and vortexed at 4°C and incubated for 90 minutes 5- 500 µl of RIA buffer is added, vortexed and centrifuged at

1700 x g for 20 minutes 6-The supernatant is aspirated off except in the total counts

tube 7- Use gamma counter to count the level of radioactivity 8- The data obtained from the gamma counter is analyzed using

the program Assayzap, Biosoft, GB, United Kingdom.

The detailed assay protocol is found in the product insert for the assay

kit (Phoenix pharmaceuticals, C# RK-011-17)

The kit we used has a cross reactivity of 41% with the mouse

BNP and total binding of 46%; calculations were made accordingly.

The detection range for this kit was 10-128 pg/ml.

Immunostaining for Non-Muscle Myosins

Two WT mice were given a bolus of BNP at 10 ng/g body weight

in 100 µl of modified Kreb’s solution followed by infusion of BNP at 1

ng/g for 30 minutes. One WT mouse was given 100 µl of vehicle and

given an infusion of vehicle for 30 minutes

34

1- The animals were then euthanized and intestinal tissue collected

and sectioned into duodenum, jejunum, ileum and colon

following anatomical demarcations.

2- The tissue is fixed in 4% paraformaldehyde over-night at 4°C.

3- The next morning the tissue is washed with phosphate buffered

saline (PBS) for 15 minutes twice at room temperature with

gentle rocking.

4- After the wash, the tissue is dehydrated and permeabilized as

follows. Tissue is placed in ascending concentration of 30, 50

and 70 % methanol each for 15 minutes. Then the tissue is

placed in a solution composed of 100 % methanol: DMSO: 30%

H2O2 made at a ratio of 4:1:1. The tissue is left in this solution

overnight at 4°C .

5- The next morning the tissue is washed in 70 % methanol for 30

minutes at room temperature with rocking.

6- Rehydration is accomplished by serially placing the tissue in 70

% methanol/PBS for 30 minutes with rocking, then 50 % of

methanol/PBS with rocking, then 1 ml of PBS for 30 minutes

with rocking, 1 ml of PBSMT (PBS, milk, Tween 20) 30 minutes

with rocking twice.

7- The tissue is then incubated overnight with 1 ml of primary

antibody diluted in PBSMT (1:250) with rocking at 4 °C.

35

8- The next morning the tissue is rinsed with PBSMT 2x with 1 ml

for 1 hour at 4°C, 4 x in 1 ml for 1 hour each at room

temperature.

9- Next the tissue is incubated overnight with 1 ml of the

Secondary antibody diluted in PBSMT (1:250) at 4°C while

rocking.

10- Next morning the tissue is washed as above with PBSMT

11- Next tissue is rinsed with PBT (PBS and Tween -20 equal

concentration).

12- Post fixing in 4 % paraformaldehyde in PBS at 4 ° C overnight.

13- Dehydration in the following sequence, 1 ml of PBT quick rinse, 1

ml PBT for 30 minutes at room temperature, 1 ml 50 %

methanol for 30 minutes, 1 ml of 70 % methanol for 30 minutes

at room temperature; 1 ml of 100 % methanol 30 minutes at

room temperature twice.

14- Plastic embedding is achieved by transferring the tissue from

100 % methanol to araldite embedding medium and kept in

medium for 3 hours. The tissue is then transferred into a fresh

embedding medium into a mold on araldite rafts and the mold is

kept overnight in 60° C oven to allow the araldite to harden.

36

15- The hardened plastic embedded tissue is then trimmed and

sectioned using a microtome at 1-3 µm thickness. The sections

pass automatically into water, the sections are transferred onto

a slide and allowed to dry for 5-10 minutes on the surface of a

hot plate. The first and last section are placed on one side and

stained with toluidine blue to guide orientation.

Details of the above technique are found in (Linask & Tsuda, 2000).

Statistical Methods

We used a one way analysis of variance (ANOVA) to compare the

average intragastric pressure at baseline, immediately after and later

than five peptide half-lives after peptide injections. Fisher’s least

significant difference (LSD) test is used as a post hoc test.

Two sample t-tests were used to compare the measures of gastric

emptying and absorption between peptide treated vs. vehicle treated

mice. Specifically percent gastric emptying between treated vs. vehicle

and average relative fluorescence units between the peptide treated

vs. vehicle groups were tested using a two sample t-test.

37

CHAPTER THREE

THE EFFECT OF NATRIURETIC PEPTIDES ON INTRAGASTRIC

PRESSURE, GASTRIC EMPTYING AND ABSOPRTION

INTRODUCTION Measurement of gastric pressure using an intragastric balloon

(manometry) offers the most direct way to quantify pressure changes

inside the stomach (Malagelada & Stanghellini, 1985; Mearin &

Malagelada, 1993). Other factors being constant, the measured gastric

pressure is directly proportional to the force of contraction of the

stomach wall (Mearin & Malagelada, 1993); therefore quantifying and

comparing the rate of change in pressure between experimental

groups yields a reliable and objective measure of gastric contractility.

However, due to the inherent variability of baseline pressure among

different animals, we further standardized the measurement by using

the difference between the baseline and peak of the gastric contraction

wave in our analysis. This makes our measurement less prone to

variability and a more objective way to compare differences in

38

pressure between or among different groups of animals in these

experiments.

Our measurements of gastric emptying and absorption were

designed to be as close to the physiological state as possible. We used

gavage feeding of fluorescence labeled dextran and let the mice roam

freely for 30 minutes, following which the surgery was performed and

measurement taken. The 70 kDa FITC-dextran was shown to be a

valid measure of emptying in prior studies as was the 4 kDa FITC

dextran for gastrointetsinal absorption (Aube et al., 2006; Thorball,

1981). Since there is a theoretic possibility that there is a potential

confounding by the action of BNP on arterial permeability and

distribution of the dextran to the third space (rather than GI tract

permeability or absorption) we measured and compared plasma

fluorescence after intravenous injection of 4 kDa FITC-dextran in BNP

treated and control animals. This avoids the potential confounding and

validates our measurement.

RESULTS

Intragastric Pressure and Gastric Contractility

A typical intragastric pressure wave pattern is shown in figure 5;

gastric contraction frequency averaged 3-7 times per minute ranging

from 0.5 to 10 mmHg in amplitude.

Figure 5. Typical pressure wave pattern of a basal gastric

contraction

A recording of a basal pressure pattern in windaq file is shown above.

Division width = 4 seconds and division height =1 mmHg. Recording

shown is a 10x compression (approximately 3 minutes) view of an

actual experiment

39

Figure 6. Simultaneous recording of blood pressure

and intragastric pressure

Two channel recording in our data acquisition system - windaq, Top

panel shows blood pressure and bottom panel shows intragastric

pressure. Note; division heights are 6.25 mmHg in top panel and 1

mmHg in bottom panel. Division width is 4 seconds in both channels.

Recording shown is 10x compression of an actual experiment.

40

The prokinetic agent gherelin was used to ascertain that these

recordings from the intragastric balloon corresponded to changes in

gastric motility. Administration of gherelin resulted in a marked and

significant increase in intragastric balloon pressure, validating that the

contraction waves recorded are that of changes in gastric contractility.

Gherelin was administered in three experiments and a typical response

is shown in figure 7.

Figure 7. The effect of intravenous administration of Gherelin

on intragastric pressure

Davison width = 8 seconds and division height = 1mmHg.

Arrow indicates point of injection of gherelin

41

Figure 8. Three individual experiments showing intragastric

pressure after a 10 ng/g iv bolus of ANP, BNP and CNP.

A N P

B N P

C N P

Division height = 1mmHg. Division width = 300 seconds

42

As shown in figure 8, intragastric pressure was decreased after

injection of any of the three natriuretic peptides, ANP, BNP or CNP and

the pressure gradually returned towards the baseline values.

Figure 9. Typical recording of post peptide injection gastric contraction wave pattern

Gastric pressure was attenuated after injection of BNP without

significant change in frequency of contraction. Also seen is a slight dip

in mean arterial pressure following BNP injection. Arrow indicates point

of injection. ‘Decompressed’ view, image shown is approximately 1

minute. A similar effect was observed for ANP and CNP. 43

Figure 10. The effect of C-ANP4-23 on gastric pressure

Individual experiment showing no change in gastric pressure or blood

pressure when the specific NPR-C ligand cANP4-23 was injected in to

wild type mice at 10 ng/g body weight iv.

Division width =80 seconds

44

Figure 11. BNP did not change gastric pressure in NPR-A knock

out mice

NPR-A knock out mice did not show decreased intragastric pressure

with any of the natriuretic peptides. Here BNP was administered at 10

ng/g body weight iv.

We quantified the change in intragastric pressure by averaging the

difference between the peak and baseline gastric pressures for three

thirty minute periods; before, immediately after and later than 5

peptide half-lives after peptide injection.

45

Figure 12. Determination of baseline and peak intragastric

pressure before and after peptide injection

Since each experimental animal had different baselines; comparing the

difference between baseline and peak (i.e., the change in amplitude of

the gastric contraction wave) was found to be more precise and

reliably comparable among different experimental animals. The basal

pressure was defined as the lowest pressure immediately before a

peak and the peak was determined by moving the cursor and

recording the highest point of the peak pressure.

46

47

Figure 13 (below) shows the pooled data on comparison of the

average gastric pressure before peptide injection (Basal), immediately

following peptide injection and later than 5 BNP half-lives (Recovery)

vs. vehicle. As shown, BNP significantly decreased intragastric

pressure from a basal value of 2.26 ± 0.29 mmHg to 1.44 ± 0.11

mmHg and gastric pressure returned to 2.08 ± 0.17 mmHg when

measured later than 5 BNP half-lives (n=5, p<0.05, ANOVA, Fisher’s

LSD test). Similar and statistically significant reduction of gastric

pressure was obtained for ANP and CNP (Figures 14 and 15). ANP

significantly decreased gastric pressure from a basal value of 2.11 ±

0.3 mmHg to 0.7 ± 0.25 mmHg and CNP decreased gastric pressure

from a basal value of 1.91 ± 0.3 mmHg to 0.85 ± 0.24 mmHg.

Average gastric contractions per minute were 4.4 ± 0.7, 3.1 ± 0.4 and

4.3 ± 0.5 for periods of BNP injection compared to 5.3 ± 1.2, 4.2 ±

0.6 and 4.3 ± 0.5 for the vehicle group (all p>0.05, ANOVA).There

was no difference in gastric pressure in BNP treated vs. vehicle treated

NPR-A KO mice.

Figure 13. Mean reduction in intragastric pressure following intravenous BNP injection

Basal Post injection Recovery

Intr

ag

ast

ric

Pre

ssu

re(m

mH

g)

0

1

2

3

4

BNP Control

*

Mean reduction in intragastric pressure (measured in mmHg) following

intravenous BNP 10 ng/g in 100 µl of vehicle vs. 100 µl of vehicle

injection to WT mice (n = 5 in each group). Intragastric pressure

measured before (Basal), immediately after (Post Injection) and more

than 5 peptide half-lives after injection (Recovery) is shown.

BNP significantly (* = p < 0.05, ANOVA, Fisher’s LSD test) decreased

intragastric pressure compared to vehicle. Gastric pressure returned

toward basal levels when measured later than 5 BNP half-lives.

48

Figure 14. Mean reduction in intragastric pressure following intravenous ANP given at a dose of 10 ng/g body weight


Intr

ag

ast

ric

Pre

ssu

re(m

mH

g)

0

1

2

3

4

ANP Control

*


intravenous ANP 10 ng/g in 100 µl of vehicle vs. 100 µl of vehicle




49

ANP significantly (* = p < 0.05, ANOVA, Fisher’s LSD test) decreased


toward basal levels when measured later than 5 ANP half-lives.

Figure 15. Mean reduction in intragastric pressure following intravenous CNP given at a dose of 10 ng/g body weight


Intr

ag

ast

ric

Pre

ssu

re(m

mH

g)

0

1

2

3

4

CNP Control

*


intravenous CNP 10 ng/g in 100 µl of vehicle vs. 100 µl of vehicle




CNP significantly (* = p < 0.05, ANOVA, Fisher’s LSD test) decreased


toward basal levels when measured later than 5 CNP half-lives.

50

Gastric Emptying

Gastric emptying was measured by gavage feeding a 0.1 ml of

2.5 mmol 70 kDa FITC labeled dextran to conscious mice immediately

after a 10 ng/g body weight bolus of BNP. The mice were sacrificed 30

minutes after gavage and the amount of fluoresce that has emptied

the stomach as a percent of total fluorescence measured in the entire

GI tract was calculated and compared between the BNP treated vs.

vehicle treated mice.

Figure 16. The effect of BNP on gastric emptying

Wild Type

Vehicle BNP

Perc

en

t G

ast

ric

Em

pty

ing

60

70

80

90

100

*

NPR-A Knockout

Vehicle BNP

Perc

en

t G

ast

ric

Em

pty

ing

60

70

80

90

100

Percent gastric emptying, measured in amount of fluorescence that

emptied the stomach as a percentage of the total fluorescence

measured in the entire gastrointestinal tract 30 minutes after gavage

feeding of 0.01 ml of 2.5 mmol 70 kDa FITC-dextran.

BNP (10 ng/g iv) significantly decreased gastric emptying in wild

type mice compared to vehicle (n =5, p < 0.05, t-test,). This effect of

BNP was absent in NPR-A knockout mice (n = 5, p > 0.05, t-test).

51

Figure 17. Dose dependent reduction of gastric emptying

Vehicle 5 ng/g 10 ng/g 100 ng/g

Perc

en

t G

ast

ric

Em

pty

ing

40

60

80

100

*

*

Figure 17 shows percent decrease in gastric emptying as a

function of BNP dose in WT mice. Measurements were taken 30

minutes after gavage feeding of 0.01 ml/g of 2.5 mmol FITC-dextran.

Progressive doses of BNP at 5, 10 and 100 ng/g iv resulted in

significant reduction of gastric emptying. (p < 0.05, n = 4 in each

group, ANOVA, Fisher’s LSD test)

52

Absorption

To measure absorption we used gavage feeding of 22ml/kg of a

solution containing 22mg/ml 4 kDa FITC-dextran to conscious mice

immediately following a 10 ng/g dose of BNP vs. vehicle. The plasma

fluoresce (taken 1 hour after gavage) was measured and compared

between the two groups. Plasma fluorescence after iv injection of FITC

dextran is also measured and shown below to ascertain that the

difference was not due to distribution rather than absorption.

Figure 18. The effect of BNP on absorption

Wild Type

Vehicle BNP

Rela

tive P

lasm

a F

luo

resc

en

ce

0

20

40

60

80

100

120

140

160

180

*

NPR-A Knock Out

Vehicle BNPRela

tive P

lasm

a F

luo

resc

en

ce

0

20

40

60

80

100

120

140

160

180

Absorption measured in relative plasma fluorescence units one hour

after gavage feeding of 4 kDa FITC-dextran in wild type mice BNP

treated (10 ng/g iv) vs. vehicle (n = 5, p < 0.05, t-test). No significant

difference in absorption was observed between BNP vs. vehicle treated

NPR-A KO mice (p > 0.05).

53

Figure 19. Plasma fluorescence following intravenous FITC- dextran, BNP vs. Vehicle

Vehicle BNP

Rela

tive P

lasm

a F

luo

resc

nce

0

50

100

150

200

250

300

Relative fluorescence of a 50µl plasma sample taken 1 hour after

intravenous administration of 100 µl 0.5mmol 4kDa FITC-dextran is

shown. BNP treated (10 ng/g iv) vs. vehicle in wild type mice, (p >

0.05, n = 4 in each groups, t-test).

54

55

Mean arterial blood pressure was continuously monitored during

all experiments using an intra-carotid catheter. There was a slight

reduction of average blood pressure from 62.9 ± 4.7 mmHg prior to

BNP injection to 59 ± 3.2 mmHg (n=5, p>0.05, ANOVA, Fisher’s LSD

test). Blood pressure returned to 61.4 ± 5.08 mmHg when measured

later than 5 peptide half-lives.

Similar drops in blood pressure were also observed during the

first few minutes after injection of both ANP and CNP with return of

blood pressure towards baseline.

Plasma BNP levels averaged 4500 pg/ml and the levels fell to

725 pg/ml at 30 minutes post injection and to undetectable levels at

90 minutes post injection.

56

DISCUSSION

These experiments show that the natriuretic peptides tested,

namely ANP, BNP and CNP all decreases intragastric pressure (gastric

contractility) in anesthetized mice. Prior studies have shown that

natriuretic peptides decrease contractility of isolated gastric and

intestinal smooth muscle cells in vitro (GuoCui et al., 2003; Yasuda et

al., 2000), this study is the first to show this effect to be true in the

whole intact animal. Furthermore, the inhibitory effects of the

natriuretic peptides appears to be dose dependent and mediated

primarily by NPR-A.

We further investigated whether this effect on contractility has a

functional significance in the whole animal. To accomplish this we

focused the subsequent studies on the effect of BNP on gastric

emptying and absorption; with its increasing utility in the diagnosis

and treatment of heart failure, further characterization of an aspect of

BNP is deemed to be potentially of important translational benefit.

Our findings confirmed that this effect of BNP on contractility is

accompanied by significant reduction in gastric emptying and

gastrointestinal absorption; clearly demonstrating that this inhibitory

effect on the gastrointestinal tract has functional significance in the

whole animal.

57

As shown in Figure 19, plasma fluorescence one hour after iv

administration of 4 kDa FITC-dextran was similar in BNP treated vs.

vehicle. This finding further strengthens our conclusion that the

reduced plasma fluorescence in gavage fed and BNP treated mice was

indeed due to decreased absorption or permeability in the GI tract and

not due to other known actions of BNP such as increased renal

excretion or change in vascular redistribution (Huxley et al., 1987).

Although there has been some indication that natriuretic

peptides are involved in inhibitory regulation of GI function (Ebert,

1988; Olsson & Holmgren, 2001), our findings are the first to

document a specific inhibitory role for BNP on emptying and absorptive

functions in the GI tract.

The average plasma BNP levels immediately after injection

averaged 4500 pg/ml. While this level is supra-physiological, it is not

uncommon in patients with heart failure (Fitzgerald et al., 2005).

Furthermore, even much higher levels of BNP are consistently seen

when recombinant BNP (Nesiritide) is administered for the treatment

of heart failure (Colucci et al., 2000). Moreover, the BNP level in our

experiments fell to levels routinely seen in heart failure patients at 30

minutes post injection. Therefore, our experimental model provides

novel insight into what would be expected in heart failure or when BNP

is exogenously administered.

58

Natriuretic peptides bind to transmembrane receptors that have

guanylyl cyclase (GC) activity. ANP and BNP bind to NPR-A where as

CNP binds to NPR-B. The ensuing peptide receptor interaction

increases intracellular cGMP with subsequent enzymatic steps that

regulate cellular functions in various tissues where these peptides

and/or receptors are expressed (Kuhn, 2005).

In our study, NPR-A KO mice did not show a significant response

with any of the peptides we used (ANP, BNP, CNP or c-ANP4-23). This

suggests that; the gastrointestinal effects of BNP and the other

natriuretic peptides are likely to be specifically mediated by the NPR-A

receptor. Since c-ANP4-23 specifically binds to NPR-C (Anand-

Srivastava, 2005), the absence of a GI effect when c-ANP4-23 was

injected into WT or NPR-A KO mice is additional evidence that NPR-A

may be the major receptor mediating the effect of BNP on gastric

emptying and absorption. Our finding is consistent with previous

studies that have shown that the inhibitory effect of CNP on isolated

gastric smooth muscle cells is mediated by a cGMP-dependent

pathway (GuoCai et al., 2003; Scotland et al., 2005).

The natriuretic peptides in general are among some of the most

evolutionarily conserved peptides across many species of the

phylogenetic tree with various functions in fluid homeostasis. Studies

done early in the discovery of natriuretic peptides have reported that

59

ANP significantly decreased jejunal fluid absorption in dogs and rats

(Morita et al., 1992; Scott & Maric, 1991). More recent studies have

shown that natriuretic peptides cause upregulation of aquaporin 3

expressions in human colonic epithelia signifying their potential role in

fluid homeostasis (Pacha, 2000). ANP has also been shown to be

important in promoting sea water adaptation in eels and decreases

intestinal sodium absorption (Tsukada et al., 2005). However, the

great majority of recent studies done on BNP have focused on its role

in modulating blood pressure, diuresis and natriuresis. Since the

primary stimulus for release of ANP and BNP is mechanical stretch of

the atrial and ventricular myocardium, their expression and release is

closely linked to body fluid volume status (Cowie & Mendez, 2002;

James et al., 2005). Moreover, the temporal pattern of expression and

release of BNP following a given stimulus, such as an acute myocardial

infarction, indicates that the endocrine heart could potentially employ

varying plasma levels of the natriuretic peptides to modulate body

fluid volume (Silver, 2006). Since natriuretic peptide receptors are

expressed on the gastric and intestinal smooth muscle cells, and we

show that intravenously administered BNP decreased gastric emptying

and absorption, it is logical to deduce that our finding may be an

indication that the endocrine heart employs natriuretic peptides to

delay or modulate the rate and amount of water and solute absorption

60

from the gastrointetsinal tract. From a physiological stand point the

effect of cardiac hormones on absorption in the GI tract is a beneficial

extension of their role in volume homeostasis. Theoretically, such a

role could extend to pathophysiological states such as heart failure

where plasma BNP levels and volume overload progressively rise

(Barclay et al., 2006) and modulation of volume status becomes even

more critical for survival.

We have shown that high plasma levels of BNP (sustained levels

of 500 pg/ml or greater), significantly decrease gastric motility,

emptying and absorption in mice. This appears to be a common effect

of the natriuretic peptides shared by ANP and CNP. The absence of this

GI effect in receptor knockout mice and the dose-response relationship

suggests a receptor mediated specific event.

While our study in the mouse model can not be generalized to

humans, our findings that BNP significantly decreased gastric emptying

and absorption offers valuable new insights into the role of the

gastrointestinal tract in fluid homeostasis, especially during heart

failure. First, symptoms of perturbed gastrointestinal function such as

nausea, dyspepsia, indigestion and malabsorption are frequently seen

in patients with heart failure where plasma BNP levels are markedly

elevated (Krack et al., 2005; Shamsham & Mitchell, 2000). Our study

suggests that some of these symptoms could at least partly be

61

attributable to the elevated BNP. Secondly, such an effect by BNP

could potentially add a new area of interest and investigation in the

role of the heart as an endocrine organ. While there are established

physiological and pathophysiological cardio-renal regulatory pathways;

a possible ‘cardio-gastric and/or cardio-intestinal’ link via natriuretic

peptides appears to be another possible pathway involving the

endocrine heart.

62

CHAPTER FOUR

THE ROLE OF BNP IN THE GASTRROINETESINAL

MANIFESTATION OF MYOCARDIAL INJURY

INTRODCUTION

Prior studies have utilized myocardial injury models to

study pathophysiological and histological effects exerted by the

natriuretic peptides on the cardiovascular system. Based on the results

of our experiments reported in the earlier chapters, mainly the fact

that the natriuretic peptides in general and BNP in particular decrease

gastric emptying and absorption; we postulated that these peptides

would have a similar effect in the face of acute myocardial infarction.

The most commonly used methods of inducing experimental

ischemia or myocardial infarction (MI) in mice are the permanent

ligation of left anterior descending (LAD) coronary artery and the

Cryoinfarction (freeze-thaw) method. In LAD ligation, the artery is tied

with surgical sutures whereas in cryoinfarction a blunt frozen metallic

probe is directly applied to a specific area of the myocardium with

resulting thermal ischemia at the point of contact and surrounding

63

myocardium. Although the cryoinfarction method was introduced as

early as 1948 (Hass & Taylor, 1948) it is only recently that it is shown

to have several advantages over LAD ligation; as the LAD method is

associated with marked variability in the size of the infarct and leads

to apical infarct resulting in ventricular aneurysm (van den Bos et al.,

2005). In the cryoinjury model the infarct area is limited to the

anterior wall of the myocardium more closely resembling what is

encountered in clinical practice in humans; where reperfusion therapy

results in limited infarct size and chances of developing apical

aneurysm are becoming increasingly less likely (Huwer et al., 1998;

Roell et al., 2002; van den Bos et al., 2005). Pathophysiologically, The

cryoinfarction method causes acute cell death probably from the

mechanical process associated with thermal injury; the infarct border

therefore corresponds to the size of the probe hence the improved

consistency of the resulting infarction as compared to the LAD ligation

method (van den Bos et al., 2005). Moreover since the cryoinfarction

method has a much lower peri and post operative mortality, fewer

numbers of animals will be needed for a given study.

We used a cryoinfarction model as the degree and distribution of

the infarct (Cell death and fibrosis) is more consistent with the

cryoinfarction as compared to the Left anterior descending artery

(LAD) ligation method (Huwer et al., 1998; Roell et al., 2002).

64

This study was designed to test whether a cryoinfarction of the

myocardium leads to change in gastric emptying and absorption in

mice and whether the difference in the plasma BNP levels between the

MI and Sham mice is responsible for this difference. The study was

also done in NPR-A knock out mice to test whether this effect is

mediated by NPR-A as shown by our previous experiments.

65

RESULTS

As shown in figure 20, our cryoinfarction model produced

myocardial cell death and fibrosis similar to what will be seen in

myocardial infarction. The infarction was limited to the area under the

application of the frozen probe and immediate surrounding myocardial

tissue without extension to the ventricular apex. AS a result no

ventricular aneurysms were observed in our model.

We tested and compared the degree of gastric emptying at one

and two week after infarction to establish the differences in plasma

BNP levels between the MI and sham mice. Subsequent experiments

on absorption were done at two weeks after infarction.

When gastric emptying was measured one week after infarction,

it was significantly decreased in the cryoinfarction group as compared

to the sham. Percent gastric emptying was 67.5% ± 5.8 for the MI

group vs. 88.7% ± 2.9 for the sham group (P<0.05, t-test) as shown

in figure 21. The plasma BNP levels were elevated in both groups but

significantly higher in the MI group as compared to the sham group.

BNP levels were 4292.2 ± 276.5 1 week after MI vs. 105.4 ±

11.3 in sham mice (n = 5, p<0.05, t test). BNP levels were 1964.7 ±

755 two weeks after MI, (n=5, p<0.05, t test compared to one week

post MI).

Figure 20. Representative histological appearance of the myocardium14 days after cryoinfarction. Stain: Hematoxylin & Eosin 4x (Top); 20x of the boxed part (Bottom)

LV Wall

LV Cavity

66

Figure 21. Comparison of percent gastric emptying in sham vs.

MI in Wild Type mice; one week after cryoinfarction

Sham MI

Perc

en

t G

ast

ric

Em

pty

ing

0

20

40

60

80

100

*

*

WT mice, 67.5% ± 5.8% for mice with MI vs. 88.7± 2.9 % for sham (n=7, P<0.05) 67

Figure 22. Comparison of percent gastric emptying in sham vs. MI Wild Type mice, two weeks after cryoinfarction Sham MI

Perc

en

t G

ast

ric

Em

pty

ing

50

60

70

80

90

100

*

WT mice, 82.2% ± 0.5% for mice with MI vs. 97.9 ± 0.4 % for sham (n=5, P<0.05) P= 0.017. As shown in figure 22 above, two weeks after infarction, percent

gastric emptying values were also significantly lower in the MI group

as compared to the sham group 82.2% ± 0.5% for MI vs. 97.9 ± 0.4

% for sham (n=5, P<0.05).

68

Figure 23. Comparison of Percent gastric emptying in sham vs. MI NPR-A KO mice, two weeks after cryoinfarction

Sham MI

Perc

en

t G

ast

ric

Em

pty

ing

50

60

70

80

90

100

KO mice, 84.6% ± 0.7% for mice with MI vs. 87.6 ± 054 % for sham (n=6, P >0.05) P= 0.07 In NPR-A knock out mice, percent gastric emptying was identical

between the MI vs. the sham group 84.6% ± 0.7% for mice with MI

vs. 87.6 ± 054 % for sham (n=6, P >0.05).

69

There was also a statically significant difference in the degree of

absorption measured in relative fluorescence units (RFU) in the plasma

following a gavage meal containing 22ml/kg of 22mg/ml 4 kDa FITC

dextran. In wild type mice, absorption was, 631.9 ± 121 (RFU) for

the MI group vs. 349.8 ± 78.6 (RFU) for the sham group; n=6, P<

0.05.

Figure 24. Comparison of absorption measured in relative Plasma fluorescence units. Sham vs. MI, WT mice Sham MI

Rela

tive F

luo

resc

en

ce U

nit

s(P

lasm

a)

0

200

400

600

800

*

Absorption measured in Relative plasma fluorescence units WT mice, 631.9 ± 121 for mice with MI vs. 349.8 ± 78.6 for sham n=6, P< 0.05 (P=0.04)

70

In NPR-A knockout mice the difference in absorption was not

statistically significant. Absorption measured in Relative plasma

fluorescence units in NPR-A KO mice was, 516.2 ± 107.3 for mice with

MI vs. 366.5 ± 39 for sham n=6, P> 0.05 (P=0.1).

Figure 25. Comparison of absorption measured in relative plasma fluorescence units. Sham vs. MI, NPR-A KO

71

Sham MI

Rel

ativ

e Fl

uore

scen

ce U

nits

(Pla

sma)

0

200

400

600

800

Absorption measured in Relative plasma fluorescence units

NPR-A KO mice, 516.2 ± 107.3 for mice with MI vs. 366.5 ± 39 for

sham n=6, P> 0.05 (P=0.1).

72

DISCUSSION

The presence of dyspeptic symptoms in heart failure and during

acute myocardial ischemia has been known for a long time. Studies

done as early as 1966 have reported what was then described as

venostatic gastritis where venous congestion is believed to be the

cause of gastric pathology (Fixa et al., 1966). The presence of nausea,

vomiting and indigestion during acute myocardial infarction and in the

course of heart failure has also been extensively reported in the

literature (Abrahamsson & Thorén, 1973; Ahmed et al., 1978;

Camurça et al., 2004; Pasini et al., 1989; Wei 1988). Studies that

have looked into mechanisms of such an association have in the past

mainly pointed to a possible chemoreceptive or neurally mediated

reflex known as the Bezold-Jarish reflex (Chianca et al., 1997; Sleight,

1981).

Both ANP and BNP have previously been shown to have

cardioprotective effect during acute myocardial infarction. For

instance, the effect of ANP and BNP on renal salt handling was shown

to be specifically enhanced during acute myocardial ischemia

irrespective of the level of activation of the renin angiotensin

aldosterone (RAS) system (Charles et al., 2003; Rademaker et al.,

2000). ANP was also shown to have important volume regulation role

73

during acute heart failure induced by ventricular pacing (Lee et al.,

1989). Both BNP and ANP are also shown to have an inhibitory effect

on regional sympathetic activity in the kidney and the heart (Brunner-

La Rocca et al., 2001). While the pathways that lead to sympathetic

activity are not clearly understood, the end result of decreased

sympathetic activity is cardioprotective. Studies that looked at the

receptors involved in such processes have consistently shown that

these effects are mediated by cGMP coupled pathways. While some of

this effect is indirectly mediated via the renin angiotensin aldoseterone

(RAS) system, protective actions of guanylyl cyclase-A that are not

mediated by the RAS have also been shown (Li et al., 2002; Nakanishi

et al., 2005).

In our experiments, we tested the effect of acute myocardial

injury on gastric emptying at one and two weeks post infarction and

compared the results along with the plasma BNP levels. There was a

significant reduction in gastric emptying at one week in mice with

myocardial infarction compared to sham. It is interesting to note that

even the sham mice had a slightly lower rate of gastric emptying at

one week compared to controls (baseline values). As the BNP levels

were higher in sham mice than in controls, and the levels were

markedly higher in the infarcted mice than the sham mice, this is an

indication that BNP levels corresponded with the degree of gastric

74

emptying. At two weeks post infarction, the sham mice had gastric

emptying statistically identical to controls where as infarcted mice

continue to show a markedly lower rate of gastric emptying, this again

indicates that the BNP levels significantly correspond with the degree

of gastric emptying adding evidence that BNP may be the major (or

one of the major) reasons for the observed differences. Since our data

shows that infarcted receptor knock out mice had no significant

difference in gastric emptying compared to sham, this validates our

assertion that the BNP difference played a role in the observed effect

and this effect is probably mediated through the NPR-A receptor.

Our absorption data for wild type mice is consistent with our

central hypothesis and infarcted mice did show significantly lower

absorption rate as compared to sham. The data in the knock out mice

in our experiment is less conclusive, since the knock out mice with MI

did show a reduced absorption compared to the sham mice. There are

several explanations for this finding. First of all, myocardial injury is

known to activate a series of systems other than the natriuretic

peptides, and secondly the process of absorption in the GI tract is also

likely to involve several different mechanisms. For instance the renin

angiotensin aldoseterone system is known to be activated with

myocardial injury, inflammatory mediators such as tissue growth

factor β and tumor necrosis factor are also a few of the cytokines that

75

are released from infarcted myocardium and circulate in the plasma

with potential effects in the GI tract. Therefore the observed effects in

NPR-A knock out mice may be manifestations of activation of these

systems that would be expected to be intact in the NPR-A knock out

mice.

It has also been shown that intravenous volume expansion

decreased gastric emptying and permeability of the mucosa to water

and solutes and vagus nerve mediated neural mechanism were

postulated as mechanisms for such an effect (Chang EB and Rao MC.,

1994). It was also reported that acute blood volume expansion with

intravenous fluids decreased net sodium absorption in the jejunum of

rats and dogs (Duffy et al., 1978; Richet & Hornych, 1969). Moreover

experiments in human volunteers have shown that body position

changes such as recumbency and simulation of hemorrhage resulted in

significant increase in intestinal water and salt absorption (Sjövall et

al., 1986). Despite this known relationship between gut motility,

gastric emptying and intestinal absorption on one hand and

intravenous venous expansion and contraction on the other, most of

the studies that have investigated this relationship were limited to the

vagal or sympathetic nervous system. Nevertheless, with the

knowledge base accumulated in the field of natriuretic peptides over

the past two decades, it is only logical to conjecture that at least some

76

of the changes observed in gastric emptying and intestinal absorption

during acute MI or heart failure may be due to natriuretic peptides.

The data supporting the relationship between volume expansion

and gastric emptying and intestinal water and salt absorption is fairly

strong. What was missing was an experiment to directly and

specifically test whether the elevated natriuretic peptide levels (caused

by volume expansion or myocardial injury) corresponded with gastric

contractility, gastric emptying and absorption. Together with our

earlier findings that all the natriuretic peptides significantly decrease

gastric contractility, our data on BNP showing significant reduction of

gastric emptying and absorption; the confirmation of these findings in

an acute myocardial infarction model is a strong indication that plasma

BNP (and natriuretic peptide) elevation during acute MI and / or heart

failure is at least partly responsible for these observed effects. More

importantly, the previously confirmed effect of increased plasma

volume on gastric emptying and absorption is validated by our data to

be mediated by natriuretic peptides in general and by BNP in

particular. This finding strengthens the case for a humoral link

between the heart and the GI tract and opens up new avenues for

heart failure research. We believe further research in this area could

identify potential new targets for the treatment of heart failure.

77

CHAPTER FIVE

BNP AND NON MUSCLE MYOSINS IN THE

GASTROINTETSINAL TRACT

INTRODUCTION

Myosins are proteins that are involved in mechanical force

generation and transduction. The characteristic myosin molecule is

composed of two heavy chain subunits measuring approximately 200

kDa each, which form a globular amino-terminal head region, and a

coiled carboxyl-terminal tail. The globular head region is non-

covalently associated with two pairs of light chains of 20 and 17 kDa

(Kelley & Adelstein, 1990). Typically, myosins interact with another

protein; actin, in a process that involves the hydrolysis of ATP.

Classically such actomyosin interaction is discussed in the context of

muscle contraction and relaxation. However myosins are also

expressed in non muscle tissue (Sellers, 2000).

Figure 26. Schematic diagram of non muscle myosin-II

Adapted from Bresnick (1999)

In non muscle cells, the prototype non muscle myosin

type-II regulates actin organization into filaments and its

functions include maintaining the cell shape, cell division and cell

movement (Bresnick, 1999). Non muscle myosin-II is also

involved in epithelial cell attachment at the tight junction

regulating paracellular permeability (Hecht et al., 1996). Tight

junctions are integral part of the intestinal villi absorptive

structures.

78

Intestinal Villi

Figure 27. Schematic depiction and electron micrograph of the structures of intestinal villi

79

Adapted from Keith R. Porter and S Clark

The electron micrograph (above) shows the microvilli of a mouse

intestinal cell. Incorporated in the plasma membrane of the microvilli

are a number of enzymes, mucous producing goblet cells and

endocrine / paracrine cells that produce a number of hormones,

among which are the natriuretic peptides and guanylins.

Figure 28. Cytoskeletal structures of the intestinal microvillus.

Adapted from Ross, Histology: A Text and Atlas, 4th ed. 80

Figure 29. Ultra structures of the microvilli cytoskeleton

A

B

Adapted from (Hirokawa & Heuser, 1981)

Actin cytoskeleton is shown by the arrow(s) in panel A, and in panel B

the quick freeze, deep etch, rotary replication images show the core of

the microvilli and its attachment at the cytoskeleton base in the

cytoplasm

81

Figure 30. Transmission Electron micrograph of intestinal villi in various state of contraction

Chicken intestinal epithelia showing increasing state of

contraction (top to bottom). Note ‘fanning’ of the microvilli as

the degree of contraction increase. Transmission electron

micrograph. Top; 6100x, middle; 5600 x and bottom 8900x.

Adapted from (Burgess, 1982)

82

Tight Junctions

Tight junctions seal adjacent epithelial cells in a narrow band

just beneath their apical surface. They perform vital functions including

regulation of the passage of molecules and ions through the space

between cells and blocking of movement of the integral membrane

proteins between the apical and basolateral surfaces.

Figure 31. Schematic depiction of tight junctions

Studies done in kidney, bladder and intestinal epithelia

have confirmed changes in permeability regulated by

Phosphorylation related contractile changes at the tight junction.

These changes primarily increase paracellular movement of

water and small molecular weight solutes (Broschat et al., 1983;

Hecht et al., 1996; Keller & Mooseker, 1982; Swanljung-Collins

& Collins, 1992). Non muscle myosin type IIB is phosphorylated

83

84

by casein kinase-II and dephosphorylated by a phosphatase that

is regulated through the action of Rho–associated kinase (Li &

Gorodeski, 2006).

Since the physiological effects of the natriuretic peptides

are mediated via cGMP dependant protein kinases, we found it

intriguing to examine whether treatment with BNP would reveal

changes in the distribution of non muscle myosins and

contractility pattern of the intestinal villi and thus shed some

light on the mechanism for the observed effect of BNP on

absorption.

We treated wild type mice with a 10 ng/g iv bolus of BNP

followed by a 1ng/minute infusion for 30 minutes. After the 30

minute infusion the mice were sacrificed and intestinal tissue

removed and sectioned into, jejunum, ileum and colon and

immunostained for non muscle myosin as described in the

methods section. We used the vehicle as an experimental

control. We also used a tissue section not treated with first

antibody as a methodological control to evaluate the potential

confounding by non specific binding.

RESULTS

Figure 32. Toluidine blue stained 3µm sections of a mouse intestinal tissues

85

Cross sections of intestinal tissue from our experiments are shown.

Top panel, Jejunum; middle panel, ileum; and bottom panel, colon.

Figure 33. Higher magnification view of intestinal villi

Images shown are 3 µm section of Jejunal villi (top) and Ileum (bottom), stained with toluidine blue. Magnification is 40x top, and 20x bottom. 86

Figure 34. Electron micrograph of the intestinal microvilli

Control vs. BNP treated

Control, 20 000x BNP treated, 20 000x Microvilli in BNP treated mice show loss of the distinctive

‘fanning’ of the microvilli that is typical of markedly contracted

state.

87

Figure 35. Jejunal villi immunostained for non muscle myosin type IIB - Control

Longitudinal section at 20x (Top) and cross section 40x bottom

88

Figure 36. Comparative images of control vs. BNP treated jejunal villi

Images show jejunal villi, immunostained for non muscle myosin

type IIB. Control (TOP) and BNP treated (bottom). Note the

marked increase in fluorescence in BNP treated as compared to

control. Immunofluorescence, 20 x magnification.

89

Figure 37. Jejunal villi and smooth muscle bundles immunostained for non muscle myosin

Cross section view of the mouse ileum is shown. Note the

circular and longitudinal smooth muscle bundles with distinct

stain (fluorescence) for non muscle myosin type IIB. The core of

the villi also shows the distinct staining pattern.

Magnification, 10 x

90

Figure 38. A 3 µm sections of a mouse jejunum immunostained for non muscle myosin type IIB. Control vs. BNP

Control top and BNP treated bottom. Note the markedly increased staining of the villus core and the crypt in BNP treated mouse. 20x

91

92

DISCUSSION Structures necessary for force generation and

mechanotransduction such as actin and myosin and their

associated proteins are integral components of the cytoskeleton

of the intestinal villi (Mooseker, 1976; Mooseker et al., 1983;

Rodewald et al., 1976; Rostgaard & Thuneberg, 1972). Although

the mechanism of contraction of intestinal villi still remains

unsettled, different studies have reported mechanisms of

contraction that are both ATP and calcium dependent and others

reporting calcium independent mechanisms of contraction

(Burgess, 1982; Mooseker, 1976; Swanljung-Collins & Collins,

1991). Still other studies have reported contraction at the tight

junctions but not of the microvilli themselves (Keller & Mooseker,

1982).

Our electron micrographic observations indicate that

elevation of BNP in the plasma appears to cause the microvilli to

loose the typical ‘fanning’ or separated appearance that is

characteristic of contraction at the crypt region of the intestinal

microvilli. Such appearance was previously shown to be due to

contraction at the tight junction regions involving a

circumferential contractile ring (Burgess, 1982). Tight junction

93

contraction in turn results in increased paracellular permeability

(Hecht et al., 1996; Turner et al., 1997). Therefore our electron

micrographic observation (decreased contraction of the tight

junction contractile ring) supports the hypothesis that elevated

BNP in the plasma makes the tight junctions of intestinal villi less

permeable to water and small molecular weight solutes. Our

fluorescence immunostaining images also support this

hypothesis as we show enhanced distribution of non muscle

myosins along the crypts of the villi; which may be an indication

that the enhanced distribution corresponds to a change in

tautness of the tight junctions thereby decreasing paracellular

movement while allowing apical transport of nutrients. Apical

transport of electrolytes and nutrients (sodium and glucose for

example) is mediated by the sodium glucose cotransporter

(SGLT1) and is accompanied by increased tight junction

permeability to small solutes (Turner et al., 1997).

Physiologically such coordination between the apical and

basolateral surfaces of the villus would be advantageous; as it

allows regulated (transporter mediated) absorption across the

apical membrane while directing water and small solutes to the

tight junction. However whenever there is a need to decreases

volume, as in the case of heart failure; decreasing free

94

paracellular movement of water while still permitting apical

transport of nutrients would allow for a more homeostatic

absorptive function; especially when modulating volume

becomes essential as in the case of heart failure. Utilizing BNP

and possibly the other natriuretic peptides for this process would

have physiological benefit for the heart; after all the heart will be

‘interested’ in getting all the nutrients it can get during heart

failure while delaying or decreasing absorption of water and salt

that could add to its stress.

Although the effect of BNP and natriuretic peptides in

general on permeability of intestinal epithelia has not been

extensively studied in the past, it is known that natriuretic

peptides in general have an effect on permeability of the

vascular epithelium (He et al., 1998; Huxley et al., 1987; Kubes,

1993; Lofton et al., 1990). Studies that have looked into this

effect of ANP and BNP on permeability of pulmonary vascular

epithelium have shown that these effects were at least partially

reproduced by cGMP or cGMP analogs (Draijer et al., 1995;

Hölschermann et al., 1997; Klinger et al., 2006; Westendorp et

al., 1994). A more direct effect that is independent of cGMP was

also shown for ANP (Kubes, 1993). Similarly, other studies that

have investigated the effect of ANP on counteracting the

95

endothelial permeability and inflammatory effect of tumor

necrosis factor (TNF) have shown that this effect is mediated by

NPR-A and cGMP and it is targeted at cytoskeletal actin

organization (Kiemer et al., 2002). An other support for this

finding comes from the observation that vascular endothelial

growth factor (VEGF) is inhibited by ANP (Pedram et al., 2002).

VEGF is also known as vascular permeability factor and is

involved in stabilization of actin association with tight junctions;

and this function was reversed by guanylyl cyclase receptor

antagonists (Pedram et al., 2002). The precise mechanism of

this process is still unclear, some studies indicated it involves

heat shock protein 27 (HSP27); a protein with known functions

in actin organization and whose actions are mediated by cGMP

dependent protein kinase (Butt et al., 2001). Other studies

suggest mechanisms that involve Phosphorylation of structural

and contractile proteins; including actin and myosin (Hecht et

al., 1996; Ma et al., 2000; Turner et al., 1997), Specifically at

the prejunctional ring of actin and myosin; sites that are

characteristic of contractile function at the tight junction (Madara

et al., 1987).

Our immunofluorescene images revealed an enhanced

distribution of non muscle myosins in the intestinal villi core and

96

along the crypts (tight junctions). While we haven’t done

biochemical analysis to correspond with these findings, previous

studies have shown that Phosphorylation related proteins such

as mitogen-activated protein (MAP) kinase and Rho of the ras

family of proteins are associated with tight junctions and this in

turn controls tight junction contractility (Murakami et al., 1994;

Zahraoui et al., 1994). Interestingly, both ANP and BNP are

known to exert anti inflammatory (thus decreased vascular

endothelium permeability) effects in vascular endothelium

through mechanisms that involve MAP-kinase and MAP kinase

phosphatase-1 (MKP-1) (Fürst et al., 2005; Weber et al., 2003).

Although more research needs to be done in this direction,

our electron micrographic imaging and immunostaining data

coupled with what is previously known about epithelial tight

junction function lends enough support to our hypothesis and

helps formulate a basis for future direction. The finding that

immunostaining of non muscle myosin in BNP treated mouse

tissue shows enhanced distribution of non muscle myosins along

the crypts and core of the intestinal microvillus appears to be a

recurring theme. Tight junctions appear to contract less and

microvilli assume the typical non-contracting appearance when

BNP plasma level was raised. These findings are associated with

97

decreased paracellular permeability at least in the case of the

vascular endothelium. Whether the biochemical signaling

mechanisms known for vascular endothelium will be the same in

the intestinal villi and whether it will have similar physiological

consequences remains to be investigated.

98

CHAPTER SIX

SUMMARY AND CONCLUSIONS

The main objective of my study was to show that there indeed is

a humoral link between the heart and the gastrointestinal system. This

humoral link appears to utilize natriuretic peptides although it is

possible that there probably are several other as yet undiscovered

peptides or even other molecules that serve this same purpose.

To this aim, we demonstrated that ANP, BNP and CNP decreased

contractility of gastric smooth muscle in the whole intact animal.

Confirming the previously done in vitro experiments that showed

natriuretic peptides decreased contractility of isolated gastric and

intestinal smooth muscle cells. This effect was shown to have a

functional significance in that; these experiments showed that gastric

emptying and absorption from the gut were decreased in the whole

animal when BNP was intravenously administered. We also presented

data showing myocardial injury and the associated elevation in plasma

BNP levels decreased gastric emptying and absorption from the GI

tract. Although the obvious reason for this result may be the effect of

BNP on smooth muscle relaxation, we also show data supporting the

99

hypothesis that BNP and natriuretic peptides have a more direct effect

on absorptive structures of the intestinal villi possibly affecting

paracellular transport of water and electrolytes.

In summary one can garner the following observations from

these experiments

a) Natriuretic peptides decrease gastric contractility and gastric

emptying in intact whole animals

b) BNP decreased gastric empting in a dose dependant manner

c) BNP decreased absorption from the GI tract when administered

at dose designed to raise the plasma level to the levels seen in

heart failure

d) Myocardial injury and elevation of BNP in the plasma was

associated with decreased gastric emptying and absorption

e) Elevated plasma BNP has en effect on contractile structures of

the intestinal villi and this effect appears to be directed at

decreasing paracellular movement of water and solutes

100

PERSPECTIVES

The next line of research should follow the intriguing questions

raised by our immunofluorescnece data. How and why does the

distribution of myosin in the microvilli appear to be increased by BNP?

What are the molecular mechanisms that are involved here? Is the

electron micrograph appearance of microvilli indication of contraction

at the tight junction? Is paracellular movement of water and solutes

decreased by BNP as suggested by our imaging data? Are there other

transporters that direct water and sodium absorption to the basal and

apical membranes; if so can they be therapeutically targeted to

regulate sodium absorption from the gut? Would that be beneficial in

heart failure treatment?

Heart failure remains to be a major public health problem. Its

annual cost to the health care field is growing every year and currently

stands at over 30 Billon US dollars. Despite remarkable advances in

the treatment of coronary disease and the risk factors associated with

it, the incidence and prevalence of heart failure has only increased. In

spite of all this, there hasn’t been a major breakthrough in heart

failure treatment for many decades. Of course targeting the gut to

101

treat the heart seems a bit far fetched at this point in time. However,

the GI tract is a complex organ, perhaps even tantamount to having

many organs in one. The experimental results presented here

strengthen the case for a direct humoral link and interplay between

the heart and the GI tract. Further understanding of this link and

characterization of the signaling process involved is likely to be a

major advance in heart failure treatment.

102

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APPENDICES

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APPENDIX A

Abbreviations Used ACE – Angiotensin converting enzyme ANF - Atrial natriuretic factor ANOVA - Analysis of variance ANP – Atrial natriuretic peptide AT1 – Angiotensin II receptor type 1A BNP – B-type natriuretic peptide cANF – c-Atrial natriuretic factor (peptide) cGMP – Cyclic guanylyl mono phosphate CNP – C-type natriuretic peptide DMSO – Dimethyl sulfoxide DNP – Deandropsis natriuretic peptide FITC – Fluorescein-isothiocyanate GC – guanylyl cyclase GI – Gastrointestinal GTP – Guanylyl tri phosphate i.d. – Internal diameter i.v. – Intravenous

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i.P. – Intraperiotneal KDa – Kilo Dalton KHD – Kinase homology domain KO – Knockout LAD – Left anterior descending artery LSD – Least square difference LV – Left ventricle MAP – Mitogen activated pathway MI – Myocardial infarction MKP – Mitogen activated kinase phosphatase NPR-A – Natriuretic peptide receptor type –A NPR-B - Natriuretic peptide receptor type –B NPR-C - Natriuretic peptide receptor type –C o.d. – Outside diameter PBS – Phosphate buffered saline PDE – Phosphodiesterase PIC- Probe induction catheter PKG – Protein kinase G RFU – Relative fluorescence units RIA – Radioimmunoassay RV – Right ventricle

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TNF – Tumor necrosis factor VEGF – Vascular endothelium derived growth factor WT – Wild type

ABOUT THE AUTHOR

Anteneh Addisu graduated with MD from Addis Ababa University

in Ethiopia and completed internal medicine residency at St. Vincent

medical center (New York) where he was also chief resident.

After six years of private practice in internal medicine, he joined

the cardiac peptide lab of Dr. John R. Dietz as a PhD student in 2004

to pursue his interest in molecular workings of the heart.

He has won numerous awards including a WHO sponsored

visiting scholarship to the University of Washington, honored as

resident of the year and chief resident, elected to fellowship of the

American College of Physicians, awarded the national science

foundation’s integrative graduate research education and training

scholarship and recently was given the new investigator travel award

from the American Heart Association.

As part of this dissertation he has authored an original article

and several abstracts and has made several presentations at regional

and national meetings.

natriuretic peptides as a humoral link between the heart

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