5T0D1ES ON THE R E U T W S H I P BETWEEN PROTON, Co" TRmSPOKV AND CKLClUtA ANTKGONIST IN HE^RT

DISSERTATION SUBMITTED FOR THE DEGREE OF

Master of Phrlosophi IN

Chemistry IN THE FACULTY OF SCIENCE THE ALIGARH MUSLIM UNIVERSITY, ALIGARH

BY

Mohammad Akram M. Sc. (Chemistry)

DIVISION OF PHYSIOLOGY CENTRAL DRUG RESEARCH INSTITUTE LUCKNOW 226 001 SEP. 1988

DS1728

Dealcatea

to those

HEARTS

from where

comes out

the essence of

HUhANITY

Telsx : 0535-286 Ttltgram : CENORUG Phone : 32411-18 PABX

No.

0TTT *ff5r5r, «Tt?e Mmi H» I 7 3

vra n; 226001 (UTTCT)

CENTRA!. DRUG RESEARCH INSTITUTE Chattar Manzil, Post Box No. 173

LUCKNOW-226001 (INDIA)

Date

P*. OnkoA N, iKipathi kAAU^tant VVLZctoK £ Head Vivi&ion oi PhtfAiology

C E R T I F I C A T E

Thi^ -c-6 to ce.fiti(,y that the. wo-tfe embod-ted -in thl^ thzM^

ha^ bzzn cafifiidd oat by Uft, Uohd Akfiam undzA. my ^pz^viM.on.

Hz ha^ didf^zd thz /lequ-c/tementi OjJ thz AUga^h UaMim

UnivZAMty AzgaA.ding thz Jta-tu-te and pKZ^cn.ibzd pzxiod o($ <n-

vz^igatlonal laoik ioi thz aviaid OjJ M.Phil. dzgKZZ.

Thz uoofik inciudzd in thi4> thz^4> i^ oKiginaJL and ha^

not bzzn ^bmittzd (Jo-t any othzA dzgizz.

inVKTHl ^. %V^ Ph.V.

SupzfLvtiox

PHONE : Office : 5515 DEPARTMENr OF CHEMISTRY ALIGARH MUSLIM UNIVERSIIY

ALrGARII-202001

Date.

VK, feAozz Ahmad Rzade.fL Vi-poAtmejit oi Chzmlitxy

C E R T I F I C A T E

Jhi-d ii to czntl^y that thz wofik embod-ced -en thii thzM.^

i^ an oKlQinaZ woik caKiiQ-d oat by M-t. Uohd Kknam, tmdzt tkz

^pzfiV'iUon 0(5 VK. Onkafi N. Tn.ipathi, A^i^fi^Uant Viizctoft and

Hzad, ViviMon oi PhyOology, CtntiaZ Viug Re4ea/ic<i In-itttute,

Lucfenow^^Jo/t thz paitisxl iuJL^^mznt o(5 M.P^-6£. Peg^ee.

Vt. Fe^ioie Ahmad Ph.V.

C O W T E M T S

Page No.

Acknowledgement

1. introduction 1 1-3

2. Review of Literature 4-15

3. Methods and Materials 16-24

4. Results 25- 34

5. Discussion 35-40

6. summary and conclusions 41-42

7. Bibliography 43-47

***** *** *

ACKNOULEVGEUENT

I am kighiy ande.^ an obligation and <ee£ thz nzcz^^ity 0($ gratitude.

acknousZzdging the. impe.n.i^hablz and intKin^ic co-ope.fLation to my z^t(i.<Lme.d

tzackdfi, Vi. Onkai N. TA.ipathi, Ph.V., A^^i-stant ViKZcton. and Hzad, Viv.

0^ Physiology, Czntial VKag Rt'bzaKcM. Jnstitutz, Lucknow, ^o^ his fids-

pZzndznt guidance, as we.lt as stfiaightf^oma^d inspifiation andzK that I

could havz got the goldzn fiays o^ my hope, thftough which I could see my

aim to be teachzd on the. peak o^ thz succzss, I, Kzally, have no woids

to zxpfizss my Indzbtzdnzss jjo-t him. I am thankful to him ^ofi his kind

initiation that tntfiusted in me the anxiousnzss to do my wofik.

My wolds also know no explanation leading to thz kind thanks and

dzzp sznsz 0^ gfiatitudz ion. VK. fznozz Ahmad, Rzadzn., Vzpan.tme.nt Oj$ Chzm-

istKy, A.M.U., A--tgafi, \xkosz ^Kizndly co-opiKation zncounagzd me to

caKKy out my Kzszaich wonk to be donz.

I am highly endowed with Vi. K.C. h^khZKjzz [Scizntist], Viv.

0(5 Physiology {^oi his silznt suppont whznzvzn I ^zlt duning my incon-

vznizncz. I wish to zxpizss my thanks also to latz Vn.. S.A. Rizvi,[Rzadzfi]

and VK. Chanan Singh [Scizntist] ^OK thzin. nzzdf^al hzlp {iiom timz to

tinK..

My pfLO^^ound thanks and dzzp lootzd obligation also go to Vn.H.M.

Vhan., FNA, jjoimei ViizctoK and Pfiof,. B.W. Vhautan, ViKZctoi, CPRI, who

piovidzd me thz nzzd^ul laboiatony {^acilitizs and othzK izsounczs.

HzvzK and nzvzK shall I ioigzt to zxpfLZSS my thanks and high obli­

gation ion. thz kzzn co-opznation oi Vn.. y.K. Singh [Scizntist] w o havz

always zncoun.aQzd me giving insight in my wonk. Also I am vzn.y macfi

Indzbtzd to Vn.. Suhail Sabin. and Vn.. Ram Kn.it Ram ion thzin. kind co-

opznation.

' I ai^o thank to my lab coltzague.6 V^AMfi^) M. Ray (ScizntiU),

V^. Rajiv Kuma^ [Sciznti^t), M -4. ^ibani, MiM Mifia, k^. Viam, Sen,

A-tun, Sanjot/ and tkinoj iox co-ope.fiatinQ me by thzii intzn^iVe.'- ^uppofit.

Hou) can I ^ofigzt thz thankialnz^A and dzzp Zovz a^ wzii a^ thz

^olidaKity that my ^^iznd^ VK. Md Salman- Ch., M-t. MP Saiman Khan, M^.J.K.

ChakaKabofLty, M^. Shahabaddin, Vi. KamKan, Wi» k^am , ManuimaKj VK. GIOVZK,

ztc. bz^owzd on my ^hoaldzn. with thziK ^oiiaitou^ co-opZKation and who

-ika^zd with mz zvzKy ^n and 'ihowzA duAing thz pzKiod o^ my fiz^zafichll

I am al4)0 highly obligzd and vziy much thank^^ul to my {^amily mzm-

be-t-i, Hon. Lt. Hohd MuAta^a, My ^athzK; UfiA. KuZ^oom Bzgam, My Mothz^;

M^4. Na^imun Ni^ (M.A.), my M-itZft; M-t. fafthat A'dh^aq [Advocatz], my

B^othzK in Law..; Lt.Col. [V^.lMohd Azam (M.P.), E^. Mohd. Maazzam IA-64-C4-

tant ViizctoK], Vn.. Mohd tMikaKfLom [Sciznti^^t], my EldZK Mothzn.^; a-6

wzll a-6 VfL. Mohd. A^^am URF Hamz^hgool [MBBS], ny youngzK bKothzK; who

constantly zncouiagzd mz and bzyond ikz-tA. ppoKt my woKk could nzvzA

havz complztzd.

I am also vzfiy than^ul to Mfi. K.L. Gupta and H.B. Hzolia ^OK

thziK nzat and clzan typing thzy pKovidzd to my woKk.

Lastly, my acknowlzdgzmznt is highly obligzd and vZKy mach thankf^ul

to thz Unichzm (Bombay) and J.C.M.R. (NZJW Vzlki) ^OK thziK financial

suppofLt in thz f^onm oi ^zllowship.

Uohd. Ntiam

INTRODUCTION

1.0 INTRODUCTION

Hydrogen is one of the most important ions in the

nature being abundantly distributed in a wide range of living

and non-living things. In the living cells it not only enters

into the composition of various subcellular constituents,

but the concentration of its ionic form also plays a vital

role in the regulation of various cellular functions.

In the aqueous solutions H exists as the hydronium

ion {H o'' ) which reflects the activity of the former and

in turn the acidity of the solution. Due to the necessity

for conveniently expressing the acidity or [H ] of the solut-

ionsj Sorensen (1909) suggested a simple scale for expressing

it in terms of pH or the negative log of [H ].

The extracellular and intracellular pH is maintained

at a very crucial level by delicately balanced local buffering

mechanisms, the details of which are not yet fully understood.

We also do not know clearly as to how the H is transported

passively across the living cell membranes and how a change

in pH affects the passive transport of other ions in the

living cells. There are, however, suggestions that an active

transport of H across the membrane is perhaps brought about

in different cells by Na"*", Ht- or Ca "*", H''' - Pumps (Roos and

Boron, 1981; Daniel et SLI. , 1986). Moreover, pH is also

implicated in maintaining the optimal conformations of the

membrane ionic channel proteins and other cellular proteins

and their activities. A decrease in pH (acidity or increased

[H"'']) is known to inhibit the membrane permeability to other

cations e.g. Na"*", K"*" and Ca " (Wada and Goto, 1975).

Among the living cells the cardiac cellular function

is reported toigpritically modulated by the cellular pH, which

has been associated with the development of myocardial dis­

orders, e.g. cardiac infarction etc. This conclusion is

based on the observations of an increase in myocardial cellu­

lar acidity during heart failure. There also exists the

+ 2+ possibility that H activity affects the Ca currents carried

2+ 2+ +

via Ca -channels and also the Ca , H -pump in cardiac cells.

Due to lack of information about the role of H in cardiac

activity it is difficult to understand, how [H ] affects

the cardiac cellular function in particular and those of

other excitable cells in general.

These ambiguities in our understanding of the mech­

anisms underlying the effects of pH on cellular functions

necessitated the present investigations encompassing the

artificial and living membrane systems. The passive membrane

ion transport which plays a crucial role in cellular functions

is strongly affected by a change in pH. Moreover, H^ is

the most active of the physiologically important ions, likely

to behave quite differently from the other ions of this cate­

gory in aqueous medium. This necessitates a study of its

transport characteristic in aqueous medium as well as the

implications thereof on-the membrane ionic channel activity.

The present studies were planned so as to provide some infor­

mation on these two different aspects of the role of H in

membrane phenomena.

In the first set of experiments studies were carried

out on artificial membrane systems to obtain information

about the transport characteristics of H vis-a-vis K and

2+ Ca . To achieve this E.M.F. with and without transport

was measured in diffusion half cells separated by a porous

partition and in half cells connected by KCl-Agar salt bridge

respectively.

In the second set of experiments the effects of pH

on transmembrane electrical activity (APs) in heart cells

were studied using the glass microelectrode technique. The

effects of pH on the passive ion transport contributing to

these APs were analysed by appropriate {techniques.

REVIEW OF LITERTURE

2.0 REVIEW OF LITERATURE

Transport of electrolytic ions across living cell mem­

branes plays a crucial role in transmembrane signalling and

cellular functions. Due to such a vital significance of mem­

brane ionic transport, its mechanisms have been studied exten­

sively by the scientists for more than a century now using

the artificial as well as natural membrane systems. since

the present study deals in particular with the transport of

H across an artificial membrane system as well as its effects

on the ionic transport in living cells, a brief review of

the salient work on the ion transport in these two systems

is given below.

Nernst in 1899 carried out his pioneer investigations

on the polarization effects at the phase boundaries between

two ionically conducting aqueous phases. He subsequently

investigated the electrical behaviour of solvent membranes

in concentration cells particularly with a view to explain

the problems associated with nerve excitation (Nernst and

Riesenfeld, 1902) which led to the development of present

concepts of bioelectrochemistry. Yet another significant

contribution in this field was made by Donnan (1911) who gave

the theory of membrane equilibrium and membrane potentials.

These investigations have provided very valuable infor­

mations on the complex mechanisms of the electrochemistry

of membranes in general and the biological membranes in parti­

cular. A two-fold impact of the electrochemistry of membranes

on biology and medicine has been proposed by Shedlovsky (1955).

Firstly, it provides powerful laboratory methods and tools

for the study of biologically important substances, such as

viruses, hormones, enzymes and other proteins. It also helps

in determination of activity, oxidation-reduction, ionic mobi­

lity, activity, diffusion, dielectric constant and dipole

moments in biological environments. Secondly, it helps to

analyse the individual membrane events in the living cells

in terms of units of complex electrochemical systems capable

of transforming chemical energy and ionic transport into elect­

rical signals.

2.1 Transport phenomena in artificial membrane systems

When an artificial membrane separates the solutions

containing different concentrations of the same electrolyte,

the nature of potential difference across it depends upon

the permeability of the membrane to the cations and anions

and the activity of the ions themselves. The transmembrane

potential difference in such systems has been described with

the help of the Nernst equation expressed as -

RT 1

^ = I F " C^ (1)

Where E, is the potential difference in volts, R, the

gas constant (8.312 Joules per degree per mole), T, the abso-

lute temperature (273.15°C ± t°C), Z, the valency of the ion,

and F, the Farady constant (96,500 coulombs per gram equi­

valent) .

Theoretically, the E.M.F. due to electrochemical gradi­

ent in the concentration half-cells is considered to be due

to the activities of the cations and anions. For the systems

without transport it can be calculated by the equation (2)

which takes account of these variables-

i ^1 •

, Where, IJ is the total number of ions, y± the number

of + or - ions produced by the ionization of one molecule

of electrolyte, a, and a- the mean ionic activity of the elec­

trolyte (where a = fc, f being the activity coefficient)

and R, T, Z and F have their usual meanings.

The E.M.F. of a concentration cell with transport,

however, requires due consideration for the transport number

of the ions and can be expressed as -

E =+V- .2.^v^^. In - ' — (3)

Where t^ is the transference number of the anion or

cation, all the other notations having the same meaning as

for equation (2).

2.2 Artificial membrane systems

Various kinds of artificial membranes have been used

for studying the ion transport mechanisms. Thus, for example,

Nagasawa and Kagawa (1956) measured the potentials of ion

exchange membrane with different concentrations of simple

electrolytes (NaCl, NaSO^).

In order to study the molecular mechanism of the pheno­

mena occurring in biological membranes, attempts have been

made to investigate the properties of artificial membranes

in terms of the known physiological processes (Tien and Diana,

1967).

Thei ion transport characteristics across the lipid

membranes, which are in many respects similar to the biological

(cellular) membranes, became a subject of intensive investi­

gations after the mid 1960's. Huang et_ £l. (1964) studied

the physical properties of the lipid membrane separating two

aqueous solutions. Such artificial membranes are produced

when a mixture of phospholipids and neutral lipids dissolved

in organic solvents is allowed to come in contact with water.

These membranes display no metabolic activity and are not

so highly selective as are biological membranes. Membrane

potentials produced by monovalent ion distribution across

artificial lipid membranes have also been measured in an attempt

to investigate the mechanisms underlying resting membrane

potentials in biological membranes (Ohki,- 1971). Mueller

8

and Rudin (1969) and later Hauser et. £l (1972) studied the

ion permeability of phospholipid bilayers and compared its

biophysical properties with those of the cellular membranes.

2.3 Permeability of artificial membranes to H

The mechanism of H^ transport across the lipid membrane

has become a subject of intensive investigations during the

last several years. Various model membranes, both of the

uni- and bi-laminar lipids, have been used for this purpose.

HCl has been shown to have a very high solubility in

hydrocarbon solvents (Linke, 1958). Chains of associated

water molecules within the bilayer would permit transport

of protons and hydroxyls via hydrogen bond exchange. Water

is considered to be present in the hydrophobic portion of

the bilayer because lipid membranes are relatively permeable

to water (Bangh et £l.f 1967; Hanai and Hayden, 1966; Cass

and Finkelstein, 1967).

Nagle and Morowitz (1978) proposed that protons cross

membranes along continuous chains of hydrogen bonds ("Proton

wire")formed from the side groups of transmembrane proteins.

Permeability of unilamillar liposomes for H"*" and 0H~ was stu­

died by Nichols and Deamer (1980). The permeability of H"*"-

through phospholipid bilayer was studied by Nozoki and Trans-

ford (1981). Gutknecnt and Walter (1981) using planar phos-

pholipid bilayers showed that net proton fluxes result from

transfer of undissociated acid molecules and that the per-

meability of phospholipid bilayers for H"*" £er £e in very low.

The uncharged acids make the major contribution to apparent

H* fluxes in planner bilayer experiments.^

Nichols and Deamer (1980) assumed that a significant

amount of water exist within the bilayer in the form of hydro­

gen bonded strands which can facilitate proton and hydroxyl

transport by the rearrangement of. hydrogen bond as in water

and ice.

The electrical measurements (e.g. E.M.F. and current

etc. ) in concentration half-cells containing electrolytic

solutions and separated by artificial membrane systems have

been frequently studied to obtain further information on the

mechanisms of ionic transport across biological membranes.

Yoshida (1972) on the other hand studied the transport pheno­

mena in a model membrane and measured the membrane potential,

ion permeability and conductance.

Investigations on these lines and on the bioelectro-

chemical aspects of the lipid membrane systems incorporated

with the functional ionic channels isolated from the living

cell membranes (Miller, 1986) have provided large amount of

valuable information on the passive ion transport mechanisms

in the living cells. Despite these advances in our knowledge

of the ion transport the fundamental processes underlying

the transport of H across the artificial and living membranes

are still far from fully understood.

10

2.4 Biological Aeinbranes

The biological membrane is a dynamic structure consist­

ing mainly of a bilayer of lipids and of proteins, which impart

a heterogeneous character to it. The membrane lipids are

composed of a polar head and two long nonpolar hydrocarbon

'tails' which possess hydrophobic properties.

In 1935 Danielli and Davson proposed a theoretical

model of the composition of biological membranes which is

called the unit membrane model. According to this concept,

the unit membrane is considered to be a bilayer of mixed polar

lipids with their hydrophobic tails extending inward and their

hydrophilic heads exposed on the surface where they are bound

to the outer monomolecular protein layer (Fig. lA).

The most convincing evidence for the structure of cell

membrane, particularly about its lipid bilayer nature was

obtained from electron microscopy (Robertson, 1964). The

organization of the unit membrane into the bilayer arrangement

of lipids was further substantiatedJ^X-ray data (Worthington,

1969).

The present concept of the structure of cell membrane .

was put forward by Singer and Nicolsoni (19 72) who expressed

it in terms of.a fluid mosaic model. According to this model

the membrane is composed of a bilayer of lipids and some pro­

tein molecules which are either in the outer, or inner half

of the membrane matrix or span the entire thickness of the

Fig.lA Structure of the unit membrane model (Davson

and Danielli, 1935). a, Lipid molecules-hydro-

phobic tail; b, Lipid molecules-hydrophilic

head; c. Protein molecules; d. Polar pore.

B Structure of the fluid mosaic model (Singer

and Nicholson, 1962). a. Lipid molecules; b.

Protein molecules.

11

membrane (Fig. IB). The lipids in the membrane behave like

liquid crystals (Singer and Nicolson-i, 19 72). It is precisely

a liquid crystal that combines high ordering with fluidity

and lability. The membrane proteins have hydrophilic surfaces

which bound to the polar end of the lipids (Quinn, 1976).

The membrane protein can be subdivided into two classes.

The first type of proteins are bound only to the membrane

surfaces like globular proteins, which have a hydrophilic

surface and function in an aqueous environment. The second

type of proteins are considered to span the width of the mem­

brane and interact with the hydrophobic tails of the lipids.

These proteins are insoluble in water because their surface

is hydrophobic in nature. They are arranged asymmetrically

in the membranes (Bergelson et al;'i970).

2.5 Bioelectrochemical aspects of heart cells

The ionic distribution gives rise to a potential diff­

erence across their membranes which have been measured by

appropriate techniques. The potential difference during the

resting state of the cell is called resting potential (or

phase 4, Fig. 2) which is negative inside with respect to

the outside and changes direction during the cellular activity.

This time dependent change in the membrane potential occurring

during the activity of the cell is termed action potential

(AP) and is due to the flow of ionic currents across the mem­

brane (Katz, 1966).

12

The most significant ions involved in myocardial cell­

ular functions are Na"*", K"*", Ca ''', H"*" and Cl". These ions

are present in different concentrations on the two sides of

the cell membrane i.e. the cytoplasm and the extracellular

fluid. During the cellular activity they carry passive mem­

brane currents giving rise to APs.

For explaining the EMF across this type of membrane

system Goldman (1943) and Hodgkin and Katz (1949) derived

an equation by modifying the Nernst equation which is called

generally as the constant-field equation.

Where, Em in the potential difference across the cell

membrane, P+> P + and P-.v- the permeability co-efficients for

K , Na and Cl" respectively, i , inside and o, outside of

the cell.

2.6 Cardiac action potentials (APs)

The classical Na -K hypothesis of Hodgkin and Huxley

(1952) explaining the ionic basis of nerve action potential

(AP) was developed on the basis of elegant electrophysiological

experiments on the giant squid axon. For the much smaller

cell like those of heart applicability of HodgKi.n-Huxley concept

was achieved later by successful recording of APs using the

glass micro-electrode technique (Ling and Gerard, 1949; Weidmann,

Na

influx Past

Fig. 2: Line drawing of ventricular APs drawn to scale

from the original record showing various para­

meters of AP. Phase 0, rapid depolarization

Phase 1, early repolarization, Phase 2, Plateau

of slow repolarization; Phase 3, rapid terminal

repolarization, phase 4, Resting potential.

APD is action potential duration in ms at -20

and -40 mV and at 90% repolarization.

13

1951; Trautwein and Zink, 1952). These experiments have shown

that the myocardial cells are also selectively permeable to

K"*" and impermeable to Na"*" during the resting state, K being

higher inside and Na"*" higher outside the cell.

The cardiac AP is characterised by rapid depolarisation

followed by very slow repolarization so that it lasts several

hundred milliseconds (ms) as compared to a few ms of the nerve

or skeletal muscle AP (Fig.2 ). The time and voltage dependent

ionic currents are responsible for generation of these APs

which could be explained initially by the bi-ionic model of

Hodgkin and Huxley (Hodgkin and Huxley, 1952; Woodbury, 1960).

We, however, now know that several J onic currents carr­

ied via specific membrane channels, in addition to the Na

and K"*" currents, are involved in the cardiac AP. A brief

description of these currents contributing to different phases

of cardiac AP (i.e. Phase 0, 1, 2," 3 and 4) are given below:

Phase 0(or depolarisation): It is characterised by a fast

reversal of membrane potential from the resting levels to

+ 20 to +30 mV due to rapid influx of Na via the fast Na

channels (Coraboeuf and Otsuka, 1956; Trautwein, 1973; Weidmann, 0

1974). The fast Na"*" inward currents (iNa) underlies this

phase of AP and is over within less than 1 ms.

Phase 1,2,3 (or Repolarisation)

Phase 1 (or early rapid repolarisation): This phase is charac­

terised by a fast but short lived repolarisation immediately

14

on the termination of upstroke. Termination of the fast Na

and a transient outward current (tto) are attributed to the

appearance of this phase (Hiraoka and Hiraoka, 1975) (Fig.

2).

Phase2.(or the plateau of repolarisation); This is character­

ised by a sustained depolarisation positive to -40 mV. Flow

2+ 2+

of the slow inward Ca current via Ca channels is considered

to be primarily responsible for this phase. Rectifying currents

carried by K^, however, are also attributed a partial role

for this- phase of repolarisation (Weidmann, 1974; Carmeliet

and Vereecke, 1979; Noble, 1984) (Fig.2:).

Phase 3(or terminal or all or none repolarisation): This

is a phase of rapid repolarisation of the cells whereby the

resting potential of the cell is restored. It is due to the

attempt of the cell to restitute the normal cellular K^ con­

centration gradient. The background K current (i ) or the

outward currents generated by electrogenic pump may also con­

tribute to this phase of repolarisation (Carmeliet and Vereecke,

1979)(Fig.2 ).

2.7 Effect of pH on cardiac AP

A change in pH significantly affects the cardiac action

potential duration (Chesnais £t £l., 1975; Carabouf et_ al.,

1976; Wada and Goto, 1975). It is reported that increase

in [H ] (or acidity) inhibits the Ca current carried via 2+

Ca channels in heart (Vogel and Sperelakis, 1977). Acidity

15

is also reported to alter the membrane Na"*", K -ATPase activity

in the myocardium (Sperelakis and Lee, 1971).

The altered pH has been attributed an effect on mem­

brane ion conductances (Harris, 1965; flutter and Warner, 1967;

Sperelakis, 1969). An acid pH decreases membrane permeability

to both anion {Cl~) and cation (K ), whereas alkaline pH in­

creases the permeability to both (Carmeliet, 1961; chesnais

et al., 1975; corabouf et_ £l., 1976). Acid pH blocks slow

cation channels rather than the fast Na channels (Chesnais

et al., 1975). In cardiac muscle acid pH has an effect on

2+ + the inward slow Ca and Na currents (vogel and Sperelakis,

1977). [H ] is considered as a new type of ion channel blocker

(Colquhoun, 1987). These observations assume greater signi­

ficance in the light of the report that the myocardial cellular

pH becomes acidic during cardiac infarction etc.

In spite of such a prevailing importance of pH in 'regu­

lating the cellular functions in general and cardiac activity

in particular, our understanding of the mechanisms of the

regulatory role of H in ion channel activity is far from

fully understood. In order to study the effects of pH on

cardiac ionic channels the effects of altered [H^] on ventri­

cular AP of guinea pig heart have been investigated in the

present study.

METHODS AND MATERIALS

16

3.0 METHODS AND MATERIALS

Two different types of experiments were carried out

in present study, involving measurements of E.M.F. due to

electrolyte concentration gradient in artificial cell systems

and those encountered in the living cells examplified by

heart cells. The former was studied in diffusion half-cells

separated by artificial membrane systems or half-cells conn­

ected by KCl-agar salt bridge. The latter on the other hand

was performed by employing the electrophysiological experi­

ments on the heart cells from the isolated guinea pig heart

tissue preparations.

3.1 Artificial membrane system

The experiments on,ionic transport in artificial mem­

brane systems were carried out by measuring E.M.F. with trans­

port in all glass diffusion half-cells containing different

concentrations of the electrolyte and separated by porous

partitions. The E.M.F.without transport was measured in

some cases for the same electrolyte systems using half-cells

connected by an agar-salt bridge. The details of these

methods (Ram et £l.., 1982) are described below:

3,1.1 Measurement of E.M.F. with transport

3.1.1.1 Diffusion half-cells: Two diffusion half-cells (7

cm length and 3 cm diameter of each half-cell) made of corning

glass with the provision of placing a filter paper partition

«LAM(

CiAMP

Fig- 3: Schematic diagram of diffusion half cells used

for measuring E.M.F. with transport. A and

B, Diffusion half cells; C and D, Side-arms

for filling electrolyte solutions; E and F,

Ag/AgCl electrodes; G and H, Glass stirrers

for stirring the solutions, pp' porous partition

(Whatman No-42 filter paper); M, Digital multi­

meter for measuring E.M.F.;

hooks; c. and H, to H., Glass

C^, Electrolyte solutions of

two different concentrations; T, thermostatic

bath filled with water (W) at 37-± 0.5°C.

17

between them and the inlet and outlet ports for filling and

removing the electrolyte solutions at two different concent­

rations. The two half-cells could be easily joined and sepa­

rated. Before pouring the electrolyte solution into the

half-cells a Whatman filter paper (No. 42) was placed between

them and they were tightened together with springs and rubber

bands. This set of half-cells was placed in a thermostatically

contolled water bath maintained at 37»o ± 0.5°C. The Ag/AgCl

electrodes and glass stirrers were placed in the half-cells

from the port for them at the top (Fig. 3 ).

3.1.1.2 Preparation of Ag/AgCl electrode: Ag/AgCl electrodes

were prepared by depositing chloride ions on the pure silver

wire (Delke, 1969)". A silver wire electrode was dipped in

a small bea)?er containing O.lN HCl solution served as the

anode for electroplating with AgCl, a Ag wire serving as

the cathode. Both these electrodes were connected with multi-

output of a D.C. power supply (Aplab 7711). A constant current

of about 3 mA at 2 mV was passed until a thin creamy white

deposit of AgCl appeared on the electrode surface. During

this electroplating process no direct light was allowed to

fall on the Ag-wires.

The prepared electrodes were stored in O.lN HCl in

the dark to avoid photochemical effects on AgCl. Before

re-using these electrodes for fresh electroplating with AgCl

for subsequent experiments the deposited AgCl was removed

each time by ammonia solution.

18

3.1.1.3 Measurement of B.M.F.: The E.M.F. with transport

was measured for different electrolytes at varying concentrat­

ion gradients in diffusion half-cells with the help of a

high input impedance Digital Multimeter (HIL-2161) Connected

to the Ag/AgCl electrodes. The electrodes and the stirrers

were placed in each half-cell through the openings on the

top of the cell. The electrolyte solutions were poured simul­

taneously through the side arms in the diffusion half-cells.

The solutions in each half-cell were agitated gently but

continuously thereafter with the help of the stirrers. The

values of E.M.F. were noted down at intervals of 30 sec

for a period of 10 min. The E.M.F. at zero time was found

by extrapolating from the graph of E.M.F. - vs - time.

3.1.2 Measurement of E.M.F. without transport

3.1.2.1 Half-cells: Two corning glass beakers (100 ml)

filled with the aqueous solutions of the same electrolyte

at different concentrations served as the two half-cells

for measuring E.M.F. without transport. Two Ag/AgCl elect­

rodes were placed in each half-cell and connected to the

digital multimeter. The two half-cells were connected to­

gether electrically with the help of the KCl-Agar salt bridge.

These half-cells were placed in the thermostatically con­

trolled water bath maintained at 37.o * 0.5°C.

3.1.2.2 Preparation of KCl-Agar salt bridge: Agar salt

bridge was prepared by making 0.8 M aqueous solution of KCl

to which Agar powder (Bacteriological grade) was added to

e- S B

^ \ 7^c ;i

E/jit--^^! -Ajt j5y[+Cj-^y^

iV H

Fig. 4 : Schematic diagram of half cells used for measur­

ing E.M.F. without transport. A and B, half

cells; C, KCl-Agar salt bridge; D, Stand; E

and F, Ag/AgCl electrodes; M, Digital multimeter

for measuring E.M.F.; c, and C-r Electrolyte

solutions of two different concentrations; T,

Thermostatic bath filled with water (W) at

37 ± 0.5°C.

19

give a concentration of 3%. This suspension of agar in KCl

solution was heated up in a small beaker on water bath till

a clear solution was obtained. This solution was then poured

while hot, in a U-tube which formed a white jelly on cool­

ing. This agar-salt bridge was stored by dipping the tips

of the U-tube in two beakers filled with the KCl solution

used for preparing the agar-salt bridge.

3.1.2.3 Measurement of E.M.F.: E.M.F. without transport

was measured with the help of the digital multimeter (HIL

2161) connected to the Ag/AgCl electrodes (Fig.4). The values

of E.M.F. were noted down at intervals of 30 sec for a period

of 10 min. The E.M.F. at zero time was found by extrapolating

from the graph of E.M.F. - vs - time.

In order to confirm the validity of E.M.F. measurement

by the digital multimeter, pilot experiments were carried

out by measuring E.M.F. in the same system using a very high

input impedence probe electrometer (M 701 W.P. Instruments,

U.S.A.). The values thus obtained were displayed on a storage

oscilloscope (Model 5113N, Tektronix, USA) for recording and

analysis as described below for the heart cells.

3.2 Electrophysiological experiments on living heart cells

3.2;1 Preparation and mounting of isolated guinea pig papi­

llary muscles

For preparing the papillary muscles from hearty young,

healthy guinea pigs (either sex, CDRI colony) were killed

Fig. 6A: Diagrammatic representation of a Sagittal

section of internal structure of mammal­

ian heart to show its internal structures.

PM, Papillary muscle; SAN, sinuatrial

node; AVN, atrio-ventricle node; SVC,

superior vena cava, IVC, inferior vena

cava; AO, arota; FO, Fossa ovales and

CS, opening of coronary sinus.

B: Low magnification electron micrograph

of papillary muscle as a blow up of

area enclosed in the rectangle of fig.

4 (A), ES, extracellular space also

depicted by arrows.

C: High magnification electron micrograph

of the area of papillary muscle enclosed

in the rectangled of Fig.4 (B) ES, extra­

cellular space. The lipid bilayer of

the plasma membrane of adjacent cells

X and Y are shown by empty arrows.

SVC

••• i ^ *

20

by a blow on the neck. The chest was opened and the heart

quickly removed and placed in a glass dissecting chamber

with sylgard floor containing HEPES-Tyrode solution (com­

position in mMol/L NaCl, 145; KCl, 4.98; CaCl22H20, 1.8;

MgCl2.6H20, 0.098; HEPES, 2.98; Glucose, 10.7 titrated to

pH 7.4 by NaOH and HCl at room temperature. (Fig. 5).

The pericardium was cut away and the right atria and

ventricles slit opened. Papillary muscle from the right

ventricle was removed and placed in the perspex electrophysio­

logical chamber and pinned to the sylgard floor of the chamber.

Oxygenated HEPES tyrode solution kept in double jacketed

graduated corning glass cylinders and maintained at 37.0

± 0.5°C with the aid of an ultrathermostatic water circulator

(Type^U-10, VEB prufgeratewerke, DDR). \

The bathing solution was adjusted to superfuse the

preparation in the electrophysiological chamber at a flow

rate of 60-80 drops/min taking care t'o maintain a constant

temperature for the preparation during the course of experi­

ment. The control experimental HEPES-tyrode solutions of

different pH were stored in the other double jacketed glass

cylinders oxygenated and maintained at identical constant

temperature in a similar manner.

A pair of Ag stimulating electrodes (suction type,

Furshpan and Potter, 1958) were attached to the base of the

papillary muscle and connected to pulse generator (160,

Fig. 6: INSET.

Photographs showing the experimental

set-up for electrophysiological studies.

Inset shows the electrophysiological

chamber (E), the micro electrode probe

(A), microelectrode (M), indifferent

electrode (I), stimulating electrode

I S ) .

21

Tektronix, Inc) via a stimulation isolation unit. Electrical

stimulation of the preparation was performed by passing supra­

maximal pulses at IHZ.. The preparations were allowed to

equilibrate for 30 min before the start of the experiment.(Fig.

6).

3.2.2 Preparation of solution of different pH

The HEPES Tyrode solutions of different pH (5.7 to

10.4) were prepared by titrating the solution with aqueous

solution of NaOH and HCl until the desired pH was achieved.

A bipolar electrode coupled to a digital pH meter (335, Sys-

tronics) was used for measuring and titrating the pH of the

solution. All the solutions were made of the salt highest

grade of purity (AR, Guarantted Reagents etc.). All glass

triple distilled water was used for this purpose.

3.2.3 Recording of action potentials

For recording the action potentials (APs) from ventri­

cular cells 3 M KCl filled glass ultramicro-electrodes were

used. These were drawn by a horizontal microelectrode puller

(H104, CF Palmer U.K.) using quickfil glass capillaries (WP

Instruments, USA). The tip resistance of microelectrode

was 10-30 M-fX . The recording microelectrode was connected

to a microelectrode holder containing Ag-AgCl pellet (W P "l)

which in turn was connected to the input stage of a high

input impedance microprobe (M701, WP). The microprobe carry­

ing the microelectrode was mounted on a micromanipulator

(Carl Zeiss, Jena) to make the ultra fine movements for cell­

ular impalements. The output of the microprobe was connected

SHIELD

Diagrammatic representation of the experimental set-up for

electrophysiological studies: B-Bath for keeping the preparation;

S-sylgard floor, T-tissue preparation, R-Recording electrode

S^-Stimulating electrode, I-Indifferent electrode, A^-Microelec-

trode input probe, SHIELD-the faraday cage in which the

whole assembly was lodged. A--microelectrode amplifier (Elec­

trometer) , OSC-storage oscilloscope for monitoring the electrode

signals obtained from the preparation, CAM-camera for making

the photographic records, P-pulse generator to stimulate the

preparation.

22

to the input stage of a suitable preamplifier (M701, WPl)

with facilities for negative capacity compensation. An Ag/

AgCl pellet (RC-2, WP i) immersed in the electrophysiological

chamber, connected to the return ground post of the micro-

electrode preamplifier served as the reference electrode.

The output of the preamplifier was connected to one channel

of a storage oscilloscope (5113N, Tektronix, Inc) to display

the APS and to an audio baseline monitor (ABM, WPJ.) for audio-

monitoring of the resting and action potentials. The micro-

probe system and its preamplifier were calibrated using a

precision millivolt source (Model 101, WPl) connected to an

equivalent circuit of the microelectrode etc(Fvq.7)»

The APs displayed on the oscilloscope screen were

recorded on a 35 mm unperforated high speed black and white

film (Eastman Kodak Co. ) by a Kymograph Camera (C4K Grass

Instruments Co., U.S.A.). The films were developed in dark

room for subsequent storage and analysis of APs. The APs

were traced on graph papers after enlarging the negatives

on an AGIL enlarger or microfilm reader (Agfa Gaevert, India).

3.2.4 Analysis of AP

From the tracings of the APs following parameters

were analysed (Fig. 2 ) .

i) Resting membrane potential (Em): The resting membrane

potential (Em) was measured taking the zero potential line

as the reference point.

23

ii) overshoot (Eov): The metRbrane potential eKcursion posi­

tive to zero mV.

iii) AP amplitude (APA): Resting membrane potential plus

overshoot indicates the APA/ i.e. the total voltage change

in one direction.

iv) AP duration at -20 mV (APD 20'): The time interval between

depolarisation and repolarization at the membrane potential

of -20 mV.

v) AP duration at -40 mV (APD 40): The time interval between

depolarization and repolarization at the membrane potential

of -40 mV.

vi) AP duration at 90% repolacisation (APD 90): The time

interval between depolarization and 90% of the repolarisation.

vii) Upstroke velocity (+vmax): The maximum rate of change

of potential during the upstroke of an AP.

3.3 The following chemicals were used in this study

i) Agar (Bacteriological Grade) (L.R., Sarabhai)

ii) Calcium chloride (CaCl-j . 2H2O) (E. Merck, Germany)

iii) Glucose (E. Merck, Germany)

iv) HEPES (N-2-Hydroxyethyl Piperazine N-2-ethanesul-

phonic acid (Sisco)

V) Hydrochloric acid (LR, BDH)

vi) Magnesium chloride (MgCl2.6H20) (AR, BDH)

24

vii) Potassium chloride (KCl) (AR, BDH)

viii) Sodium chlotide (NaCl) (AR, Glaxo)

ix) Sodium hydroxide (NaOH) (AR, Sisco)

x) Sylgard (Dow corning).

RESULTS

25

4.0 RESULTS

4.1 Artificial Membrane System

4.1.1 E.M.F. with transport

The observed E.M.F. with transport in different electro­

lyte systems was found to decrease with decreasing concentrat­

ion ratio C-ZC, (Tables 1, 2 and 3). At the higher concent­

ration ratio of 0.1/0.01 M the measured E.M.F. estimated

for zero time for the System containing KCl was found to

be 50.5 mV and for those containing CaCl- was 54.5 mV. At

the lower concentration ratio of 0.08/0.03 M the E.M.F. for

KCl and CaCl, containing systems was 23 mV and 21.5 mV res-

^pectively. The values of E.M.F. gradually declined with

time at all the concentration gradients approaching 22.8

mV for KCl and 19.0 mV for CaCl- at 120 sec.

The E.M.F. changed with variations in the electrolyte

species even at the same concentration gradient. For example,

the E.M.F. for KCl, CaCl2 and HCl at concentration ratio

of 0.1/0.01 M at zero time was 55.5, 34.0 and 82.0 mV res­

pectively. The values for the system containing KCl or CaCl^

were close to the one predicted by the equation (1). In

the case of HCl, this was not the case as the E.M.F. obtained

experimentally could not be predicted by the equation (1).

Instead, the E.M.F. due to concentration gradient of HCl

could be closely compared to the one obtained theoretically

from the equation (3), which also predicts the values for

KCl and caci- system.

u

o 1

c c 0 O O -H C J

O (0 u ^

a;

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u

CL)

26 c: 1

-H M 0)

d) -p o c C -rl u Q) O j j oj r -<D e n X-4 -H w ^ C «J 4J w C +J <U

u x: (u 4J U-l •H IW ? -H

x> •

fa JJ • 10

s • >

W E

0) 1 o c C -H (U M (U

AJ to

-—« •

o Q) to —'

w rH (0 >

(U 6 U <W -HO 03 4J c rO 4J

h-ro

M C -U 4 j <y

M JJ (U 3 <W O »w x: -H j j -O • H

? -u

OJ

— O 0) M

«—'

w nS -H

• fa > • E

s • c

ro > u Q) 4J

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O VO

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i n

C>) i n

o

n i n

CT»

n i n

o i n i n

00

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CM

CN i n

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n «*

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o t ^ <*

r-<-i ^

iH

CM

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o

r~ ro

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r n

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n n

c

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VO

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VO

r CM

o\

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<-i

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m

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00

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s EH

C 1

<u Q) 4J

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a\

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tn iH O

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r^

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u J5 <U 4J M-) • H <M ^ - H

TD •

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4J Q> U

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r--ro

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a

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c <u S -o o x; c CO O

rtJ • 4J

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s o • o

u

29

0 0

in

in

B • -H

in o i n

i n

in

c 0

• H

-U ro U

4J

• H

u \

CN

u J

c \ (U 0 c 0 u

cr cq

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w

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Q. X w

CN ro

LOOT

SO

60

EM.F.

40

20-

^

L^ KCL

m i r

Ca^CL.

^

nci

Fig. 8 Bar diagram showing E.M.F. in mV with

and without (hatched) transport at

zero time for different electrolyte

systems e.g. KCl, CaCl2 and HCl for

concentration ratio of 0.1/0.01 M

at 37 C.

30

4.1.2 E.M.F. without transport

The observed E.M.F. without transport was also found

to change with time particularly for the initial 2 minutes

as evident from Tables 1,2 & 3. E.M.F. without transport,

however, changed directly with a change in the concentration

ratio. It was higher at high concentration ratios and lower

at low concentrations ratios.

E.M.F. without transport also depended on the electro­

lyte species used as in the case of E.M.F. with transport.

For the systems containing HCl, KCl and Cacl- at concentration

ratio of 0.1/0.01 M E.M.F. was found to be 57.2, 53.5 and

52.8 (mV) at 37°C.

The above observations showed that E.M.F. with and

without transport depends upon concentration ratio and nature

of the electrolyte.

Moreover, E.M.F. without transport at zero time for

the systems containing KCl and CaCl^ at different concent­

ration ratios (0.1/0.01, 0.095/0.015, 0.09/0.02, 0.085/0.025,

0.08/0.03) and HCl at the concentration ratio of 0.1/0.01

was found to follow t.he order HCl KCl Cacl-. The E.M.F.

with transport for these concentration ratios of the same

electrolytes on the other hand, followed the order: HCl KCl

CaCl2 Tables 1, 2 and 3).

4.2 Biological Membrane System

4.2.1 Effects of different pH of APs of myocardial cells

The guinea pig papillary muscle APs showed prominent

o <u '^

C I nj x: Q u &(

<

(D o CncN c

O Q

a*

o

I

Q

<

o I <i)

Q W

<

o 04

Di _» C > •rW

43

w (U «

e v.^

e w

o o M M

> o

a:

31

<T\ in in

03 (N

+

1 f^ ro +

<N

n +

0 ,

cs 1

1 r-04

1

in

9

+

in

+

in

CO

+

00

I

00 rH I

in in

r-n (N

0 0 ro

0

r-rH

CN 0 rg

(N <y\ -H

• ^

in CM

r-in CM

in 0 fS

in n CM

0 <Ti .H

in

r •H CM

0

r ro CM

in

CM in -H

in

1^

r- -H

0

0 00 M

in

1^ ro r>i

0

CM ro CM

0

0 cr> rH

0

CM rH CM

0

CM

r~ .H

in 00 1

0 00

1

<Tl

1

in

1

ro 00 1

CM 00 1

rH 00

1

CM 00 1

00 00 00 I

0 00

+

0 ro +

in OJ

+

• ^

CM

+

0 ro +

ro ro +

0 ro +

.H ro +

0 ro +

0 ro +

• ^

( r in

• ^

1^

• < *

V£>

"

r--• ^

00

^

r-xij'

(Ti

'S*

r-•^

0

32

xi

> O

<u c en o c u m x: o

<*p

4J •H S

W <u o c 0) v< 0

M-l U-\ •H Q

-H O u 4J C o o

e

o in o n •^ CN (N • * VO <JD

+ I I I I

n ro 00 n

VD ro M OO LD (N 00 i n 03 00

I + I I I I

ro CO

ro o o

o o 00

o in

00

-^ r

r-i n

•^ vc

M*

00

• *

en

• *

o

A •*]

looms

Fig. 9: Action potentials ,APS, lecoided fro.

guinea pig papniary .uscle showing

the effects of different pH. The records

Show control .PS I„ „„,,,, ^^^^^^^^

those in the solutions of pH 5.7 ,Ab,

. V ' " ' '•' ''''' '•' ' - ' -<^ "-^ 'E«'- The upper trace in each ..„=,

2m5

Fig.10: APs recorded from guinea pig papillary

muscle showing the effects of different

, pH (7.4, 5.7 and 10.4) on the upstroke

of APs recorded at fast sweep speed.

The upstroke of APs recorded in the

solution of pH 7.4 (A&C), 5.7 (B) and

10.4 (D). The uppertrace in each Panel

shows OmV reference line.

33

changes in different parameters following changes in the

pH of the superfusing solution from 5.7 to 10.4 as evident

from Figs. S) and ^and Tables 5 and 6. Figs. 9 andio show

the effects of alterations in the pH on the APs recorded

10 min after the change over to these solutions.

4.2.2 Effects of higher pH (8.4, 9.4 and 10.4)

An increase in the pH from 7.4 to 8.4 brought about

an increase in the duration of AP at -20 (APD20) and at -

40 (APD40) mV. A further increase of pH from 7.4 to 9.4

or 10.4, however, caused a decrease in APD20 and APD40.

The other parameters of membrane electrical activity e.g.

resting potential, overshoot and AP amplitude remained un­

altered under these conditions. The maximal upstroke velo­

city (+Vmax) was reduced progressively by increasing levels

of pH as evident from Fig. 9 and Table 6. It may be pointed

out here that the changes in AP durations at the enhanced

pH were somewhat erratic for the initial period ( 10 min)

of change over to the higher pH (not illustrated).

The above effects of increase in pH disappeared within

10 min of replacing the experimental solution with the control

one of pH 7.4,

4.2.3 Effectsof lower pH (5.7 and 6.4)

A decrease in pH of the Tyrode solution from 7.4 to

6.4 and 5.7 brought about a slight depolarization of the

cells as evident from decrease in resting potential (Fig.

34

9 and Table 5). AP duration was drastically increased at

pH 5.7, the values of APD2() and APD40 being increased signi­

ficantly (Fig., 9 and Table 5). The overshoot remained un­

altered but +Vinax was increased by about 20% at pH 5.7 (Fig.

lo and Table 6).

All these effects of lowering the pH of the bathing

solution disappeared withiri 10-15 min of resuperfusion with

the solution of normal pH (i.e. 7.4).

Recovery of the control activity under these conditions,

however, took longer time i.e. 20 to 30 min than in the experi­

ments with higher pH.

DISCUSSION

35

5.0 DISCUSSION

Although H^, the smallest ion with high membrane per­

meability (Pu = 100 Pj ) (Gilbert and Lowenberg, 1964;Woodbury,

1966; Izutsu, 1972) plays an important role in cellular funct­

ions, the characteristics of its transport across the plasma

membrane are not yet fully understood. Since it has to move

across the lipid bilayer membrane of the living cells to

participate in the cellular functions, it was considered

useful to know about its transference behaviour in an arti­

ficial system using uncharged porous partition with and with­

out lipid coating. With these objectives in mind present

study was carried out on E.M.F. due to H in artificial mem­

brane system and its effects on the transmembrane potential

changes in the living cells of heart.

5.1 Studies on artificial membrane systems

It was observed presently that,in the artificial mem­

brane systems separated by uncharged porous partition-Whatman

filter paper-E.M.F. changed with change in concentration

ratio of the chloride salts of cations i.e. H^, K^ and Ca

the value of E.M.F. with transport obtained by this technique

followed the following order for these cations HCl^KCl^CaCl^,

for the same concentration ratios. The higher values of

E.M.F. observed for the system containing KCl than

for Cacl2 are in agreement with those observed earlier

*PH and Pj( are membrane permeabilities for H^ and K"*" res­pectively.

36

for a similar system (Ram et al., 1982). It is interesting

to note that the E.M.F. due to HCl was much higher than that

due to the other two cations. This is likely to be due to

the higher mobility of H"*" than K"** and Ca "*" (Glasstone, 1947,

Rao, 1973). These differences in the behaviour of different

physiologically important cations used in present study could

be due to differences in their crystal ionic radii which

follow the order H"*'» K^\. Ca "*" (Lange, 1967) which would re­

verse the order of their mobility in the aqueous medium due

to the shell of hydration around them .(Katz, 1966).

The finding that E.M.F. without transference due to

these ions at the same very concentrations are lower than

the corresponding values of E.M.F. with transference (Table 1,2

3 ,Fig. 8 ) demonstrates that the movement of the cations

across the membrane without any impedence contributes to

the differences in the values under these two experimental

conditions. These findings are supported by the earlier

reports using similar experimental techniques (Ram et^ al. ,

1982). It is of particular interest to note that E.M.F.

with transference' for H was substantially higher than that

without transference the latter being closer to the values

+ 2+ obtained for the K and Ca containing systems. This further

confirms that the higher value of E.M.F. with transference

due to H as compaired to the other cations was due to its

greater mobility.

5.2 Studies on living cell membrane system of heart cells

The passive membrane ion transport which plays a cru­

cial role in cellular ' functions is known to be strongly

37

affected by a change in pH as the altered pH has an effect

on membrane ion conductances (Harris, 1965; flutter and Warner,

1967; Sperelakis, 1969). An acid pH decreases membrane per­

meability while alkaline pH increases it (Carmeliet, 1961;

Chesnais et al,, 1975; Corabocuf et al., 1976). H inhibits

+ + 2+ the membrane permeabilities of cations (Na , K and Ca )

causing the various reported effect's of pH on the electrical

and mechanical properties of the myocardium (Wada and Goto,

1975). Changes in [H^] or pH has been reported to affect

the cardiac action potential duration by altering the ionic

channel activity etc. (Chesnais £t a].., 1975; Coraboeuf et

al., 1975). In most of these earlier studies pH was altered

by altering the HCO~ content of the solutions outside the

cells.

In present study we have tried to see the effect of

pH on the transmembrane electrical activity in heart cells,

the pH having been altered by using HEPES buffer which is

not normally present in the cellular systems. When pH was

changed from 7.4 to 5.7 and 6.4 a change in the resting pot-

ential, APD-20, APD-40 overshoot and +Vmax of the ventricular

AP was noticed. Resting potential showed a decrease while

APD-20, APD-40 and +'Vmax were increased at pH 5.7 but +Vmax

showed a definite decrease at pH 6.4. There was no change

in overshoot. At higher ipH (5.4, 9.4 and 10.4), a decrease

in the APD-20 and APD-40 was observed while the overshoot

and resting potential remained unaltered; +Vmax was decreased

38

under these conditions. These changes suggest opposing effects

of decrease in pH (or increase in [H"^]) and increase in pH

(or decrease in [H'*']) on the transmembrane E.M.F. of the heart

cells. Since different phases of cardiac AP are due to diff­

erent ionic currents flowing across the membranes (Carmeliet

and Vereecke, 1979), x the effects of changes in [H ] could

be due to H dependent ionic currents and/or thfeir effects

on the other ionic channels and ionic pumps. Increase in

[H ] or acidosis has been shown to decrease the slow inward

2+ 2+ Ca currents by inhibiting Ca channels in heart (Vogel

2+ and Sperelakis, 1977). Since the Ca channel activity contri­butes to the APD-20 and APD-40 (C.-:tn)eliet and Vereecke, 1979)',

a decrease in these parameters in acidic medium shows that

2+ activation and mactivation parameters of Ca channels- were

slowed down by excessive H in the external solution. This

could be due to protonation of the corresponding functional

groups of the channel protein (Martel and Smith, 1977).

Possibly involving the carboxylic terminals at the inner and

outer 'gates' of the channel (Kostyuk, 1984). This increased

[H ] has been found to cause inhibition of cells. Since

an overlapping K outward current also contributes to the

APD-20 and APD-40 of cardiac APs (Carmeliet and Vereecke,

1979; Noble, 1984), it is likely that H"*" have also an effect

on these ionic channels.

A decrease in +Vmax at low pH of 6.4 is suggestive

of an inhibition of the fast Na" channels by excessive H"*".

It is, however, interesting to note that even at higher pH

of 8.4, 9.4 and 10.4 (Table 6) +Vmax was reduced drastically

39

indicating of an inhibition of the fast Na channels. When

viewed in the light of reports that H inhibited the membrane + + • + permeability of cations Na , K and Ca (Wada and Goto, 1975)

and altered the membrane ionic conductance (Harris, 1965,

Hutter and Warner, 1967; Sperelakis, 1969), the above conclu-

+ 2+ +

sion about the effects of altered pH on Na , Ca and K cha­

nnels in heart cells is justified by the direction of changes

in different parameters of AP following alterations of [H ].

The effects of H on the electrical activity of living heart

cells is complicated by its effects on various cellular pro­

teins including enzymes etc. Moreover, the cellular buffering

between extra-and intra-cellular pH would also contribute

to some uncertainty in correlating the observed effects with

+ +

the extracellular pH ([H 1 ) and intracellular pH ([H ].)

since buffering in the cell plays an important role in determin-

ing [H ]. and [H ] (Gilbert and Lowenberg, 1964; Woodbury,

1966; Izutsu, 1972; Thomas, 1984).

It is probably because of these reasons that the investi­

gators have reported a wide range of effects of pH on membrane /

ionic permeability, ionic conductances and AP configuration

as discussed above. The effects of altered pH observed in

the present study offer findings for a system devoid of extra­

cellular HCOZ as a buffer since HEPES, an unnatural agent

was used as the buffer for artificial solutions in the electro­

physiological experiments. Further experiments are required

to show the relevance of these findings in terms of [H"* ]

and [H J vis-a-vis HCO~ buffer system in the living cells

40

and their relationship to ionic channel activity. In addition,

it will also be useful to study the E.M.F. due to H in

artificial systems, consisting of diffusion half cells sepa­

rated by lipijd coated membranes. Studies on these lines are

in progress at the moment both on the heart cells and arti­

ficial membranes..

SUMMARY AND CONCLUSIONS

41

6.0 SUMMARY AND CONCLUSIONS

The objective of this study was to investigate the

transference characteristics of H and some other physiologi-

+ 2+ cally important ions, e.g. K and Ca and the effects of

H"*" (i.e. pH or [H"*"]) on the ionic channel activity in living

animal cell membrane. These objectives were achieved by meas­

uring E.M.F. with and without transference in diffusion half

cells separated by Whatman filter paper, an uncharged porous

partition, and in half-cells connected by KCl-Agar salt bridge

respectively. . The effects of [H ] on ionic channels was

studied by observing the effects of altered pH on the trans-V

membrane E.M.F. in mammalian heart cells using the glass ultra-

microelectrode technique.

Present observations on the artificial membrane system

showed that E.M.F. with transport changed with the concentrat­

ion ratios as well as the electrolyte species. E.M.F. without

transport was also found to change with the concentration

ratio and the electrolyte species but was different from the

E.M.F. with transport, the magnitude of difference depending

on the ionic species.

In the heart cells where AP configuration in an indicat-

+ 2+ + ion of the passive ion transport of Na , Ca and K , through

specific membrane bound ionic channels, alteration in extra­

cellular pH (or [H"*"] ) caused significant effects on AP confi­

guration. A decrease in [H"*"] (higherpH) reduced the APD-

20, • APD-40 and +Vmax. Increase in [H"*"] (lower pH) brought

42

about increase in APD-20, APD-40 and . +Vniax. These findings

suggest that altered [H ] strongly affects the ionic channel

activity.

Further work to study the mechanism of the above effects

of pH on cardiac membrane ionic channels as well as its relat­

ionship with the transference characteristics of H as studied

in artificial membrane system is in progress.

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43

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