pressor responses to ephedrine are mediated by a direct...

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Pressor Responses to Ephedrine are Mediated by a Direct

Mechanism in the Rat

John T. Liles, Paul A. Dabisch, Keith E. Hude, Leena Pradhan, Kurt J. Varner,

Johnny R. Porter, Alyssa R. Hicks, Conni Corrll, Syed R. Baber, and Philip J.

Kadowitz

Department of Pharmacology, Tulane University Health Sciences Center School of

Medicine, New Orleans, LA 70112 (JT Liles, PA Dabisch, KE Hude, L Pradhan, SR

Baber, PJ Kadowitz) ; Department of Pharmacology, Louisiana State University Health

Science Center, School of Medicine (KJ Varner, AR Hicks) and Department of

Physiology, Louisiana State University Health Science Center, School of Dentistry(JR

Porter, C Corrll)

JPET Fast Forward. Published on July 7, 2005 as DOI:10.1124/jpet.105.090035

Copyright 2005 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on July 7, 2005 as DOI: 10.1124/jpet.105.090035

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Running title page

a) Running title: Responses to Ephedrine are Directly Mediated

b) Corresponding author: Dr. Philip J. Kadowitz

1430 Tulane Avenue

Department of Pharmacology, SL83

New Orleans, LA 70112

Tel: (504) 988-2638

Fax: (504) 988-5283

Email: [email protected]

c) Number of text pages: 23

Number of tables: 4

Number of figures: 8

Number of references: 40

Words in Abstract: 191

Words in Introduction: 400

Words in discussion: 1,416

d) Abbreviations: AMPT, α-methyl-p-tyrosine; NET, norepinephrine reuptake

transporter

e) Recommended section: Cardiovascular


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Abstract

The mechanism of the pressor response to ephedrine is controversial. In the

present study iv injections of ephedrine increased systemic and pulmonary

arterial pressure, and ia injections decreased hindlimb blood flow in a dose-

related manner. Responses to ephedrine were inhibited by α-receptor blocking

agents and were not attenuated by blockade of the norepinephrine reuptake

transporter (NET) or by catecholamine depletion using reserpine or a

combination of reserpine and α-methyl-p-tyrosine, whereas responses to

tyramine and amphetamine were inhibited by these treatments. The magnitude

of the pressor response to ephedrine was similar in anesthetized and conscious

rats. Tachyphylaxis developed to pressor responses to ephedrine and

amphetamine with sequential injections, however, ephedrine tachyphylaxis

differed in that subsequent responses to α-receptor agonists were attenuated.

These results suggest that the systemic and pulmonary pressor and hindlimb

vasoconstrictor responses to ephedrine are mediated by direct action on α-

adrenergic receptors and that the release of norepinephrine from adrenergic

terminals plays no significant role. These results provide support for the

hypothesis that responses to ephedrine are directly mediated in the intact rat

whereas responses to amphetamine are mediated in a large part by the release

of norepinephrine from adrenergic terminals.


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INTRODUCTION

Ephedrine is chemically and structurally related to amphetamine and

methamphetamine and is a substrate for the adrenergic nerve terminal membrane

monoamine transporter (Kraemer and Maurer, 2002;Abdallah et al., 1967).

Ephedrine has actions on α- and β- adrenergic receptors and displaces

norepinephrine from adrenergic terminals (Abdallah et al., 1967: Rothman et al.,

2001; Mcmahon and Cunningham, 2003). The indirect actions of ephedrine are

reported to be the mechanism by which ephedrine increases arterial pressure,

cardiac output, and peripheral resistance, however direct effects have also been

reported (Vansal and Feller, 1999; Waldeck and Widmark, 1985; Kawasuji et al.,

1996). Ephedrine has been linked to serious cardiovascular toxicity including

hypertension, vasospasm, angina, coronary artery disease, arrhythmia, myocardial

infarction, stroke, and death (Mcmahon and Cunningham, 2003; Jones, 2000; Zahn

et al., 1998; Foxford et al., 2000; Haller and Benowitz, 2000; Josefson, 1996;

Cockings and Brown, 1997; Gedevanishvili et al., 2004; Adamson et al., 2004 ).

Cardiovascular events result from both acute and chronic ephedrine use and occur

in individuals with no history of cardiovascular disease (Jones, 2000; Onuigbo and

Alikhan, 1998; Cockings and Brown, 1997; Grzesk et al., 2004). Ephedrine, like other

drugs of abuse, has stimulant and hallucinogenic actions (Mcmahon and

Cunningham, 2003). In addition, experienced drug users report that ephedrine-


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induced euphoria is qualitatively similar to that induced by other amphetamine-like

sympathomimetics (Martin et al., 1971). Despite a recent FDA ban on ephedrine-

containing dietary supplements, ephedrine preparations are still available through the

mail and on the internet and ephedrine use is not likely to change because of the high

demand for these preparations (FDA and HHS, 2004; Ashar et al., 2003; Kim and

LeBourgeois, 2004; Kim and LeBourgeois, 2004).

The release of catecholamines and subsequent activation of adrenergic

receptors is believed to be the primary mechanism responsible for the cardiovascular

response to ephedrine and isolated tissue studies support the concept of an indirect

mechanism of action (Burn and Rand, 1958; Fleckenstein and Burn, 1953;DeMoraes

et al., 1968;Cairolli et al., 1962). Other studies in isolated tissues have shown that

ephedrine acts by direct stimulation of adrenergic receptors (Tye et al., 1967;Bao et

al., 1990;Waldeck and Widmark, 1985;Kawasuji et al., 1996). It has recently been

reported that despite evidence that ephedrine has direct effects in isolated tissues,

the pressor response is completely indirectly mediated in vivo in the rat (Kobayashi et

al., 2003).

The effects of ephedrine-like agents such as amphetamine

methamphetamine), and methylenedioxymethamphetamine (MDMA) have been

shown to be reduced by pretreatment with reserpine as well as with a combination of

reserpine and α-methyl-p-tyrosine (AMPT) (Cadoni et al., 1995; Urabe et al., 1997,

Spector, 1965; Sabol and Seiden, 1998; Yuan et al., 2002; Uehara et al., 2003).

Few studies have examined the mechanisms involved in the cardiovascular


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responses to acute ephedrine administration in the intact rat. Since there is little

information available on mechanisms in vivo, the present study was undertaken

to investigate the mechanisms by which this agent increases arterial pressure in

the rat. Catecholamines were depleted by administering reserpine and by

administering reserpine and α-methyl-p-tyrosine together in order to determine if

the release of catecholamines mediates the responses to ephedrine and

amphetamine. The effect of acute ephedrine administration on arterial pressure,

hindlimb blood flow, and on pulmonary arterial pressure as well as the adrenergic

receptors involved in these responses were examined in this study.


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MATERIALS AND METHODS

General Methods. Experiments were performed on male Sprague-Dawley rats

weighing 300-400 grams anesthetized with thiobutabarbital sodium (Inactin®)

(100 mg/kg i.p.) with supplemental doses given as needed to maintain a uniform

level of anesthesia. The trachea was cannulated to maintain airway patency and

the animals breathed room air spontaneously. An external jugular vein was

catheterized for the intravenous (i.v.) administration of drugs. The common

carotid artery was catheterized for the measurement of systemic arterial

pressure. Systemic arterial pressure was measured with a Statham transducer

and recorded on a Grass Model 7D polygraph and the data were digitized by a

Biopac MP100 data acquisition system.

Hindlimb vascular response to ephedrine. For hindlimb blood flow

measurements a Transonic flow probe (Transonic Systems, Inc.) was placed

around the right iliac artery just below the aortic bifurcation and hindlimb blood

flow was measured with a Transonic Systems T-106 small animal flowmeter. A

catheter in the left iliac artery was advanced to the aortic bifurcation for the i.a.

administration of drugs into the right hindlimb circulation. Systemic arterial

pressure and right iliac flow data were recorded on a PC using a Biopac MP100

acquisition system. Agonists were injected directly into the right hindlimb


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circulation in small volumes (10 – 30 µl) so that changes in right iliac blood flow

could be measured with minimal changes in systemic arterial pressure.

Pulmonary response to ephedrine. For measurement of pulmonary arterial

pressure, a 3F radiopaque catheter was passed from the left external jugular vein

into the main pulmonary artery under fluoroscopic guidance. Pressures were

recorded on a Grass model 7 polygraph, and mean pressures were derived by

electronic integration. Catheter positions were verified at postmortem

examination and these methods have been described previously (Baber et al.,

2003)

Hemodynamic data are expressed as mean ± SE and were analyzed

using ANOVA, paired and unpaired t-tests. The duration of the response is

defined as the period of time from injection of the agonist to the time at which the

measured parameter (either mean arterial pressure, or hindlimb blood flow)

returned to the pre-injection value. A P value of < 0.05 was used as the criterion

for statistical significance.

Responses to ephedrine in the conscious rat. Mean arterial pressure (MAP)

and heart rate were recorded in conscious, unrestrained rats in their home cages

using a radio telemetry system (Dataquest A.R.T. 2.2; Data Sciences

International, St. Paul, MN.). Briefly, under ketamine/xylazine (90 mg/kg and 10

mg/kg i.p., respectively) anesthesia, the arterial pressure cannula of a battery


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powered radio telemetry probe (TL11M2-C50-PXT, Data Sciences International,

St. Paul, MN) was inserted and secured into the descending abdominal aorta

rostral to the femoral bifurcation. The probe was then placed in the abdominal

cavity and secured to the abdominal musculature. In all rats, a polyurethane

cannula (Micro-renathane, 0.33-inch o.d. x 0.014-inch i.d.; Braintree Scientific,

Braintree, MA) was inserted into the femoral vein and the free end tunneled

subcutaneously to the nape of the neck and exteriorized. After surgery, fluids

and penicillin (60,000 units, i.m.) were administered. Buprenorphine (2.5 mg/kg,

i.p., b.i.d.) was administered for 2 days. The rats were allowed to recover from

the surgical procedures for 7–10 days before beginning any experimental

protocol. In these experiments, the daily weight was measured and baseline

MAP and heart rate were recorded for a minimum of 20 min prior to the

administration of any drugs. Ephedrine (10 mg/kg, 0.10 ml) or saline (0.10 ml)

was administered and followed by a 0.1 ml saline flush. Cardiovascular

parameters were allowed to return to baseline before administering the next dose

The output from the telemetry probes was recorded (250 Hz) using

receivers placed under the home cages. The data was sent to a consolidation

matrix before being stored on a personal computer. Data acquisition was

controlled using Data Sciences Dataquest acquisition software. The data were

averaged into 2-s bins and displayed. The magnitude of the peak changes in

MAP and heart rate elicited by the drugs were calculated off-line as the


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difference between the baselines and peak drug response using the Dataquest

analysis program. Response duration was calculated off-line using the

Dataquest analysis program. The duration of the systemic arterial pressure

response was calculated as the interval between drug administration and the

point at which the systemic arterial pressure returned to within 7 mmHg of

baseline value.

Experimental Protocols. In the first set of experiments, a dose-response curve

for ephedrine and amphetamine was obtained and tachyphylaxis was observed

when ephedrine or amphetamine was administered as sequential injections in

the same animal. Therefore, midrange doses of ephedrine (10 mg/kg i.v.) and

amphetamine (3 mg/kg i.v.) were used for further evaluation of acute responses.

The l-ephedrine, d-amphetamine, and dl-norepinephrine stereoisomers were

used in the present study.

In a second set of experiments, animals were treated with cocaine (5

mg/kg i.v.) and blockade of reuptake following cocaine administration was

assessed by analyzing responses to tyramine (100 ug/kg i.v.) and norepinephrine

(3 µg/kg i.v.).

In a third set of experiments, the effect of treatment with reserpine (2.5

mg/kg i.p.) or with reserpine and α-methyl-p-tyrosine (AMPT 200 mg/kg i.p.) was

investigated. Reserpine was administered 18 hours prior to the experiment while

AMPT was given at least 2 hours before the experiment. The extent of


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catecholamine depletion was functionally assessed by evaluating responses to

norepinephrine and to tyramine and tissue levels were measured. The

administration reserpine caused a decrease in baseline pressure from 111± 2 to

97 ± 2 while the combination of reserpine and AMPT resulted in a baseline

pressure of 106 ± 3.0. Responses to ephedrine, amphetamine, norepinephrine

and angiotensin II were not changed during experiments where an infusion of

phenylephrine (1 µg/kg/min) was used to restore baseline systemic arterial

pressure to control value.

In a fourth set of experiments animals were pretreated with propranolol

(0.5 mg/kg i.v.) or with phentolamine (0.5 mg/kg i.v.) prior to the evaluation of

ephedrine responses in the systemic, hindlimb, or pulmonary vascular bed.

Blockade of alpha- and beta- receptors was assessed by evaluating responses to

phenylephrine and isoproterenol. Phentolamine administration decreased

baseline arterial pressure to 86 ± 4 mmHg, while propranolol had no significant

effects on baseline systemic arterial pressure.

High-performance liquid chromatography (HPLC) determination of tissue

catecholamines. Following the completion of experiments measuring the

systemic or hindlimb responses to ephedrine the brain, heart, and adrenal glands

were removed and homogenized in a buffer of 0.1M citrate, 10% ethanol, and

250mg/L sodium octyl sulfate (SOS) at pH 4. Tissues were stored in the buffer at

a temperature of –80°C until analysis. An internal standard (IS) of 3,4-


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dihydroxybenzylamine hydrobromide (DHBA) was added such that its final

concentration was 1 ng/100 ul of the HPLC injectate. The homogenate was

centrifuged in a RC2B Sorvall centrifuge at 12, 000 X G and the supernatant was

frozen at -80 degrees C until assayed. Recoveries ranged from 75-90% in these

and other studies. Our chromatographic system consisted of the following

hardware and software. Dual Rainin Rabbit HP pumps equipped with a self-

washing piston pump heads (capable of maximum flows of 10 ml/min) provided a

flow rate of 1.5 ml/min. Injection of samples was accomplished automatically

using an Alcott model 728 autosampler. This allowed us to run as many as 40

samples overnight. The HPLC column consisted of a Rainin microsorb (5uM)

C18 column (25cmX4.6mm). A 1.5 cmX4.6mm guard column packed with

microsorb C18 preceded the analytical column. Chromatographically separated

monoamines were assayed by electrochemical detection utilizing an ESA model

5100 Coulchem multielectrode array. This electrode array consisted of a model

5020 guard cell and a model 5010 dual analytical electrode cell. The guard cell

voltage was + 0.4 Volts. The two analytical cells were set at -0.04 V (Detector 1)

and +0.32 V (Detector 2). Norepinephrine (NE), epinephrine (Epi), dopamine

(Dop), 5-hydroxytryptamine (5HT); serotonin, and 5-hydroxyindoleacetic acid

(5HIAA) were determined in unknown and standard samples by comparison to

retention times and integrated area of the peak. Co-chromatography of spiked

standards of each compound were initially run with brain tissue in order to


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determine that the peaks identified as monoamines were indeed authentic NE,

Epi, Dop, 5HT, and 5HIAA.


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Results

Responses to ephedrine and amphetamine

Responses to ephedrine and amphetamine were compared and injection of

ephedrine in doses of 1- 30 mg/kg i.v. produced dose-related increases in

systemic arterial pressure when each injection was given in a separate animal

(Fig1A, Left). Injections of ephedrine in doses of 1 – 30 mg/kg i.v. also caused

dose related increases in systemic arterial pressure when injections were given

at 30 minute intervals in the same animal (Fig1A). I.V. Injections of

amphetamine in doses of 1 – 30 mg/kg increased systemic arterial pressure

(Fig1B). However, pressor responses to amphetamine in doses of 1 – 30 mg/kg

i.v. were only dose-related when single injections were administered to separate

animals (Fig 1B, Left). Figures 1A-B also illustrate the effect of repeated

injections of ephedrine (3 mg/kg i.v.) and amphetamine (3 mg/kg i.v.). The

pressor response to ephedrine was reduced after the fourth injection and further

injections resulted in a measurable but diminished response, indicating the

development of tachyphylaxis. The pressor response to amphetamine was

reduced after the second injection and responses to further injections were

greatly attenuated.


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Effects of ephedrine and amphetamine on responses to phenylephrine and

norepinephrine

Tachyphylaxis to the pressor response to ephedrine (3 mg/kg i.v.) or

amphetamine (3 mg/kg i.v.) occured following sequential injections of each

agent. The effect of ephedrine or amphetamine induced tachyphylaxis on

responses to norepinephrine (0.3 – 3 µg/kg i.v.) and phenylephrine (1-10 µg/kg

i.v.) were investigated (Fig 2). Injections of norepinephrine and phenylephrine

were administered following five sequential injections of ephedrine or

amphetamine to induce tachyphylaxis. The increase in arterial pressure in

response to norepinephrine was not changed by ephedrine tachyphylaxis,

however the area under-the-curve of the norepinephrine response at lower doses

(0.3 and 1 ug/kg i.v.) was reduced (Fig 2A). However, amphetamine

tachyphylaxis enhanced the increase in arterial pressure and the area-under the

curve of the response to norepinephrine (Fig 2A). Following the development of

ephedrine tachyphylaxis the increase in arterial pressure in response to

phenylephrine was diminished, as was the area under-the-curve of the response

(Fig 2B). Following administration of amphetamine the increase in mean arterial

blood pressure in response to phenylephrine was not changed, however the area

under-the curve of the responses was increased (Fig 2 B). Ephedrine or

amphetamine induced tachyphylaxis had no effect on the pressor response to

angiotensin II or on the vasodepressor response to bradykinin (Table 1).


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Effects of cocaine, reserpine, and AMPT

The effect of cocaine, reserpine and reserpine plus AMPT on pressor responses

to ephedrine and amphetamine were compared and these data are summarized

in figures 3 - 5. Following administration of cocaine (5 mg/kg i.v.) the increase in

systemic arterial pressure in response to ephedrine (Fig 3A, Left) was not

changed whereas responses to amphetamine (Fig 3A, Right) and tyramine (Fig

3B, Left) were decreased significantly. The pressor response to norepinephrine

(Fig 3B, Right) was enhanced by cocaine whereas the pressor responses to

angiotensin II (Fig 3C, Left) and depressor responses to acetylcholine (Fig 3C,

Right) were not altered by cocaine administration. The increase in systemic

arterial pressure in response to ephedrine and area under-the-curve of this

response were not altered by treatment with reserpine (2.5 mg/kg i.v.) or

reserpine plus AMPT (200 mg/kg i.p.) (Fig 4A). However treatment with reserpine

or reserpine plus AMPT significantly decreased the pressor response and the

area under-the curve of the response to both amphetamine (Fig 4B) and

tyramine (Fig 4C). Reserpine or reserpine plus AMPT significantly increased the

pressor response and the area under-the curve of this response to

norepinephrine (Fig 4D). Pressor responses to angiotensin II and phenylephrine

were not changed by treatment with reserpine or by reserpine plus AMPT (Table

2). Table 3 shows that there were significant decreases in catecholamine levels

in the brain, heart, and adrenal glands of rats treated with reserpine and AMPT.


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Hindlimb vascular response

In the hindlimb vascular bed of the rat direct i.a. injections of ephedrine (100-

1000 µg) and amphetamine (10- 30 µg) caused dose-related decreases in

hindlimb blood flow and with smaller doses of the amines tachyphylaxis was not

observed. The decreases in hindlimb blood flow in response to ephedrine were

not altered by reserpine or by treatment with reserpine and AMPT (Fig 5A). In

contrast, the decreases in hindlimb blood flow in response to i.a. injections of

amphetamine (Fig 5B) and tyramine (Fig 5C) were significantly reduced by

pretreatment with reserpine and with reserpine and AMPT. Decreases in

hindlimb blood flow in response to i.a. injections of norepinephrine were

enhanced (Fig 5D).

Responses to ephedrine in the conscious and anesthetized rat

Responses to ephedrine were compared in anesthetized and conscious, freely

moving rats and these data are presented in Figure 6. The magnitude of the

increase in mean arterial blood pressure following the systemic injection of

ephedrine (10 mg/kg) in the anesthetized rat was similar to that observed in the

conscious rat (Fig 6A). However, the duration of the pressor response to

ephedrine was significantly shorter in the conscious animal (Fig 6B). The

administration of ephedrine increased heart rate in the anesthetized animal, but

resulted in a significant decrease in heart rate in the conscious rat (Fig 6C).


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Effect of phentolamine on systemic and hindlimb responses

The effect of the phentolamine on the pressor response to ephedrine is shown in

figure 7A. Following administration of phentolamine (0.5 mg/kg i.v.) the

magnitude of the pressor response to ephedrine was decreased significantly (Fig

7A, Right). In contrast, the increase in heart rate in response to ephedrine was

not decreased by phentolamine (Fig. 7A, Middle). Similar results were obtained

with the alpha-1 selective receptor antagonist prazosin (Table 4). Phentolamine

attenuated the pressor response to phenylephrine (Table 4). The role of α-

adrenergic receptors in mediating the hindlimb vasoconstrictor response to

ephedrine was investigated (Fig 7A, Right). When ephedrine (100-1000 µg i.a.)

was administered following phentolamine (0.5 mg/kg i.v.) the decrease in

hindlimb blood flow was abolished and an increase in hindlimb blood flow was

observed(Fig 7A, Right).

Effect of propranolol on systemic hindlimb responses to ephedrine

The effect of the propranolol on the response to ephedrine is shown in figure 7B.

Propranolol (0.5 mg/kg i.v.) did not affect the magnitude of the pressor response

(Fig. 7B, Left), however, the heart rate response was decreased. The duration of

the pressor response to ephedrine was also significantly increased by

propranolol (Fig. 7B, Middle). Propranolol attenuated the effects of the β-

adrenergic receptor agonist isoproterenol (Table 4). The role of β-adrenergic


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receptors was evaluated and the decrease in hindlimb blood flow in response to

ephedrine (100-1000 µg i.a.) was not changed after propranolol (0.5 mg/kg i.v.)

(Fig. 7B, Right). Subsequent administration of phentolamine inhibited the

decrease in hindlimb blood flow in response to ephedrine.

Pulmonary vascular responses to ephedrine

The effect of ephedrine (1- 10 mg/kg i.v.) on pulmonary arterial pressure is

shown in figure 8. Ephedrine produced dose-related increases in pulmonary

arterial pressure. The magnitude of the increases in pulmonary pressure were

significantly decreased following treatment with phentolamine (0.5 mg/kg i.v.)

(Fig 8, Left) and were not changed by propranolol (0.5 mg/kg i.v.) (Fig 8, Middle)

However, the duration of the pulmonary pressor response was enhanced by

propranolol (Fig 8, Right). The effect of ephedrine following catecholamine

depletion was investigated. The increases in pulmonary pressure in response to

ephedrine after the administration of reserpine plus AMPT were not changed (Fig

8B, Left). The pulmonary pressor response to tyramine was inhibited (Fig 8B,

Middle) and responses to norepinephrine were enhanced (Fig 8B, Right)

following catecholamine depletion with reserpine and AMPT.


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Discussion

The present results show that systemic and pulmonary pressor responses

and hindlimb vasoconstrictor responses to ephedrine are mediated by direct

activation of α-receptors and that release of norepinephrine from adrenergic

terminals plays no significant role in the rat. Systemic pressor and hindlimb

vasoconstrictor responses to ephedrine were attenuated by phentolamine and

were increased in duration by propranolol suggesting that responses are

mediated by α-receptors and modulated by β-receptors. Responses to

ephedrine were not attenuated by cocaine in a dose that blocked the response to

tyramine. The hypothesis that responses to ephedrine are not mediated by an

indirect mechanism is supported by experiments with reserpine and AMPT

showing that ephedrine induced systemic and pulmonary pressor responses and

hindlimb vasoconstrictor responses were not reduced by catecholamine

depletion. In contrast, the increase in systemic arterial pressure and the

decrease in hindlimb blood flow in response to amphetamine were abolished

following catecholamine depletion with reserpine or with a combination of

reserpine and AMPT.

The mechanism of action of ephedrine action is controversial. Isolated

tissue studies suggest that ephedrine acts through an indirect mechanism.

Ephedrine induced contraction of the nicitating membrane, constriction of the

pupil, forelimb, and coronary vasoconstriction are mediated by an indirect


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mechanism (Burn and Rand, 1958; Fleckenstein and Burn, 1953; DeMoraes et

al., 1968; Cairolli et al., 1962). However, direct stimulation of adenergic receptors

in the guinea pig trachea, portal vein, and reserpinized rat atrium has been

reported (Tye et al., 1967; Bao et al., 1990; Waldeck and Widmark, 1985;

Kawasuji et al., 1996). Ephedrine has been shown to bind to β1, β2, and β3

receptors expressed on chinese hamster ovary cells (Vansal and Feller, 1999).

It has been reported that despite evidence that ephedrine has direct effects in

isolated tissues, the pressor response was shown to be indirectly mediated in

vivo in the rat (Kobayashi et al., 2003). In contrast, the present results

demonstrate that responses to ephedrine are directly mediated whereas

responses to amphetamine are dependent on an indirect mechanism.

Reserpine depletes catecholamines from central and peripheral

adrenergic terminals by interfering with vesicular transmitter storage (Sabol and

Seiden, 1998). Therefore, the actions of indirect-acting agents are inhibited by

reserpine while responses to direct-acting amines are not reduced and may be

enhanced (Sabol and Seiden, 1998). The literature suggests that there are two

distinct norepinephrine pools within the adrenergic terminal, a cytoplasmic and a

vesicular pool (Forin et al., 1995; Simpson, 1980). An exchange diffusion model

suggests that only cytoplasmic catecholamines are released by amphetamine-

like drugs (Fischer and Cho, 1978). Since reserpine is selective for vesicular

stores, many studies have employed co-administration of α-methyl-p-tyrosine


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(AMPT), an inhibitor of tyrosine hydroxylase, with reserpine to deplete both

stores of catecholamines (Abrahams et al., 1996; Yuan et al., 2002). In the

present experiments responses to phenylephrine and angiotensin II were

unchanged by treatment with reserpine or reserpine and AMPT (Table 4). The

present results are the first to show that ephedrine has significant direct pressor

and vasoconstrictor effects in vivo following treatment with reserpine and AMPT.

The systemic, pulmonary, and hindlimb vascular responses to ephedrine were

not reduced by reserpine and AMPT whereas responses to amphetamine and

tyramine were abolished. The present data are not in agreement with the

concept that responses to ephedrine are indirectly mediated (Kobayashi et al.,

2003). Pretreatment with reserpine and with reserpine and AMPT at doses that

depleted catecholamines as measured by High Performance Liquid

Chromatography (HPLC) and that inhibited responses to tyramine and

amphetamine did not reduce the magnitude or the area under-the-curve of the

response to ephedrine. The observation that responses to amphetamine are

reduced by reserpine (Sabol and Seiden, 1998; Yuan et al., 2002; Florin et al.,

1995) and reserpine and AMPT (Abrahams et al., 1996; Lin et al., 1992) is in

agreement with the observation that responses to amphetamine are reduced by

catecholamine depletion (Sabol and Seiden, 1998; Abrahams et al., 1996). In

the present study both reserpine and reserpine and α-methyl-p-tyrosine inhibited

responses to amphetamine. Although norepinephrine is believed to be stored in

distinct pools, there is evidence that these pools are in flux (Simpson, 1980).


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Additionally, amphetamine can alter the integrity of the vesicular pool, allowing

transmitter leakage into the cytoplasmic pool where it is available for release

(Sulzer and Rayport, 1990). The present results show that responses to

ephedrine and amphetamine are mediated by different mechanisms.

The present data are not in agreement with a recent report showing that the

pressor response to ephedrine in the rat is entirely due to the release of

norepinephrine from adrenergic terminals (Kobayashi et al., 2003). The reason

for the difference in results is uncertain but could be due to the doses of

ephedrine administered as well as to the use of different catecholamine depletion

regimens. It is possible that 6-hydroxydopamine causes non-specific depression

of vascular responses (Purdy et al., 1981). The i.v. doses of ephedrine used in

the present study are within the range of the doses used in ephedrine-dependent

individuals and in animal studies (Miller and Waite, 2003; Miller et al., 1998;

Young and Glennon, 1998). However, it is possible that ephedrine may act

through an indirect mechanism in some organ systems and in the present study

catecholamine depletion reduced the heart rate increase in response to

ephedrine.

Ephedrine increases systemic and pulmonary arterial pressure and

decreases hindlimb blood flow in the anesthetized rat by directly stimulating α-

adrenergic receptors while β-receptors modulate the responses. Phentolamine

attenuated responses to phenylephrine and significantly reduced the increase in


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arterial pressure but not the increase in heart rate in response to ephedrine.

Phentolamine also reduced the decrease in hindlimb blood flow and the increase

pulmonary arterial pressure in response to ephedrine. These results suggest

that the increase in systemic and pulmonary arterial pressure and the decrease

in hindlimb blood flow in response to ephedrine are predominately due to direct

activation of α-adrenergic receptors. Propranolol, in a dose that decreased

responses to isoproterenol, attenuated the increase in heart rate but not the

pressor response to ephedrine, indicating that the increase in heart rate is due to

the stimulation of ß-receptors and is in agreement with previous studies

(Schindler et al., 1992). In addition, propranolol increased the duration of the

systemic and pulmonary pressor responses to ephedrine. The pressor response

to ephedrine involves concurrent α and β-2 receptor activation, with α-receptor

induced vasoconstriction opposing the β-2 receptor mediated vasodilation

(Lambrecht et al., 2002). Thus, β-receptor inhibition allows unopposed

stimulation of α-receptors, which could result in a longer duration of the response

to ephedrine. Moreover, when α-receptors were blocked, a β-receptor mediated

vasodilator mechanism was unmasked in the hindlimb vascular bed, providing

further evidence in support of the concept that the response to ephedrine results

from activation of both α- and β-adrenergic receptors.

The present study demonstrates that the response to ephedrine in the rat

systemic vascular bed exhibits tachyphylaxis when multiple doses are given at


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short intervals (20 minutes) in the same animal. However, tachyphylaxis to

ephedrine was different than tachyphylaxis induced by repeated doses of

amphetamine. The present observation that the pressor response to ephedrine

was reduced but not abolished following repeated administration is consistent

with studies that reported different patterns of tachyphylaxis with ephedrine and

amphetamine (Takasaki et al., 1971; Takasaki et al., 1972; DeMoraes and

Carvalho, 1967). In the present study, pressor responses to phenylephrine and

norepinephrine were reduced following the development of ephedrine

tachyphylaxis but not reduced by amphetamine tachyphylaxis. These findings

are in agreement with isolated tissue studies showing that α-receptors are

inhibited by repeated administration of ephedrine (Morishita and Furukawa, 1975;

Patil et al., 1966; Furukawa and Morishita 1975). The present data provide

support for the hypothesis that tachyphylaxis to the pressor response to

ephedrine may involve α-receptor inhibition while tachyphylaxis to amphetamine

may involve catecholamine depletion, in that norepinephrine and phenylephrine

pressor responses are preserved during amphetamine tachyphylaxis.

Responses to ephedrine were compared in anesthetized and in

conscious, freely moving rats and the magnitude of the pressor response in the

anesthetized and conscious rat was similar. However, The duration of the

pressor response in the conscious rat was significantly shorter and ephedrine

induced a decrease in heart rate in contrast to the increase in heart rate


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observed in the anesthetized rat. These differences are most likely due to

anesthesia induced alterations in baroreflex function (Schwartz et al., 1988).

In summary, the present results demonstrate that systemic and pulmonary

pressor and hindlimb vasoconstrictor responses to ephedrine are mediated by

direct α-adrenergic receptor stimulation while β-receptors modulate the

responses. The increase in systemic and pulmonary arterial pressure and the

decrease in hindlimb blood flow in response to ephedrine were not reduced by

catecholamines depletion. In contrast, the increase in systemic arterial pressure

and the decrease in hindlimb blood flow in response to amphetamine were

abolished following treatment with reserpine and with a combination of reserpine

and AMPT. These results provide support for the hypothesis that ephedrine has

significant direct receptor mediated effects in the intact rat whereas responses to

amphetamine are indirectly mediated, suggesting that responses to structurally

similar phenylethylamines may be mediated by different mechanisms. These

data also demonstrate that responses to phenylephrine and norepinephrine are

decreased by ephedrine tachyphylaxis while these responses were unchanged

by amphetamine tachyphylaxis. Theses data suggest that pressor response to

ephedrine is mediated by direct α-adrenergic receptor stimulation. The present

data show that responses to ephedrine and amphetamine are mediated by

different mechanisms and that cardiovascular responses to ephedrine-like drugs

are complex.


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Acknowledgements

The authors thank Ms. Janice Ignarro for editorial assistance.


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Footnotes

A) This work was supported by National Heart, Lung, and Blood Institute Grants

HL62000 and HL77421 from the National Institutes of Health.

B) Reprint requests: Dr. Philip J Kadowitz 1430 Tulane Ave. SL83 New Orleans, LA 70117 [email protected]


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Legends for Figures

Fig 1. A: (left) Effect of iv injections of ephedrine on mean systemic

arterial pressure when doses are administered non-sequentially as a single

injection in separate animals and when all injections were administered

sequentially in the same animal 20-30 minutes apart. (right) Effect of repeated

injections of ephedrine on mean systemic arterial pressure. B: Effect of

intravenous injections of amphetamine on mean systemic arterial pressure when

doses are administered non-sequentially as a single injection in separate animals

and when all injections were administered sequentially in the same animal 20-30

minutes apart, and the effect of repeated injections of amphetamine on mean

systemic arterial pressure. n indicates number of experiments, and the asterisk

indicates that the response is significantly different than control.

Fig 2. A: Effect of ephedrine and amphetamine induced tachyphylaxis on

the increase in mean systemic arterial pressure and the area under-the-curve of

the response to norepinephrine. B: Effect of ephedrine and amphetamine

tachyphylaxis on the increase in mean systemic arterial pressure and the area

under-the-curve of the response to phenylephrine. n indicates number of

experiments, and the asterisk indicates that the response is significantly different

than control.

Fig 3. A: Effect of cocaine (5 mg/kg i.v.) on the increase in mean arterial

pressure in response to IV injections of ephedrine and amphetamine. B: Effect

of cocaine (5 mg/kg i.v.) on the increase in mean arterial pressure in response to

intravenous injections of tyramine and norepinephrine. C: Effect of cocaine (5

mg/kg i.v.) on the increase in mean arterial pressure in response to iv injections

of angiotensin II and on the decrease in mean arterial pressure in response to iv


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injections of acetylcholine. n indicates number of experiments, and the asterisk

indicates that the response is significantly different than control.

Fig 4. A: Effect of reserpine (2.5 mg/kg i.v.) and reserpine plus AMPT

(200 mg/kg i.v.) on the increase in mean systemic arterial pressure and the area

under-the-curve of the response to iv injections of ephedrine; B: the increase in

mean systemic arterial pressure and the area under-the-curve of the response to

iv injections of amphetamine; C: the increase in mean systemic arterial pressure

and the area under-the-curve of the response to iv injections of tyramine; and D:

the increase in mean systemic arterial pressure and the area under-the-curve of

the response to iv injections of norepinephrine. n indicates number of


than control.

Fig 5. A: Effect of reserpine (2.5 mg/kg i.p.) and reserpine plus AMPT

(200 mg/kg i.p.) on the decrease in hindlimb blood flow and the area under-the-

curve the response to iv injections of ephedrine; B: the decrease in hindlimb

blood flow and the area under-the-curve of the response to iv injections of

amphetamine; C: the decrease in hindlimb blood flow and the area under-the-

curve of the response to iv injections of tyramine; and D: the decrease in

hindlimb blood flow and the area under-the-curve of the response to iv injections

of norepinephrine. n indicates number of experiments, and the asterisk indicates

that the response is significantly different than control.

Fig 6. A: Change in mean systemic arterial pressure in response to IV

injection of ephedrine in the conscious rat. B: Duration of the increase in mean

systemic arterial pressure in response to IV injection of ephedrine in the

conscious rat. C: Change in heart rate in response to IV injection of ephedrine in


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the conscious rat. n indicates number of experiments, and the asterisk indicates

that the response is significantly different than control.

Fig 7. A: Effect of phentolamine (0.5 mg/kg i.v.) on the increase in mean

systemic arterial pressure (left); the increase in heart rate (middle); and the

decrease in hindlimb blood flow (right) in response to iv injections of ephedrine.

B: Effect of propranolol (0.5 mg/kg i.v.) on the increase in mean systemic arterial

pressure (left); the increase in heart rate (middle); and the decrease in hindlimb

blood flow (right) in response to iv injections of ephedrine. n indicates number of


than control.

Fig 8. A: Effect of phentolamine (0.5 mg/kg i.v.) on the increase in mean

pulmonary arterial pressure in response to iv injections of ephedrine (left); and

the effect of propranolol (0.5 mg/kg i.v.) on the increase in mean pulmonary

arterial pressure (middle) and the duration of the response (right) in response to

IV injections of ephedrine. B: Effect of reserpine (2.5 mg/kg i.v.) and AMPT (200

mg/kg i.v.) on the increase in pulmonary arterial pressure response to iv

injections of ephedrine, tyramine, and norepinephrine. n indicates number of


than control.


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Table 1 - The effect of ephedrine or amphetamine induced tachyphylaxis on

responses to angiotensin II and bradykinin

Effect of ephedrine or amphetamine tachyphylaxis on the increase in

mean systemic arterial pressure in response to angiotensin II and on the

decrease in mean systemic arterial pressure in response to bradykinin. The

responses to angiotensin II and bradykinin were not decreased following

ephedrine or amphetamine tachyphylaxis. n indicates number of experiments.

n = 8 *= p < 0.05

DRUG DOSE (ug/kg i.v.)

Treatment Change in Arterial Pressure (mmHg)

Control 30 ± 3

Ephedrine 28 ± 4

0.1

Amphetamine 29 ± 5

Control 47 ± 4

Ephedrine 48 ± 3

Angiotensin II

0.3

Amphetamine 52 ± 5

Control -20 ± 4

Ephedrine -22 ± 3

Bradykinin 10

Amphetamine -18 ± 4


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Table 2- The effect of reserpine or reserpine plus AMPT on the pressor

responses to phenylephrine and angiotensin II

Effect of reserpine (2.5 mg/kg i.v.) and reserpine plus AMPT (200 mg/kg

i.v.) on the increase in mean systemic arterial pressure in response to iv

injections of angiotensin II and phenylephrine. The pressor responses to

angiotensin II and bradykinin were not decreased following treatment with

reserpine or with reserpine and AMPT. n indicates number of experiments.

n = 6 –10 *= p < 0.05

DRUG DOSE (µg/kg i.v.)

Treatment Change in Arterial Pressure (mm/Hg)

Control 16 ± 2

Reserpine 14 ± 3

1

Reserpine + AMPT 13 ± 2

Control 35 ± 4

Reserpine 39 ± 3

Phenylephrine

3


Control 30 ± 3

Reserpine 28 ± 4

0.1


Control 48 ± 3

Reserpine 45 ± 7

Angiotensin II

0.3



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Table 3 -The effect of reserpine plus AMPT on tissue catecholamine levels

in the rat brain, heart, and adrenal gland

Effect of reserpine (2.5 mg/kg i.v.) plus AMPT (200 mg/kg i.v.) on tissue

catecholamine levels in the rat brain, heart, and adrenal gland. There was a

significant decrease in norepinephrine, epinephrine, and dopamine levels in all

tissues. n indicates number of experiments, and the asterisk indicates that the

response is significantly different than control.

Levels expressed as ng/mg wet tissue * p < 0. 05

TISSUE TREATMENT NE EPI 5-HIAA DA 5-HT

Control 0.090 ± 0.005

0.0043 ± 0.0006

0.025 ± 0.001

0.064 ± 0.004

0.026 ± 0.002 BRAIN

Reserpine + AMPT

*0.011 ± 0.001

*0.0015 ± 0.0003

*0.015 ± 0.003

*0.009 ± 0.001

*0.011 ± 0.002

Control 0.129 ± 0.010

0.0038 ± 0.0005

0.0013 ± 0.0003

0.0028 ± 0.0012

0.023 ± 0.003 HEART

Reserpine + AMPT

*0.012 ± 0.003

*0.0022 ± 0.0004

*0.0005 ± 0.0001

*0.0007 ± 0.0002

*0.003 ± 0.0006

Control 22.13 ± 0.830

44.24 ± 0.855

0.011 ± 0.001

0.010 ± 0.0009

0.058 ± 0.017 ADRENAL

GLAND Reserpine +

AMPT *7.24 ± 0.80

*18.86 ± 3.16

0.010 ± 0.002

*0.0059 ± 0.0028

0.052 ± 0.001


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Table 4 – The effect of phentolamine and prazosin on reponses to

phenylephrine, ephedrine, and isoproterenol

Effect of phentolamine (0.5 mg/kg i.v.) and prazosin (0.5 mg/kg i.v.) on the

increase in mean systemic arterial pressure in response to iv injections of

phenylephrine and ephedrine; and the effect of propranolol (0.5 mg/kg i.v.) on the

decrease in mean systemic arterial pressure in response to iv injections of

isoproterenol. Responses to phenylephrine and ephedrine were inhibited by

phentolamine or prazosin whereas responses to isoproterenol were inhibited

following the administration of propranolol. n indicates number of experiments,

and the asterisk indicates that the response is significantly different than control.

n = 6 –10 * p < 0.05

DRUG DOSE (ug/kg i.v.)

Treatment Change in Arterial Pressure (mm/Hg)

Control 30 ± 2 Phentolamine *1.0 ± 0

3 (mg/kg i.v).

Prazosin *0.8 ± 0 Control 40 ± 2 Phentolamine *3 ± 1

Phenylephrine

10 (mg/kg i.v.)

Prazosin *2 ± 1 Control 50 ± 4 Phentolamine *24 ± 2

Ephedrine 10 (mg/kg i.v.)

Prazosin *18 ± 2 Control -11 ± 4 0.1 (mg/kg i.v.) Propranolol *0.0 ± 0 Control -30 ± 5

Isoproterenol

0.3 (mg/kg i.v.) Propranolol *-1.8 ± 1


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