QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.105.090035v1 316/1/95    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Liles, J. T. Articles by Kadowitz, P. J. Search for Related Content PubMed PubMed Citation Articles by Liles, J. T. Articles by Kadowitz, P. J.

CARDIOVASCULAR

Pressor Responses to Ephedrine Are Mediated by a Direct Mechanism in the Rat

Department of Pharmacology, Tulane University Health Sciences Center School of Medicine, New Orleans, Louisiana (J.T.L., P.A.D., K.E.H., L.P., S.R.B., P.J.K.); and Departments of Pharmacology, School of Medicine (K.J.V., A.R.H.), and Physiology, School of Dentistry (J.R.P., C.C.), Louisiana State University Health Science Center, New Orleans, Louisiana

Received May 24, 2005; accepted July 6, 2005.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
The mechanism of the pressor response to ephedrine is controversial. In the present study. i.v. injections of ephedrine increased systemic and pulmonary arterial pressure, and i.a. injections decreased hindlimb blood flow in a dose-related manner. Responses to ephedrine were inhibited by -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 -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 -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 -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.

The release of catecholamines and subsequent activation of adrenergic receptors is thought 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 (Fleckenstein and Burn, 1953; Burn and Rand, 1958; Cairolli et al., 1962; de Moraes et al., 1968). Other studies in isolated tissues have shown that ephedrine acts by direct stimulation of adrenergic receptors (Tye et al., 1967; Waldeck and Widmark, 1985; Bao et al., 1990; 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 have been shown to be reduced by pretreatment with reserpine as well as with a combination of reserpine and -methyl-p-tyrosine (AMPT) (Sabol and Seiden, 1998; Uehara et al., 2004). Few studies have examined the mechanisms involved in the cardiovascular 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 -methyl-p-tyrosine together to determine whether the release of catecholamines mediates the responses to ephedrine and amphetamine. The effect of acute ephedrine administration on arterial pressure, hindlimb blood flow, and pulmonary arterial pressure as well as the adrenergic receptors involved in these responses were examined in this study.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
General Methods. Experiments were performed on male Sprague-Dawley rats, weighing 300 to 400 g, 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., Ithaca, NY) 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 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).

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 preinjection 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 and 10 mg/kg i.p., respectively) anesthesia, the arterial pressure cannula of a battery powered radio telemetry probe (TL11M2-C50-PXT; Data Sciences International) 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 to 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 before 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 were 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 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 mm Hg 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 after cocaine administration was assessed by analyzing responses to tyramine (100 µg/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 AMPT (200 mg/kg i.p.) was investigated. Reserpine was administered 18 h before the experiment, whereas AMPT was given at least 2 h before the experiment. The extent of 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, whereas 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.) before the evaluation of ephedrine responses in the systemic, hindlimb, or pulmonary vascular bed. Blockade of -and -receptors was assessed by evaluating responses to phenylephrine and isoproterenol. Phentolamine administration decreased baseline arterial pressure to 86 ± 4 mm Hg, whereas propranolol had no significant effects on baseline systemic arterial pressure.

High-Performance Liquid Chromatography (HPLC) Determination of Tissue Catecholamines. After 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 0.1 M citrate, 10% ethanol, and 250 mg/liter sodium octyl sulfate at pH 4. Tissues were stored in the buffer at a temperature of –80°C until analysis. An internal standard of 3,4-dihydroxybenzylamine hydrobromide was added such that its final concentration was 1 ng/100 µl of the HPLC injectate. The homogenate was centrifuged in a RC2B Sorvall centrifuge at 12,000g, and the supernatant was frozen at –80°C until assayed. Recoveries ranged from 75 to 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 (5 µm) C18 column (25 cm x 4.6 mm). A 1.5 cm x 4.6-mm guard column packed with microsorb C18 preceded the analytical column. Chromatographically separated monoamines were assayed by electrochemical detection using 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 V. 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. Cochromatography of spiked standards of each compound were initially run with brain tissue to determine that the peaks identified as monoamines were indeed authentic NE, Epi, Dop, 5HT, and 5HIAA.

View larger version (33K): [in this window] [in a new window]   Fig. 1. A, left, effect of i.v. injections of ephedrine on mean systemic arterial pressure when doses are administered nonsequentially as a single injection in separate animals and when all injections were administered sequentially in the same animal 20 to 30 min 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 nonsequentially as a single injection in separate animals and when all injections were administered sequentially in the same animal 20 to 30 min 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.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Responses to Ephedrine and Amphetamine. Responses to ephedrine and amphetamine were compared and injection of ephedrine in doses of 1 to 30 mg/kg i.v. produced dose-related increases in systemic arterial pressure when each injection was given in a separate animal (Fig. 1A, left). Injections of ephedrine in doses of 1 to 30 mg/kg i.v. also caused dose related increases in systemic arterial pressure when injections were given at 30-min intervals in the same animal (Fig. 1A). Intravenous injections of amphetamine in doses of 1 to 30 mg/kg increased systemic arterial pressure (Fig. 1B). However, pressor responses to amphetamine in doses of 1 to 30 mg/kg i.v. were only dose-related when single injections were administered to separate animals (Fig. 1B, left). Figure 1, A and B, also illustrates 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.

View larger version (35K): [in this window] [in a new window]   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.

Effects of Cocaine, Reserpine, and AMPT. The effects of cocaine, reserpine, and reserpine plus AMPT on pressor responses to ephedrine and amphetamine were compared, and these data are summarized in Figs. 3, 4, 5. After 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.

View larger version (25K): [in this window] [in a new window]   Fig. 3. A, effect of cocaine (5 mg/kg i.v.) on the increase in mean arterial pressure in response to i.v. 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 i.v. injections of angiotensin II and on the decrease in mean arterial pressure in response to i.v. injections of acetylcholine. n indicates number of experiments, and the asterisk indicates that the response is significantly different than control.

View larger version (28K): [in this window] [in a new window]   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 i.v. injections of ephedrine B, increase in mean systemic arterial pressure and the area under the curve of the response to i.v. injections of amphetamine C, increase in mean systemic arterial pressure and the area under the curve of the response to i.v. injections of tyramine. D, increase in mean systemic arterial pressure and the area under the curve of the response to i.v. injections of norepinephrine. n indicates number of experiments, and the asterisk indicates that the response is significantly different than control.

View larger version (31K): [in this window] [in a new window]   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 i.v. injections of ephedrine. B, decrease in hindlimb blood flow and the area under the curve of the response to i.v. injections of amphetamine. C, decrease in hindlimb blood flow and the area under the curve of the response to i.v. injections of tyramine. D, decrease in hindlimb blood flow and the area under the curve of the response to i.v. injections of norepinephrine. n indicates number of experiments, and the asterisk indicates that the response is significantly different than control.

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 Fig. 6. The magnitude of the increase in mean arterial blood pressure after 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 it resulted in a significant decrease in heart rate in the conscious rat (Fig. 6C).

View larger version (13K): [in this window] [in a new window]   Fig. 6. A, change in mean systemic arterial pressure in response to i.v. injection of ephedrine in the conscious rat. B, duration of the increase in mean systemic arterial pressure in response to i.v. injection of ephedrine in the conscious rat. C, change in heart rate in response to i.v. injection of ephedrine in the conscious rat. n indicates number of experiments, and the asterisk indicates that the response is significantly different than control.

View larger version (34K): [in this window] [in a new window]   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 i.v. 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 i.v. injections of ephedrine. n indicates number of experiments, and the asterisk indicates that the response is significantly different than control.

Effect of Propranolol on Systemic Hindlimb Responses to Ephedrine. The effect of the propranolol on the response to ephedrine is shown in Fig. 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 -adrenergic receptor agonist isoproterenol (Table 4). The role of -adrenergic 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 Fig. 8. Ephedrine produced dose-related increases in pulmonary arterial pressure. The magnitude of the increases in pulmonary pressure was significantly decreased after 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 after 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) after catecholamine depletion with reserpine and AMPT.

View larger version (36K): [in this window] [in a new window]   Fig. 8. A, effect of phentolamine (0.5 mg/kg i.v.) on the increase in mean pulmonary arterial pressure in response to i.v. 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 i.v. 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 i.v. injections of ephedrine, tyramine, and norepinephrine. n indicates number of experiments, and the asterisk indicates that the response is significantly different than control.

	Discussion

Top Abstract Materials and Methods Results Discussion References

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 mechanism (Fleckenstein and Burn, 1953; Burn and Rand, 1958; Cairolli et al., 1962; de Moraes et al., 1968). However, direct stimulation of adenergic receptors in the guinea pig trachea, portal vein, and reserpinized rat atrium has been reported previously (Tye et al., 1967; Waldeck and Widmark, 1985; Bao et al., 1990; Kawasuji et al., 1996). Ephedrine has been shown to bind to 1, 2, and 3 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, whereas 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 (Simpson, 1980). An exchange diffusion model suggests that only cytoplasmic catecholamines are released by amphetamine-like drugs (Fischer and Cho, 1979). Since reserpine is selective for vesicular stores, many studies have used coadministration of 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 after 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 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) and reserpine and AMPT (Abrahams et al., 1996) is in agreement with the observation that responses to amphetamine are reduced by catecholamine depletion (Abrahams et al., 1996; Sabol and Seiden, 1998). In the present study, both reserpine and reserpine and -methyl-p-tyrosine inhibited responses to amphetamine. Although norepinephrine is thought to be stored in distinct pools, there is evidence that these pools are in flux (Simpson, 1980). 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 nonspecific 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 et al., 1998; Young and Glennon, 1998; Miller and Waite, 2003). 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 -adrenergic receptors, whereas -receptors modulate the responses. Phentolamine attenuated responses to phenylephrine and significantly reduced the increase in 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 -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 -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 and -2 receptor activation, with -receptor induced vasoconstriction opposing the -2 receptor mediated vasodilation (Lambrecht et al., 2002). Thus, -receptor inhibition allows unopposed stimulation of -receptors, which could result in a longer duration of the response to ephedrine. Moreover, when -receptors were blocked, a -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 - and -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 short intervals (20 min) in the same animal. However, tachyphylaxis to ephedrine was different from tachyphylaxis induced by repeated doses of amphetamine. The present observation that the pressor response to ephedrine was reduced but not abolished after repeated administration is consistent with studies that reported different patterns of tachyphylaxis with ephedrine and amphetamine (de Moraes and Carvalho, 1967; Takasaki et al., 1972). In the present study, pressor responses to phenylephrine and norepinephrine were reduced after the development of ephedrine tachyphylaxis but not reduced by amphetamine tachyphylaxis. These findings are in agreement with isolated tissue studies showing that -receptors are inhibited by repeated administration of ephedrine (Furukawa and Morishita, 1975; Morishita and Furukawa, 1975). The present data provide support for the hypothesis that tachyphylaxis to the pressor response to ephedrine may involve -receptor inhibition, whereas 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 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 -adrenergic receptor stimulation, whereas -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 catecholamine depletion. In contrast, the increase in systemic arterial pressure and the decrease in hindlimb blood flow in response to amphetamine were abolished after 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, whereas these responses were unchanged by amphetamine tachyphylaxis. Theses data suggest that pressor response to ephedrine is mediated by direct -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.

	Acknowledgements

 
We thank Janice Ignarro for editorial assistance.

	Footnotes

 
This work was supported by National Heart, Lung, and Blood Institute Grants HL62000 and HL77421 from the National Institutes of Health.

doi:10.1124/jpet.105.090035.

ABBREVIATIONS: AMPT, -methyl-p-tyrosine; MAP, mean arterial pressure; HPLC, high-performance liquid chromatography; NE, norepinephrine, Epi, epinephrine, Dop, dopamine, 5HT, 5-hydroxytryptamine; 5HIAA, 5-hydroxyindoleacetic acid.

Address correspondence to: Dr. Philip J. Kadowitz, Department of Pharmacology, SL83, 1430 Tulane Ave., New Orleans, LA 70112. E-mail: pkadowi{at}tulane.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Abdullah AH, Tye A, and Patil PN (1968) Steric aspects of adrenergic drugs. X. Tachyphylaxis to the ephedrine isomers in the isolated rabbit heart. Arch Int Pharmacodyn Ther 174: 454–468.[Medline]

Abrahams TP, Faust ML, and Varner KJ (1996) The depletion of monoamines blocks the sympathoinhibitory response to cocaine. J Auton Nerv Syst 58: 170–176.[CrossRef][Medline]

Ashar BH, Miller RG, Getz KJ, and Pichard CP (2003) A critical evaluation of Internet marketing of products that contain ephedra. Mayo Clin Proc 78: 944–946.[Medline]

Baber SR, Hyman AL, and Kadowitz PJ (2003) Role of COX-1 and -2 in prostanoid generation and modulation of angiotensin II responses. Am J Physiol 285: H2399–H2410.

Bao JX, Wang B, and Yang ZC (1990) Effects of ephedrine on postsynaptic alpha-adrenoceptors and presynaptic beta-adrenoceptors in isolated guinea pig portal veins. Zhongguo Yao Li Xue Bao 11: 130–133.[Medline]

Burn JH and Rand MJ (1958) The action of sympathomimetic amines in animals treated with reserpine. J Physiol (Lond) 144: 314–336.[Free Full Text]

Cairolli VJ, Reilly JF, and Roberts J (1962) Effect of reserpine pretreatment on the response of isolated papillary muscle to ephedrine. Br Pharm Chemother 18: 588–594.

Cockings JG and Brown M (1997) Ephedrine abuse causing acute myocardial infarction. Med J Aust 167: 199–200.[Medline]

de Moraes S and Carvalho FV (1968) Reciprocal pressor effects between amphetamine and some sympathomimetic amines. Pharmacology 1: 129–134.[CrossRef][Medline]

Food and Drug Administration, HHS (2004) Final rule declaring dietary supplements containing ephedrine alkaloids adulterated because they present an unreasonable risk. Final rule. Fed Regist 69: 6787–6854.[Medline]

Fischer JF and Cho AK (1979) Chemical release of dopamine from striatal homogenates: evidence for an exchange diffusion model. J Pharmacol Exp Ther 208: 203–209.[Free Full Text]

Fleckenstein A and Burn JH (1953) The effect of denervation on the action of sympathomimetic amines on the nictitating membrane. Br J Pharmacol 8: 69–78.[Medline]

Foxford RJ, Sahlas DJ, and Wingfield KA (2003) Vasospasm-induced stroke in a varsity athlete secondary to ephedrine ingestion. Clin J Sport Med 13: 183–185.[CrossRef][Medline]

Furukawa T and Morishita H (1975) Modifications of responses to adrenergic drugs in arterial strip by treatment in vivo with ephedrine and reserpine. Jpn J Pharmacol 25: 441–451.[Medline]

Grzesk G, Polak G, Grabczewska Z, and Kubica J (2004) Myocardial infarction with normal coronary arteriogram: the role of ephedrine-like alkaloids. Med Sci Monit 10: CS15–CS21.[Medline]

Haller CA and Benowitz NL (2000) Adverse cardiovascular and central nervous system events associated with dietary supplements containing ephedra alkaloids. N Engl J Med 343: 1833–1838.[Abstract/Free Full Text]

Kawasuji T, Koike K, and Saito H (1996) Chronotropic effects of optical isomers of ephedrine and methylephedrine in the isolated rat right atria and in vitro assessment of direct and indirect actions on beta 1-adrenoceptors. Biol Pharm Bull 19: 1423–1428.[Medline]

Kim TJ and LeBourgeois HW 3^rd (2004) Banned, but not forgotten: a case of ephedrine-induced psychosis. Prim Care Companion J Clin Psychiatry 6: 136–137.[Medline]

Kobayashi S, Endou M, Sakuraya F, Matsuda N, Zhang XH, Azuma M, Echigo N, Kemmotsu O, Hattori Y, and Gando S (2003) The sympathomimetic actions of l-ephedrine and d-pseudoephedrine: direct receptor activation or norepinephrine release? Anesth Analg 97: 1239–1245.[Abstract/Free Full Text]

Lambrecht JE, Hamilton W, and Rabinovich A (2002) A review of herb-drug interactions: documented and theoretical. US Pharm 25: 8.

Martin WR, Sloan JW, Sapira JD, and Jasinski DR (1971) Physiologic, subjective, and behavioral effects of amphetamine, methamphetamine, ephedrine, phenmetrazine and methylphenidate in man. Clin Pharmacol Ther 12: 245–258.[Medline]

McMahon LR and Cunningham KA (2003) Discriminative stimulus effects of (–)-ephedrine in rats: analysis with catecholamine transporter and receptor ligands. Drug Alcohol Depend 70: 255–264.[CrossRef][Medline]

Miller DK, McMahon LR, Green TA, Nation JR, and Wellman PJ (1998) Repeated administration of ephedrine induces behavioral sensitization in rats. Psychopharmacology 140: 52–56.[CrossRef][Medline]

Miller SC and Waite C (2003) Ephedrine-type alkaloid-containing dietary supplements and substance dependence. Psychosomatics 44: 508–511.[Free Full Text]

Morishita H and Furukawa T (1975) The vascular changes after ephedrine tachyphylaxis. J Pharm Pharmacol 27: 574–579.[Medline]

Purdy RE, Ashbrook DW, Hurlbut DE, Reidy JP, Stratford RE, and Watanabe MY (1981) Effects of reserpinization, surgical denervation and in vitro chemical denervation with 6-hydroxydopamine on the contractile response of isolated rabbit ear artery to propranolol. Blood Vessels 18: 153–160.[Medline]

Rothman RB, Vu N, Partilla JS, Roth BL, Hufeisen SJ, Compton-Toth BA, Birkes J, Young R, and Glennon RA (2003) In vitro characterization of ephedrine-related stereoisomers at biogenic amine transporters and the receptorome reveals selective actions as norepinephrine transporter substrates. J Pharmacol Exp Ther 307: 138–145.[Abstract/Free Full Text]

Sabol KE and Seiden LS (1998) Reserpine attenuates D-amphetamine and MDMA-induced transmitter release in vivo: a consideration of dose, core temperature and dopamine synthesis. Brain Res 806: 69–78.[CrossRef][Medline]

Schindler CW, Zheng JW, Tella SR, and Goldberg SR (1992) Pharmacological mechanisms in the cardiovascular effects of methamphetamine in conscious squirrel monkeys. Pharmacol Biochem Behav 42: 791–796.[CrossRef][Medline]

Schwartz AB, Boyle W, Janzen J, and Jones RT (1988) Acute effects of cocaine on catecholamines and cardiac electrophysiology in the conscious dog. Can J Cardiol 4: 188–192.[Medline]

Simpson LL (1980) Evidence that there are subcellular pools of norepinephrine and that there is flux of norepinephrine between these pools. J Pharmacol Exp Ther 214: 410–416.[Abstract/Free Full Text]

Sulzer D and Rayport S (1990) Amphetamine and other psychostimulants reduce pH gradients in midbrain dopaminergic neurons and chromaffin granules: a mechanism of action. Neuron 5: 797–808.[CrossRef][Medline]

Takasaki K, Urabe M, and Yamamoto R (1972) Tachyphylaxis of indirectly acting sympathomimetic amines. III. Influence of acutely and chronically administered reserpine. Kurume Med J 19: 153–168.

Tye A, Baldesberger R, LaPidus JB, and Patil PN (1967) Steric aspects of adrenergic drugs. VI. Beta adrenergic effects of ephedrine isomers. J Pharmacol Exp Ther 157: 356–362.[Abstract/Free Full Text]

Uehara T, Sumiyoshi T, Itoh H, and Kurachi M (2004) Inhibition of dopamine synthesis with alpha-methyl-p-tyrosine abolishes the enhancement of methamphetamine-induced extracellular dopamine levels in the amygdala of rats with excitotoxic lesions of the entorhinal cortex. Neurosci Lett 356: 21–24.[CrossRef][Medline]

Vansal SS and Feller DR (1999) Direct effects of ephedrine isomers on human beta-adrenergic receptor subtypes. Biochem Pharmacol 58: 807–810.[CrossRef][Medline]

Waldeck B and Widmark E (1985) The interaction of ephedrine with beta-adrenoceptors in tracheal, cardiac and skeletal muscles. Clin Exp Pharmacol Physiol 12: 439–442.[Medline]

Young R and Glennon RA (1998) Discriminative stimulus properties of (–)ephedrine. Pharmacol Biochem Behav 60: 771–775.[CrossRef][Medline]

Zahn KA, Li RL, and Purssell RA (1999) Cardiovascular toxicity after ingestion of "herbal ecstacy". J Emerg Med 17: 289–291.[CrossRef][Medline]

This article has been cited by other articles:

				G. Ma, S. A. Bavadekar, Y. M. Davis, S. G. Lalchandani, R. Nagmani, B. T. Schaneberg, I. A. Khan, and D. R. Feller Pharmacological Effects of Ephedrine Alkaloids on Human {alpha}1- and {alpha}2-Adrenergic Receptor Subtypes J. Pharmacol. Exp. Ther., July 1, 2007; 322(1): 214 - 221. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.105.090035v1 316/1/95    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Liles, J. T. Articles by Kadowitz, P. J. Search for Related Content PubMed PubMed Citation Articles by Liles, J. T. Articles by Kadowitz, P. J.