Dual Effect of Clonidine on Isolated Rabbit Pulmonary Arteries

laboratory and animal investigations Dual Effect of Clonidine on Isolated Rabbit Pulmonary Arteries* Tai-Shion Lee, MD, FCCP; and Xiuhua Hou, MD Clonidine, a partially selective agonist for a 2-adrenoceptors, has been increasingly used in anesthesia. Its direct effect on pulmonary arteries has not yet been clearly characterized. This in vitro study was performed to determine the vasoactive effects of clonidine on isolated rabbit pulmonary arteries. Responses of pulmonary artery rings from New Zealand white rabbits were assessed in the presence and absence of intact endothelium and with or without precontraction by norepinephrine (NE, 3Xl0-6M) or potassium chloride (KCl, 3Xl0-2M). Using tissue bath preparation, cumulative concentration response curves of clonidine were obtained at different concentrations (10-S, I0-7, 10-6, 10-5, 104 M) after a period of stabilization. Clonidine caused vasoconstriction of isolated pulmonary arteries without any pretreatment. The magnitude of the constriction was dose related at lower concentrations and reached

maximum of 300 g/g wet tissue when above 10-6M. On KCI-precontracted pulmonary arteries, clonidine caused significant dose-related vasoconstriction. On the NEprecontracted vessel rings, it elicited significant dosedependent vasodilation up to 80% relaxation at 10'4M. All the above effects were endothelium independent. In conclusion, clonidine has dual endothelium-independent vasoactive effects, causing vasoconstriction on isolated rabbit pulmonary arteries, either untreated or precontracted with KCl, and vasodilation on those precontracted with NE. Clonidine may act as a competitive a-adrenoceptor blocking agent. (Chest 1995; 107:793-97)

clonidine, an imidazoline-derivative a2-agonist, has only recently become an important adjunct in anesthesia practice. 1 Other than for its potent antihypertensive properties, it has been increasingly used perioperatively for its sedative and anesthesia/ analgesic effects in either general or regional anesthesia.1·2 When given systemically, epidurally, or intrathecally, clonidine is a potent analgesic with no clinically significant respiratory depression. 3 It can significantly reduce inhalational and narcotic anesthetic requirement while maintaining better h emodynamic condition against surgical stress. 1·3.4 Clinically, clonidine reduces mean systemic arterial pressure, cardiac output, heart rate, and stroke volume with variable changes of total peripheral resistance.5·6 It is well known that clonidine attenuates sympathetic outflow and thereby decreases blood pressure via activation of a2-receptors at the depressor site of the vasomotor center and at the intermediolateral column of the spinal cord in the central nervous system. 2·6•7 Pharmacological! y, clonidine acts on multiple potential levels: (1) central a2-adrenore-

ceptors; (2) presynaptic a 2-adrenoreceptors on peripheral noradrenergic neurons; and (3) postjunctional or extrajunctional a2-adrenoceptors. 2 However, because of the coexistence of postsynaptic a land a2-adrenoceptors in both the arterial and venous vasculature and the various proportions of a 1- and a 2-adrenoceptors,8 administration of clonidine, which is not a highly selective a 2-agonist,1 may produce more complicated responses than expected. As far as the pulmonary circulation is concerned, previous studies suggested that there was no evidence of decreased myocardial contractility or an increase in pulmonary arterial pressure. 6•9.1° However, Hermiller et al 11 reported a 20% reduction of mean pulmonary artery pressure with negative inotropic effects in patients with congestive heart failure. Since pulmonary circulation also contains a functional adrenergic innervation and neuroeffector system,12 it is essential to know the effects of clonidine on the pulmonary circulation and the potential of clonidine to be used for pulmonary hypertension. So far, the direct effects of clonidine on the pulmonary artery have not been well characterized. This study investigated the direct effects of clonidine on isolated rabbit pulmonary arteries with or without precontraction by potassium chloride (KCl) or norepinephrine (NE) and with or without intact endothelium.

*From the Department of Anesthesiology, Harbor-UCLA Medical Center, Torrance, Calif. Manuscript received October 19, 1993; revision accepted Max 26. Reprint requests: Dr. Lee, Dept. of Anesthesiology, HarborUCLA Medical Center, 1000 W. Carson St., Torrance, CA 90509-2910

I NE=norepinephrine I

Key words: clonidine; pulmonary vasoconstrictor; pulmonary vasodilator

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METHODS AND MATERIALS

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Ten New Zealand white rabbits, weighing2 to2.5 kg, were used for the experiment. The rabbits were anesthetized with pentobarbital sodium (40 mg/kg). A segment of main pulmonary artery just distal from the bifurcation was removed through midsternotomy immediately following respiratory arrest. Precaution was exercised to avoid damage of the intimal surface. The vessel was freed from the connective tissue and cut into 3-mmwide rings. Each ring of the pulmonary artery was immersed in a 10-mL tissue bath containing a continuously oxygenated (95% 02/5% C02) Krebs solution at 37°C and a pH of 7.4. The solution consisted of 118 mmol of NaCI, 0.8 mmol of KCI, 2.5 mmol of CaCI2, 25 mmol of NaHCOs, 1.18 mmol of KH2P0 4, 1.19 mmol of MgC04, and 11 mmol of glucose. The ring was suspended between a fixed hook and an isometric transducer (Grass FT03). The contractions were recorded on a polygraph (Beckman R612). The resting tension of each ring was adjusted to 3. 75 g based on the prior search of the optimal resting tension in rabbit pulmonary arteries. At least 1 h was allowed for equilibration and stabilization. Two groups of vessels were prepared, one with and one without intact endothelium. Both were tested under the same conditions for comparison. The de-endothelialization was performed by gently rubbing the intimal surface with forceps and confirmed pharmacologically by standard test with NE (3Xl0' 7 M) and acetylcholine (10· 6M). Two (one intact and one denuded) pulmonary artery rings were obtained from each animal. Ten rings were prepared for each set of the experiment ; totally 20 rings were used.

600

Protocol Step 1: Following stabilization , cumulative concentrationresponse curves of clonidine were constructed by adding increasing concentrations of clonidine in logarithmic increments (lo· 8 to 10'4 M). Step 2: The rings were washed after step 1 until complete re-

covery of the resting tension was reached. Another hour was allowed for equilibration. The submaximal precontraction of the vessels was induced by KCI (3X10- 2M). When contractile responses reached a plateau, cumulative concentration-response curves of clonidine were obtained in the same manner as in step 1. Step 3: The procedures were repeated as in step 2, replacing the KCI (3X10- 2M) with NE (3X10- 6 M). The order of using nonpretreated, KCl- or NE-pretreated pulmonary artery rings in each step was randomized. After each assay, each pulmonary artery ring was removed from the organ bath, blotted dry, and weighed. All results were expressed as the mean± SEM. The difference between mean values was analyzed by analysis of variance and Student's t test. The difference was considered statistically significantwhen p was less than 0. 05. RESULTS

Effect of Clonidine on Isolated Unpretreated Pulmonary Arteries With or Without Intact Endothelium. Without any pretreatment, clonidine caused vasoconstriction of isolated pulmonary arteries. There was no difference in groups with or without intact endothelium. The magnitude of the constriction was dose related in lower concentrations. However, it seemed to have a ceiling effect reaching to 300 to 320 g/g wet tissue when the concentration was higher 794

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Effect of Clonidine on Isolated KCl-Precontracted Pulmonary Arteries With or Without Intact Endothelium. Clonidine caused vasoconstriction in the KCl (30 mM)-precontracted pulmonary arteries in a dose-related manner (Fig 2). At 10-4 M, it increased constriction by almost 20%. This vasoconstriction was endothelium independent.

Effect of Clonidine on Isolated, NE-Precontracted Pulmonary Arteries With or Without Intact Endothelium Clonidine elicited significant dose-dependent vasodilatation in NE-precontracted pulmonary arteries (Fig 3). At 10- 4 M, it produced 80% relaxation from the control-contraction by NE (3 JLM). There was no difference between groups with or without intact endothelium. DISCUSSION

Clonidine, according to our study, has dual vaso100~------------------------------~

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FIGURE 2. Cumulative concentration responses of clonidine in isolated, KCl-precontracted rabbit pulmonary arteries with (+)or without (-) endothelium (E). P values of all E (+) vs E (-) are >0.05. Dual Effect of Clonidine on Isolated Rabbit Pulmonary Arteries (Lee, Hou)

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active effect on isolated rabbit pulmonary arteries (Fig 4) . In vivo, administration of clonidine usually has been associated with hypotension and bradycardia.13·14 Although the hypotensive effect is thought to be due to an attenuation in sympathetic activity, the exact mechanism is not fully elucidated.15 Nevertheless, it had been shown earlier that intravenous clonidine in anesthetized animals caused biphasic blood pressure response with a transient initial increase followed by a lasting decrease in arterial pressure. 6 There seemed to be a brief increase in systemic vascular resistance that was dose related and could be abolished by phentolamine, a nonselective a2-adrenoceptor antagonist. 6 Davies et aJl 6 reported that clonidine produced hypotensive effects at plasma concentrations of less than 2 ng/ ml, with a less than expected reduction at 2 to 10 ng/ ml. Our study showed that the vasoconstrictive effect of clonidine on isolated rabbit pulmonary arteries started at w-8M (2.26 ng/ ml) . The constriction reached the maximum plateau at w- 6 M (226 ng/ ml), which was about 55% of the force pro40 ,.-...

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4. Comparative effects of clonidine on isolate, KCI- and NE-precontracted rabbit pulmonary arteries. Dual effect (vasoconstriction on KCl-precontracted and vasodilation on NEprecontracted vessels) is shown. P values of all E (+) vs E (-) are >0.05. FIGURE

duced by equivalent dose of NEP The vasoconstrictive role of postsynaptic a2-adrenoceptors had been unrecognized until relatively recently. 5.lO,ll This vasoconstriction may act as a modulator of arterial circulation protecting it from oversupply in normal conditions. 10 Pharmacologically, clonidine is classified as a partially selective agonist for a2-adrenoceptors, with a ratio of 200:1 (a2:a1).l Because of coexistence of postsynaptic a 1 and a2-adrenoceptors in the vasculature, through which vasoconstriction is mediated independent of the nerve supply, 18 it is not surprising to observe vasoconstrictive response of clonidine on the pulmonary artery. Activation of a2-adrenoceptors induces influx of extracellular ca++, whereas activation of aradrenoceptors elicits not only ca++ influx but also the release on intracellularly bound Ca ++ 19 The efficacy of an a-adrenoceptor agonist in causing contraction more heavily depends on its ability to stimulate Ca ++ influx. 19 This current study suggests the existence of postjunctional a 2-adrenoceptors in pulmonary arteries as in systemic arteries. In contrast to vasodilatation involving the release of endothelial-derived relaxation factor in previous in vivo studies, 20-22 clonidine in our study induced vasoconstriction in isolated rabbit pulmonary arteries that was independent of the existence of the intact endothelium. It is also in conflict with the reports that intact endothelium impaired contractile responses in rat aorta to clonidine.19 The discrepancy may be explained by the fact that the distribution of a-adrenoceptors varies between species and within the same species, particularly different parts of circulation. 22 Several studies reported no consistent changes in systemic vascular resistance following clonidine administration, and it is suggested that the reduction in blood pressure is thus due to the decreased cardiac output. 6•23 Although most studies suggest that the reduction of blood pressure is of central origin secondary to activation of postsynaptic a2-adrenoceptors in the depressor site of the vasomotor center, the direct effects of clonidine on blood vessels, particularly the pulmonary artery, has not been well defined. Even though it is commonly used for antihypertension, clonidine has been shown to cause vasoconstriction in the rat coronary artery and canine saphenous vein. 4J 0·24 However, it has also been reported that only at lower plasma concentration is the hypotensive effect of clonidine dose related.l·6 Occasionally, clonidine causes hypertension when larger doses are used .7•14 Recently, the existence of the postsynaptic as well as extrasynaptic a2-adrenoceptors has been found both inside and outside the central nervous system in many organ/ tissue, including blood vessels. 2·10•14 This may partially explain the CHEST / 107/3/ MARCH, 1995

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disparity. Apparently, moreover, the net vasoactive effect of clonidine is the balance between central sympatholytic effect (az-adrenoceptors) and peripheral vasoconstriction (a 1- and a 2-adrenoceptors). 25· 26 In tetraplegic patients in whom there is no significant peripheral sympathetic tone, clonidine does not affect mean resting blood pressures or reduce plasma NE and renin. 9·14·25 Likewise, in an isolated pulmonary artery preparation, vasoconstriction is the direct effect and there is no central neurohumoral influences of clonidine. In our study, clonidine caused vasoconstriction on KCl-precontracted pulmonary arteries in a dose-related manner. KCl is known to induce contraction of the blood vessel by causing membrane depolarization. It thereby causes Ca2+ influx through voltageoperated channels resulting in vasoconstriction. Since it is independent of adrenoceptors, there seems no direct interaction with clonidine in our study. Clonidine has been suggested to suppress Ca2+ entry into the nerve terminals by activating a2-adrenoceptormediated inhibition of Ca2+ channels.27 However, Jim and Matthews 24 reported that activation of a2-adrenoceptor results primarily in an influx of extracellular calcium to produce contraction. The increases in vasoconstriction on KCl-precontracted pulmonary arteries in our study seems to rule out the possibility of inhibition of Ca2+ channels by a 2adrenoceptor activation in vascular smooth muscles. It also excludes the potential antagonism against a-adrenoceptors by clonidine. Besides, clonidine also stimulates a1-adrenoceptor that produces contraction by releasing calcium from sarcoplasmic reticulum as well as causing influx of extracellular calcium.24 It has been reported that the hemodynamic responses of clonidine are related to the level of preexisting sympathetic tone , and the various changes in blood pressure in response to clonidine in patients with autonomic dysfunction was inversely proportional to the level of NE. 2-5 Our study consistently demonstrated that clonidine caused a significant dose-dependent but endothelium-independent vasodilatation in NE-precontracted pulmonary arteries. Plasma NE, increased with sympathetic discharge, induces contraction by causing Ca2+ influx through a1-adrenoceptor-operated mechanism as well as direct or indirect release of intracellular Ca2+ . Since NE has been known to be equally effective as an agonist at a1- and az-adrenoceptor sites,8 we speculate that there is a competitive mechanism involved between NE and clonidine. Clonidine replaces NE at the receptor sites and produces vasodilatation of NE-precontracted vessels by attenuating the efficacy of NE on a1-adrenoceptor. Elevated sympathetic 796

activity is an important compensatory mechanism in a highly stressed situation; profound hypotension may develop if clonidine is administered due to its central sympatholytic and peripheral competitive effects. 28 Because of its action on postsynaptic azadrenoceptor, clonidine probably should not be simply classified as a centrally acting az- and weak peripheral aradrenergic agonist as suggested. 29 In summary , our experiments indicate the following: (1) clonidine has dual vasoactive effects, causing vasoconstriction on pulmonary arteries, either untreated or precontracted with KCl, vasodilation on those precontracted with NE ; and (2) other than central sympatholytic effect, clonidine causes vasoconstriction of the pulmonary artery via a1- and azadrenoceptor. It may also have a competitive a-adrenoceptor blocking effect depending on plasma level of NE. REFERENCES

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