Effects of verapamil on sympathetic tone in normal ...

Effects of veraparnil on sympathetic tone in normal and chronic sinoaortic-denervatedconscious dogs CHRISTINE BAMASE-MICHEL, MARIE-ANTOINETTE TRAN,AND JEAN-LOUIS MONTASTRW@~ Labsratoire de pharmcasologie midicade et Clinique INSERM U3P 7 , Facult&de midecine, 37 aklkes Jules-Guesde, 31073 Tsubuse CLdex, France

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Received August 8, 1988 DAMASE-MICHBL, C., TRAN,M.-A., and MONTASTRUC, So-L.1989- Effects of verapamil on sympathetic tone in nomal and chronic sinoaortic-$enervated conscious dogs. Can. J. Physiol. Phmacol. 67: 428-430. Previous studies have shown that calcium channel blockers from the dihydropyridine group (such as nicardipine) induce an increase in sympathetic tone from a central origin in chronic sinoaortic-denervated (SAD) dogs. In the present study, we investigated to see if verapamil possesses such properties. The effects of acute injection of verapamil (0.2 mglkg i.v.) were compared in nomal and SAD conscious dogs. Verapamil induced a decrease in blood pressure in the two groups of animals, and an increase in heart rate and plasma catecholamines (noradrenaline and adrenaline) in normal but not in SAD dogs. Contrary to the dihydropyridine studies (nicardipine), we did not find any evidence for a centrally mediated sympsthoexcitatory effect of verapabmil in conscious SAD dogs. Key words: verapamil, plasma catecholamines, hypertension, sinoaortic denervation. DAMASE-MICHEL, C., TRAN,M.-A., et MONTASTRUC, J.-L. 1989. Effects of verapapnil on sympathetic tone in normal and chronic sinoaortic-denervated conscious dogs. Can. S. Physiol. Phmacol. 67 : 428-430. k s travaux prbliminaires ont d6montrC que les anticalciqjues du groupe des dihydropyridines (comme la nicardipine) dtteminaient une activation du systkme nerveux sympathique d'origine centrale chez le chien en dtnervation sinoaortique chronique (SAD). Dans cette Ctude, nous avons rechercht si Ie vCrapmil rnanifestait de telles propribtks. Ainsi, les effets d'une injection aiguC de v e r a p d l (0,2 rnglkg i.v.) ont CtC cornparts chez le chien Cveillt nomal et ehronique SAD. Le verapmil diminue la pression artCrielle dans les deux groups d'animaux. I1 tlkve Ia frtquence cardiaqjue et les cat6cholamines plasmatiques (noradrknaline et adknaline) chez le chien normal mais pas chez le canien SAD. Contrairernent aux dihydropyridines (nicardipine), le v e m p h l ne rCv&lepas d'effets centraux sympathoexcitateurs chez %echien SAD vigile.

Introduction The antihypertensive effect of calcium channel blockers is mainly explained by vasodilatation following the inhibition sf transmembrane calcium influx (Gdfraind et al. 1986). After acute administration, these drugs elicit a positive chronotropic response mediated by the baroreflex pathways (Millard et al. 1982). Moreover, recent studies from our group (Chelly et al. 1986; Msntastmc et al. 1988) showed that intracisternal injection sf low doses (ineffective by the peripheral route) of calcium channels blockers (such as dihydropyridines, diltiazem, or verapamil) induced an increase in blood pressure and heart rate mediated by a rise in sympathetic tone in nomotensive anesthetized dogs. We were able to confirm these results in chronic sinsaortie-denervated (SAD) conscious dogs (i.e., animals deprived of baroreceptsr pathways) when intravenous injection of nicardipine, a dihydropyridine derivative, still increased the sympathetic tone, demonstrating that this sympathoexcitatory effect does not only involve bmreflex pathways (Damase-Michel et al. 1987). The aim of the present study was to investigate whether such results are found under similar experimental conditions (SAD dogs) with the non-dihydropyridine agent, verapamil (Singh et al. 1978). Measurement of plasma catecholamine levels allowed us to explore the involvement of the sympathetic nervous system in normal md SAD dogs. Methods Sinoaoreic denervatisn Chronic sinoaortic denervation was performed in seven mongrel dogs of either sex weighing 12-20 kg, as previously described (Dmase-Michel et al. 1987). Briefly, carotid and aortic nerves were cut under chloralose anesthesia ($0 mglkg i.v.1 during two successive procedures, one at time O md the second one 7 weeks later. During the ' ~ u t h o rto whom all correspondence may be addressed. Printed in Canada I Imprime au Canah

surgery, care was taken to keep intact both vagal and sympathetic fibers in the vagus. The effectiveness of baroreceptor denervation was checked by the failure of phenylephine (8.1, 1, and 10 yglkg i. v.) to g to induce bradycardia, and of nitroglycerin (1,3, 10, and 30 ~ g l k i.v.) induce tachycardia. ~ ~ Y ~ ~ O V Qparameters S C U ~ Q ~

Systolic and diastolic blood pressures were recorded by means of a catheter introduced into the abdominal aorta via the left femoral artery (under local anesthesia with Xylocaine) and connected to a Gcsuld P23 ID transducer on one channel of a Honeywell recorder. Heart rate was obtained using a heart period (pulse interval) meter triggered by the eiectrocardiogram (lead 11).

Catecholamine assays Fresh blood was collected, mixed with lithium hepain and 18 mhl sodium metabisulfite, and centrifuged at 20Wg for 18 min at 0°C. Plasma was stored at -88°C. Catecholamines were selectively isolated from the sample at OQC,in the darkness, by adsorption to activated dumina, then eluted with 0. B M perchloric acid. Dihydroxybenzylamine was used as an internal standard to monitor recovery from this extraction step. Noradrenaline and adrenaline were assayed by high pressure liquid chromatography using electrochemical (amperesmetric) detection: the working electrode potential was set at 0.65 V against a Ag- AgCl reference electrode. Catecholamines were separated on a C18 Waters column (3.9 x 150 m) at a constarat flow rate of 1 d l r n i n . The electrochemical detector response was linear for concentrations ranging from 0.17 to 0.68 p M ;r = 0.997 for noradrenaline, r = 0.992 for adrenaline. In these conditions'the detection limit was 0.3 nM. Intercoefficients of variance for plasma catecholamines performed over 5 days were, for these high levels, 11%for noradrenaline and 12% for adrenaline. The intraeoefficients of variance perfowned on three assays were 1% for noradrendine and 7% for adrenaline. Blood smpies were obtained from the femoral artery, and the volume (10 d) was immediately replaced by 10 mL isotonic saline. General pr~cedure Afer dogs had rested on a Pavlov table for at least 30 min after


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FIG. 1. Effect of verapmil(0.2 mglkg i.v.) on blood systolic (solid squares) and diastolic (solid circles) blood pressures (in mmHg; 1 mmHg = 133.3 Pa) (upper panel), heart rate (beatshin) (inset), and plasma catecholamines (NA, noradrenaline; AD, adrenaline, in pg/mL) (lower panel) in seven normal and seven SAD conscious dogs. Plasma catecholamines were measured before and 1 min after verapamil injection. Statistical analysis was made using Wilcoxon's test for paired comparisons. Mean values + SEM are given. * p < 0.05; NS, not significant. &rial and venous caarnulations, they were injected (acute i.v. saphenous injection for 5 s) with 8.2 mgkg verapamil hydrochloride (Biosedra, Malakoff, France). Dogs were fasted for at least 12 h. This dose of verapamil was selected based on both our previous data (Montastmc et al. 1987) and the fact that higher doses (0.3 mglkg) induce atrioventricular blocks in dogs. Two groups of dogs were used: each of the seven SAD (and seven normal dogs) received verapamil. Saline was administered to these two groups of animals as a sham injection. Respective administrations of verapamil and saline were made according to a randomized protocol. These experiments were performed at least 3 months after SAD. The c~diovascularparameters were measured before, at 1 min, and every 5 razin for 30 min; the plasma catecholamine levels were measured at time 8 and 1 min (i.e., at the peak effect on blood pressure and heart rate). Statistical analysis Results are presented as mean values + SEM. Statistical analysis was performed with the Wilcoxon's test for paired comparison. Values of p higher than 0.05 were not considered to be significant.

Only the data with verapmil ape presented because sham experiments (using saline as a vehicle) (not shown) failed to show any significant change in blood pressure, heart rate, and plasma catecholamines in normal as well as in SAD conscious dogs during 38 min. Blood pressure and heart rate In n o m d dogs, acute injection of verapamil(0.2 mgikg i.v.) induced a decrease in blood pressure (significant at time 1 and 5 min) accompanied by a marked tachycardia for 25 min (Fig. 1). In SAD dogs, verapamil induced a more marked and longer decrease in blood pressure but no significant variation in heart rate.

Plasma catecholamines In normal dogs, plasma catecholamines (noradrenaline as well as adrenaline) significantly increased 1 min after verapamil injection (Fig, 1). In SAD dogs, plasma noradrenaline (but not adrenaline) resting values were significantly higher than those in normal dogs: 533 + 147 vs. 251 + 49 pgimL (p < 0.05). Verapamil failed to induce any significant change in plasma catecholamine levels. The importance of baroreceptor pathways in the chronotropic responses to verapamil was investigated in several studies. In chloralose-anesthetized cats, verapamil augmented baroreceptor sensitivity (Abdel-Rahman and Ingenito 1985). Goldman and Saum (1981) and Heesch et al. (1982) found that baroreflex gain was decreased by verapamil. Millard et al. (1982) found that verapamil did not interfere with the arterial baroreflex pathways, whereas Taylor and Kowalski ( 1984) indicated that the reflex vagal bradycardic ratio elicited by noradrenalineinduced pressor responses was reduced by verapamil . Some authors have investigated the effect of verapamil in SAD neurogenic hypertensive models: in acute anesthetized rats (Vasquez et ale 1984) or dogs (Montastrue et al. 1987), verapamil significantly decreased heart rate. In the present experiments, the (expected) positive chronotropie response elicited by verapamil in normaldogs (i.e., with intact baroreflex pathways) was not observed in SAD conscious dogs, demonstrating that this induced tachycardia is only from a baroreflex origin. In fact, a slight bradycardia can occur in some animals, resulting from the negative chronotropic effect of the drug (Singh et al. 1978). It is interesting to compare the present results with veragamil


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with the data obtained under similar conditions with nicadipine (Damase-MicheH et al. 1987). In SAD dogs, nicardipine increased both plasma catecholamine levels and heart rate. In fact, plasma catecholamine concentrations can be useful indices of the stimulation of the sympathetic nervous system, as recently discussed by Fernandez et al. (1988). This led us to suggest the involvement sf central sympathetic activation in this effect of nicxdipine (Damase-Michel et al. 1987). In fact, this action cannot be explained by a peripheral effect ow the sympathetic nervous system, because nicardipine failed to modify adrenal catecholamine release from the gland (%ran et al. 1988). By contrast, no such evidence for a similar central effect was found in the present study with verapamil, since the calcium channel blocker failed to modify heart rate or plasma catecholmines in SAD dogs. In fact, when we compare the present results with those concerning nicardipine (Bamase-Michel et a%.1987), we can assume that the lack of effect of verapamil cannot be due to the high Bevels of catechslamines or to an inability to raise them further, since the dihydropyridine increased these two parameters. We did notice that the resting levels of adrenaline were relatively high, although the animals were trained for several days and remained quiet on the table. We have no explanation for this finding, but it could explained (at least partly) by the fasting state of the animals. Moreover, it is interesting to note that high levels of noradrenaline (but not adrenaline) characterized the SAD model (a twofold increase). In conclusion, the present study did not find any evidence for a central effect of verapamil in SAD dogs. These results and the previous data from Damase-Michel et al. (1987) suggest that differences exist in the central effects of calcium channel blockers, especially according to the level of the sympathetic tone; thus, when this tone is n s m d (nomotensive dogs), acute dihydropyridine as well as verapamil injections induce an increase in sympathetic tone though both a baroreflex and a direct central effect (as shown by studies with intracistemal injections) (Montastmc et al. 1988). In contrast, in SAD animals, i.e., when the sympathetic tone is high (as shown by the level of plasma catecholamines), only nicardipine, but not verapmil, induces a centrally mediated sympathetic activation. Thus, we suggest that the magnitude of central effects of verapamil depends on the level of the sympathetic tone.

Acknawledgements The authors gratefully acknowledge the technical help of Mrs. G. Portolan and Mr. J. P. Bernou. Mrs. Bontemps prepared the manuscript. This work was supported by the "Fondation pour la recherche m6dicale."

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