Endothelium-Independent Contractions of Human Cerebral Arteries in Response to Vasopressin

Transcription

1 1689 Endothelium-Independent Contractions of Human Cerebral Arteries in Response to Vasopressin E. Martin de Aguilera, PhD, J.M. Vila, PhD, A. Irurzun, PhD, M.C. Martinez, BSc, M.A. Martinez Cuesta, BSc, and S. Lluch, MD We studied the effects of vasopressin in isolated segments from branches ( fim in external diameter) of human middle cerebral arteries obtained during autopsy of 15 patients who had died 3-8 hours before. Paired segments, one normal and the other de-endothelized by gentle rubbing, were mounted for isometric recording of tension in organ baths. In 11 normal segments, vasopressin produced concentration-dependent contractions with an EC 50 of 7.0 x 10" l0 M. Removal of the endothelium from 12 segments did not significantly affect vasopressin-induced contractions. Vasopressin produced further contractions in arterial segments with («=4) or without (n=5) endothelium precontracted with KC1. In segments precontracted with prostaglandin F 2a, acetylcholine caused relaxation only of those with endothelium. At 10~ 8 M (n=ll), the vasopressin V-l receptor antagonist d(ch 2 ) 5 Tyr(Me)AVP produced a 60-fold shift to the right of the control response curve for vasopressin. Increasing the concentration of the receptor antagonist to 10~ 6 M («=7) further displaced the control curve in a parallel manner. These results indicate that vasopressin exerts a powerful constrictor action on isolated human cerebral arteries by direct stimulation of V-l receptors located predominantly on smooth muscle cells. It appears that this contractile response is not modulated by the presence of an intact endothelial cell layer. (Stroke 1990;21: ) Vasopressin causes powerful constriction in a variety of vascular regions 1-2 and is now thought to serve as a major fast-acting pressure control system in hypotensive states. 3-4 The vasopressin receptor designated V-l seems to mediate the cardiovascular actions of the peptide, whereas its antidiuretic action is mediated through V-2 cyclic adenosine monophosphate-dependent receptors. 5-6 Some cardiovascular actions of vasopressin are mediated by V-2-like receptors in both humans 7 and dogs. 8 A common finding in the vascular effects of this peptide is the heterogeneity of responsiveness, depending on region and species With regard to the cerebral vessels, the debate goes on whether pharmacologic studies of vasopressin in various animal species can be extrapolated to humans. 11 Integrity of the endothelial cell layer is a factor that may account for variations in the re- From the Departamento de Fisiologfa, Universidad de Valencia, Valencia, Spain. Supported by Comision Asesora de Investigation and Ministerio de Sanidad. Address for reprints: S. Lluch, MD, Departamento de Fisiologi'a, Facultad de Farmacia, Universidad de Valencia, Avenida Blasco Ibariez 13, Valencia, Spain. Received March 28, 1990; accepted July 31, sponses of different vascular beds to neurohumoral agents Therefore, we designed this study to determine the direct effects of arginine vasopressin on isolated human cerebral arteries, with special emphasis on endothelium-dependent responses. We also studied the concentration-response relation of the cerebral arteries to vasopressin after treatment with d(ch 2 ) 5 Tyr(Me)AVP, a compound that is devoid of agonistic pressor activity and antagonizes the vasopressor effects of vasopressin Materials and Methods Human cerebral arteries were obtained during autopsy of 15 patients (nine men and six women, aged years) who had died 3-8 hours before. The cause of death varied; eight patients were victims of automobile accidents, four had died of myocardial infarction, and three had died of cancer of the stomach. The arteries were immediately placed in chilled Krebs-Henseleit solution, and cylindrical segments 3 mm long, were cut from branches of the middle cerebral artery for isometric recording of tension. Outside diameter of the segments was measured using an ocular micrometer within a Wild M8 zoom microscope (Heerbrugg, Switzerland) and ranged from 500 to 700 (mean±sem 623 ±37) jam.

2 1690 Stroke Vol 21, No 12, December 1990 In some arterial segments the endothelium was removed mechanically by inserting a roughened stainless steel wire into the lumen and gently rolling the segment on wet filter paper. Two stainless steel pins 100 /tin in diameter were introduced through the arterial lumen. One pin was fixed to the wall of the organ bath, while the other was connected to a strain gauge. The recording system included a Universal transducing cell (UC3, New Orleans, La.), a Statham microscale accessory (U15, Glen Burnie, Md.) and a Beckman recorder (Type RS, Fullerton, Calif.). Each arterial segment was set up in a 4-ml bath containing modified Krebs- Henseleit solution of the following millimolar composition: NaCl 115, KC1 4.6, KH 2 PO 4 1.2, MgSO 4 1.2, CaCl 2 2.5, NaHCO 3 25, glucose 11.1, and disodium ethylenediaminetetraacetic acid The solution was equilibrated with 95% O 2 and 5% CO 2 to give a ph of Temperature was held at 37 C. To establish the resting tension for maximal force development, the arterial segments were exposed repeatedly to 60 mm KC1. Basal tension was increased gradually until contractions were maximal. The optimal resting tension was 1 g. The segments were allowed to attain a steady level of tension during a 2-hour accommodation period before testing. After each experiment the arterial segments were carefully opened flat and stained with AgNO 3 to visualize the endothelium. 16 Only those with >70% of the endothelium were considered as normal segments. Segments from which the endothelium had been removed never showed more than 5% of their intima covered with endothelium either before or after the experiment. Concentration-response curves for vasopressin were determined in a cumulative manner, and control [in the absence of d(ch 2 ) 5 Tyr(Me)AVP] and experimental [in the presence of d(ch 2 ) 5 Tyr(Me) AVP] responses were obtained from different segments. The antagonist (10~ 8 or 10" 6 M) was added to the organ bath 15 minutes before the concentrationresponse curve was determined. Concentrations of vasopressin that produced 50% of the maximum contraction (EC 50 values) were determined for each arterial segment, and from these values the geometric means for EC 50 with 95% confidence intervals were calculated. 17 Responses to the cumulative addition of acetylcholine (10~ 7 to 10" 6 M) were determined in some arterial segments after submaximal 1O0On VASOPRESSIN (-logm) FIGURE 1. Cumulative concentration-response curves for vasopressin determined in human cerebral arterial segments with ( ) and without (o) endothelium. Data are shown as mean±sem. Number of experiments is given in parentheses. contractions induced by 10 6 to 3x10 6 M prostaglandin F 2a (PGF 2a ). Drugs used were acetylcholine chloride, arginine vasopressin, [/3-mercapto-/3,/3-cyclopentamethylenepropionylVO-Me-tyr^Arg 8 ] vasopressin [d(ch 2 ) 5 Tyr (Me)AVP], papaverine hydrochloride, and PGF 2a (Sigma Chemical Co., St. Louis). Drugs were dissolved in distilled water and added to the organ bath in volumes of <70 fil. Stock solutions of the drugs were freshly prepared every day. Data are expressed as mean±sem. The results were evaluated statistically by means of Student's t test. A probability value of less than 0.05 was considered significant. Results Cumulative application of vasopressin produced a constrictor response that was concentration-dependent (Figure 1). The maximum tension developed as well as the EC 50 were similar (/?>0.05) in arterial segments with and without endothelium (Table 1). There was no significant difference in the contraction TABLE 1. ECso Values and Maximal Responses of Vasopressin in Human Cerebral Artery Segments ECso (M) With endothelium Without endothelium With d(ch 2 ) 5 Tyr(Me)AVP. n Geometric mean 7.0X10" x10"' 95% confidence interval 2.6x10-' to 1.8x10"' 1.9X10" 10 to 9.2x10"' Maximal response (mg) 821 ± ±84 10" 8 M xlO" 8 * 1.5X10-8 to 6.8X *119 10" 6 M 7 1.9xlO" 6 t LOxlO" 6 to 3.5X ±75 *1p<0.05, 0.001, respectively, different from segments with or without endothelium by Student's t test.

3 Martin de Aguilera et al Vasopressin in Human Cerebral Arteries VP(-logM) VASOPRESSIN (-logm) FIGURE 2. Concentration-response curves for vasopressin determined in human cerebral arterial segments in the absence ( ) and presence of d(ch 2 ) 5 Tyr(Me)AVP(o, lo' 8 M; A, 10' 6 M). Values are shown as mean±sem. Number of experiments is given in parentheses. of segments with and without endothelium to the addition of 60 mm KC1 (904 ±325 versus 805 ±207 mg,p>0.05). Because they did not differ significantly, the concentration-response curves of arterial segments with and without endothelium were combined to determine the control curve for vasopressin. The presence of 10~ 8 M d(ch 2 ) 5 Tyr(Me)AVP in the organ bath displaced the control curve for vasopressin to the right, but differences in the maximal tensions developed were not significant (p>0.05) (Figure 2, Table 1). Increasing the concentration of d(ch 2 ) 5 Tyr(Me)AVP to 10" 6 M further displaced the control curve for vasopressin (Figure 2, Table 1). To determine whether vasopressin induces relaxation in previously contracted arteries, increasing concentrations of this peptide were added to nine segments precontracted with KC1. Figure 3 shows that vasopressin caused further contractions in both normal and endothelium-denuded arterial segments previously contracted with 60 mm KC1. In these segments the ability of the smooth muscle to relax was tested by adding 10" 4 M papaverine when maximal contractions had been attained with vasopressin. Under these conditions papaverine caused almost complete relaxation of both normal and endothelium-denuded segments (Figure 3). Acetylcholine was added during steady-state contractions produced by PGF 2o (Figure 4). Relaxation was observed in 14 of 20 normal arterial segments tested even though examination of whole-mount sections confirmed the presence of >80% of the endothelium in all 20 segments. In 12 rubbed arterial FIGURE 3. Contractile effects of vasopressin (VP) in human cerebral arterial segments with (above) and without (below) endothelium previously contracted with 60 mm KCl. Papaverine (PV, 10~ 4 M) caused almost complete relaxation of arterial segments. segments with >90% of the endothelial cell layer removed, acetylcholine had no effect on the contraction induced by PGF 2a. Discussion Our results indicate that vasopressin is a potent agonist for the contraction of cerebral smooth muscle through activation of specific V-l vasopressinergic receptors. The maximal tensions attained with vasopressin are similar to those induced with KCl and the EC 50 values for vasopressin are lower than those for 2-, Ach(-iogM) 6 Ach(-logM) 20t FIGURE 4. Contractile effects of acetylcholine (Ach) in human cerebral arterial segments with (above) and without (below) endothelium previously contracted with 10~ 6 M prostaglandin F 2a (PGF 2a ). W, washout.

4 1692 Stroke Vol 21, No 12, December hydroxytryptamine, angiotensin II, and norepinephrine in the same vascular preparations. 18 Our data also show that the vasopressin-induced contraction is not linked to the presence of an intact endothelium, thus indicating that vasodilator substances secreted by endothelial cells under basal, spontaneous conditions 19 do not counteract the potent contractile effects of vasopressin on smooth muscle cells. In agreement with our results, previous experiments have shown that vasopressin causes endothelium-independent contraction in human mesenteric arteries 20 and in the aorta and renal and carotid arteries of rats. 21 In contrast, in experiments in isolated basilar and circumflex coronary arteries from dogs, vasopressin caused concentration-dependent relaxations during contractions evoked by PGF 2<r ; after removal of the endothelium these inhibitory responses were significantly reduced. 22 These latter findings indicate that the relaxation induced by vasopressin in canine basilar and coronary arteries may be mediated by the activation of specific V-l vasopressinergic receptors on endothelial cells, resulting in the release of an endothelium-derived relaxing factor (EDRF). Conversely, we observed only constriction in human cerebral arteries, even when the segments were precontracted with KC1. At 60 mm KC1, one can assume that the vascular muscle was well depolarized. Presumably, vasopressin acts by increasing Ca 2+ influx into the muscle to above and beyond the influx produced by depolarization. Integrity of the endothelium had no effect on this response. Our results indicate the lack of specific V-l receptors on endothelial cells or the absence of an effective release of EDRF induced by vasopressin. Therefore, it is likely that the contractile effects of vasopressin on human cerebral arteries are due to direct stimulation of specific receptors located on smooth muscle cells. In addition to regional and species differences, size of the vessels used may be a factor in variations in the responses of these studies. The dog basilar and left circumflex coronary arteries used by Katusic et al 22 are large main arteries compared with the small human arteries that we analyzed. In relation to this, previous studies in anesthetized cats have shown that vasopressin selectively dilates large cerebral arteries (the circle of Willis and its major branches) and raises the resistance of small vessels. 23 In anesthetized goats vasopressin decreases blood flow in a branch of the middle cerebral artery. 24 It is concluded that vasopressin produces contraction only in small cerebral arteries, particularly those near the site at which extracranial vessels become intracranial vessels. 23 In contrast to the absence of a modulatory role for endothelium in the vasopressin-induced contraction, acetylcholine caused endothelium-dependent relaxation with a pattern similar to that originally described in most mammalian arteries The lack of relaxation in response to acetylcholine observed in cerebral vessels without endothelium does not reflect a nonspecific effect on smooth muscle cells since the responsiveness to K + and papaverine was similar in normal and endothelium-denuded arterial segments. The acetylcholine-induced relaxation in middle cerebral artery segments that we observed confirm similar findings in human basilar artery rings. 26 Antagonists of arginine vasopressin have been used to study the cardiovascular effects of vasopressin and to characterize the receptors involved. 27 d(ch 2 ) 5 Tyr(Me)AVP has been reported to be a potent inhibitor of the pressor responses to vasopressin in anesthetized rats Furthermore, this compound does not interfere with the vasoconstrictor response to angiotensin II or norepinephrine We demonstrate that d(ch 2 ) 5 Tyr(Me)AVP is an effective antagonist of the response to arginine vasopressin in human cerebral arteries. At 1(T 8 M, the antagonist produced a 60-fold shift to the right of the control concentration-response curve for vasopressin, presumably due to a competitive agonist-antagonist interaction. Our results parallel those previously observed in human mesenteric arteries using the same V-l vasopressin antagonist 20 and in human cerebral veins using dpvdavp, 30 another arginine vasopressin antagonist. 31 Our results are, however, somewhat different from those of Fox et al, 29 who reported the absence of a rightward shift of the concentration-response curves for arginine vasopressin in the rat tail artery after exposure to d(ch 2 ) 5 Tyr(Me)AVP. The absence of competitive antagonism in the latter report probably depended on the conditions of the experiments and the type of artery used. These factors are particularly relevant in the assessment of the vascular effects of vasopressin and emphasize the need for caution in extrapolating the effects from experimental animals to humans. Immunocytochemical studies have provided strong evidence for the role of arginine vasopressin as a putative neurotransmitter or neuromodulator within the brain. 32 The question of whether this neuropeptide can be released in amounts sufficient to influence arterial lumen or blood flow under physiologic or pathologic conditions is as yet hypothetical. Indeed, there are experimental observations showing that a sudden increase in intracranial pressure in cats, produced by either the injection of blood into the subarachnoid space or cerebral compression, leads to a rapid rise in the concentration of vasopressin in the blood. 33 It has also been reported that 24% of a series of 42 patients with subarachnoid hemorrhage had an increased concentration of vasopressin in either the plasma or the cerebrospinal fluid, and sometimes in both fluids. 34 Moreover, in patients with severe congestive heart failure, the plasma levels of arginine vasopressin can be up to 20 times normal. 35 Consequently, the cerebral vasoconstriction that we observed should be taken into consideration in certain pathophysiologic states in which vasopressin is released in amounts that could interfere with the blood supply to the brain.

5 Martin de Aguilera et al Vasopressin in Human Cerebral Arteries 1693 In conclusion, we demonstrate that vasopressin exerts a powerful constrictor action on isolated human cerebral arteries by direct stimulation of V-l vasopressin receptors predominantly located on smooth muscle cells. It appears that in human cerebral arteries the contractile response to vasopressin is not modulated by the presence of an intact endothelium. References 1. Altura BM, Altura BT: Vascular smooth muscle and neurohypophyseal hormones. Fed Proc 1977;36: Nakano J: Cardiovascular actions of vasopressin. Jpn Circ J 1973;37: Cowley AW Jr, Switzer SJ, Guinn MM: Evidence and quantification of the vasopressin arterial pressure control system in the dog. Circ Res 1980;46: Schwartz J, Reid IA: Effect of vasopressin blockade on blood pressure regulation during hemorrhage in conscious dogs. Endocrinology 1981;109: Michell RH, Kirk CJ, Billah MM: Hormonal stimulation of phosphatidylinositol breakdown, with particular reference to the hepatic effects of vasopressin. Biochem Soc Trans 1979;7: Penit J, Faure M, Jard S: Vasopressin and angiotensin II receptors in rat aortic muscle cells in culture. Am J Physiol 1983;244:E72-E82 7. Bichet DG, Razi M, Lonergan M, Arthus MF, Papukna V, Kortas C, Barjon JN: Hemodynamic and coagulation responses to 1-desamino 8-D-arginine vasopressin in patients with congenital nephrogenic diabetes insipidus. N Engl J Med 1988;318: Liard JF: Peripheral vasodilatation induced by a vasopressin analogue with selective V 2 -agonism in dogs. Am J Physiol 1989;256:H1621-H Schmid PG, Abboud FM, Wendling MG, Ramberg ES, Mark AL, Heistad DD, Eckstein JW: Regional vascular effects of vasopressin: Plasma levels and circulatory response. Am J Physiol 1974;227: Altura BM, Altura BT: Actions of vasopressin, oxytocin, and synthetic analogs on vascular smooth muscle. Fed Proc 1984; 43: Share L: Role of vasopressin in cardiovascular regulation. Physiol Rev 1988;68: Furchgott RF: Role of endothelium in responses of vascular smooth muscle. Circ Res 1983;53: Vanhoutte PM, Rubanyi GM, Miller VM, Houston DS: Modulation of vascular smooth muscle contraction by the endothelium. Annu Rev Physiol 1986;48: Kruszynsky M, Lammek B, Manning M: l-(/3-mercapto-/3,j3- ciclopentamethylenepropionic acid),2-(o-methyl)tyrosine arginine-vasopressin and l-(/3-mercapto-/3,/3-ciclopentamethylenepropionic acid)arginine-vasopressin, two highly potent antagonists of the vasopressor response to argininevasopressin. / Med Chem 1980;23: Liard JF, Spadone JC: Hemodynamic effects of antagonists of the vasoconstrictor action of vasopressin in conscious dogs. / Cardiovasc Pharmacol 1984;6: Caplan BA, Schwartz CJ: Increased endothelial cell turnover in areas of in vivo Evans blue uptake in the pig aorta. Atherosclerosis 1973;17: Fleming WW, Westfall DP, de La Lande IS, Jellet LB: Log-normal distribution of equieffective doses of norepinephrine and acetylcholine in several tissues. J Pharmacol Exp Ther 1972;181: Conde MV, Gomez B, Lluch S: Contractile responses of isolated human middle cerebral artery to vasoactive agents. / Cereb Blood Flow Metab 1981;l:S350-S Martin W, Furchgott F, Villani M, Jothianandan D: Depression of contractile responses in rat aorta by spontaneously released endothelium-derived relaxing factor. J Pharmacol Exp Ther 1986;237: Ohlstein EH, Berkowitz BA: Human vascular vasopressin receptors: Analysis with selective vasopressin receptor antagonists. J Pharmacol Exp Ther 1986;239: Katusic ZS, Krstic MK: Vasopressin causes endotheliumindependent contractions of the rat arteries. Pharmacology 1987;35: Katusic ZS, Shepherd JT, Vanhoutte PM: Vasopressin causes endothelium-dependent relaxations of the canine basilar artery. Circ Res 1984;55: Faraci FM, Mayhan WG, Schmid G, Heistad DD: Effects of arginine vasopressin on cerebral microvascular pressure. Am J Physiol 1988;255:H70-H Lluch S, Gomez B: Vasopressin and the cerebral circulation, in Edvinsson L, McCulloch J (eds): Peptidergic Mechanisms in the Cerebral Circulation. Chichester, England, Ellis Horwood Ltd, 1987, pp Furchgott RF, Zawadzki JV: The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980;288: Kanamaru K, Waga S, Fujimoto K, Itoh H, Kubo Y: Endothelium-dependent relaxation of human basilar arteries. Stroke 1989;20: Sawyer WH, Grzonka Z, Manning M: Neurohypophysial peptides. Design of tissue-specific agonists and antagonists. Mol Cell Endocrinol 1981;22: Pang CCY, Leighton KM: Prolonged inhibition of pressor response to vasopressin by a potent specific antagonist,l-(/3- mercapto-j3,/3-ciclopentamethylenepropionic acid),2-(omethyl)tyrosine arginine-vasopressin. Can J Physiol Pharmacol 1981;59: Fox AW, Karapanos G, Mitch WE: Noncompetitive blockade of vasopressin-1 receptors in rat tail artery. / Pharmacol Exp Ther 1987;243: Conde MV, Diez-Tejedor E, Frank A, Gomez B, Lluch S: Responses of isolated human cerebrocortical veins to norepinephrine and vasopressin. J Cereb Blood Flow Metab 1983;3: S522-S Manning M, Lowbridge J, Stier CT Jr, Haldar J, Sawyer WH: (l-deaminopenicillamine,4-valine)-8-d-arginine-vasopressin, a highly potent inhibitor of the vasopressor response to arginine-vasopressin. J Med Chem 1977;20: Buijs RM: Intra- and extrahypothalamic vasopressin and oxytocin pathways in the rat. Cell Tissue Res 1978;192: Rap CM, Chwalbinska-Moneta J: Vasopressin concentration in blood during acute short-term intracranial hypertension in cats. Adv Neurol 1978;20: Mather HM, Ang V, Jenkins JS: Vasopressin in plasma and SCF of patients with subarachnoid hemorrhage. J Neurosurg Psychiatry 1981;44: Nicod P, Waeber B, Bussien JP, Goy JJ, Turini G, Nussberger J, Hofbauer KG, Brunner HR: Acute hemodynamic effect of a vascular antagonist of vasopressin in patients with congestive heart failure. Am J Cardiol 1985;55: KEY WORDS vasopressins cerebral arteries endothelium, vascular