Acta Physiol Scand 1991, 142, 525-526
ADONIS
00016772910015 1L
Endothelium-independent vascular relaxation by histamine in the rabbit aorta
A.W E N N M A L M and A.-S. P E T E R S S O N Department of Clinical Physiology, University of Gothenburg, Gothenburg, Sweden
The obligatory role of the endothelial cells in the relaxation of arterial smooth muscle by acetylcholine was originally described by Furchgott & Zawadzki (1980). Subsequently, a number of vasodilators have been shown to operate by stimulating the formation of the so-called endothelial-derived relaxing factor (EDRF, see e.g. Bassenge & Busse 1988), later identified as nitric oxide (Palmer et al. 1987). Among these vasodilators are histamine, which was proposed to elicit endothelium-dependent relaxation in aortic ring segments from rat and rabbit (Van de Voorde & Leusen 1984). This observation of endotheliumdependent relaxation of the rabbit aorta to histamine has been cited frequently but-to our knowledge-not confirmed by others. Here we report that strips of rabbit aorta relax to histamine, even after rubbing of the endothelial surface. Helically cut strips of thoracic aorta, taken from 13 New Zealand rabbits (2.2-2.7 kg) of mixed sexes, were used for the experiments. They were mounted in 5 ml organ baths filled with aerated Tyrode solution kept at 37 "C. Isotonic (preload 2 g) contractions and relaxations of the strips were recorded via Harvard smooth muscle transducers on a Grass polygraph. In some strips the endothelial surface was gently rubbed with a forceps before the strip was mounted in the bath. Fifty ,d additions of acetylcholine (Sigma, final conc. 0.5-50 p ~ ) ,N-monomethyl-I-arginine (1-NMMA, Sigma, 50-100 PM), cimetidine (SKF, 0.2 mM), clemastine (Sandoz, 20 p ~ ) ,histamine , noradrenaline (Sigma, 0.1(Sigma, 10-100 p ~ )and 1 ,UM) were made directly to the bath. The strips were precontracted with 0.1-1 ,UM noradrenaline. Acetylcholine elicited dose-dependent relaxations in unrubbed strips, but contraction in strips with the endothelial layer rubbed (Fig. 1). Also in unrubbed strips treated with 1-NMMA acetylcholine induced contraction. In strips responding to acetylcholine (0.5-5 ,UM) with relaxation histamine Received 27 April 1991, accepted 1 May 1991. Key words: acetylcholine, endothelium, histamine, rabbit aorta. Correspondence : Dr Ake Wennmalm, Department of Clinical Physiology, Sahlgrenska Hospital, S-413 45 Goteborg, Sweden.
(10-50 p ~ also ) elicited relaxation (Fig. l), while higher concentrations of histamine in most cases caused a contraction. In strips responding to acetylcholine (0.5-50 ,UM) with contraction histamine (l& 100 p ~in)most cases caused relaxation (Fig. I), but occasionally contraction. Relaxation responses to histamine were reversed into contraction by addition of cimetidine (0.2 mM) to the bath, both in strips responding to acetylcholine with relaxation and in those responding to acetylcholine with contraction. The contractile responses to histamine were reversed into relaxation by clemastine , in strips responding to acetylcholine (20 p ~ ) both with relaxation and to those responding to acetylcholine with contraction.
Ach
Hist.
50wM
50pM
NA 0.01 @4
Fig. 1. Representative tracings of the isotonic responses of helically cut rabbit aortic strips to different agonists. The strips were precontracted with noradrenaline, after which acetylcholine was added. In the unrubbed strip acetylcholine elicited relaxation, indicating that the endothelium was intact. In the rubbed strip acetylcholine induced contraction, indicating absence of endothelium. In either of the strips histamine elicited a pronounced relaxation.
525
11I s cc s s 10s In the present experiments histamine elicited relaxation of rabbit aortic strips, both in those responding to acetj-lcholine with relaxation and in those responding to acet?-lcholine with contraction. Gentle rubbing of the intimal surface of vascular tissue, as performed in the present experiments, removes the endothelial cells (Furchgott & Zawadzki 1980) and thereby the ahilitj- ofthe \-essel \vall to form nitric oxide and to respond to acetj-lcholine a i t h relaxation. In the present experiments the responses of the rabbit aortic strips to acetylcholine, i.e. relaxation or contraction, nere used to judge whether the strips had an intact endothelium or not. Strips responding to acetylcholine with relaxation also responded to moderate doses of histamine uith relaxation, Such a response might be taken to indicate that histamine stimulated endothelium-dependent formation of nitric oxide, therebj- causing relaxation of the strip. Howeier, strips responding to acetylcholine with contraction - i.e. strips in which the endothelial cells had been rubbed off also relased to histamine. This observation does certainl!- not disfivour the esistence of endothelium-dependent relaxation to histamine in the rabbit aorta per se; it does, hoa-e\er, strong11 indicate that endothelium-independent relaxation to histamine ma! (also) occur in the tissue. Addition of cimetidine to the organ bath reversed the relaxation response to histamine to a contraction, indicating that the tascular smooth muscle receptor responsible for the relaxation was of the €I,-type. The contractile response to histamine (100 pv) was blocked by clemastine, indicating that it was caused b!- activation of H,-receptors in the \-essel uall. The present esperiments are in contrast to those discussed above (\-an de Yoorde & Ixusen 1984) in which only contractile responses to histamine were obtained after endothelial denudation. Our results do not, however, not allow an!- conclusions as to whether histamine stimulates endothelial formation of nitric oxide or not. Recent11 Nishimoto rt ul. (1990) reported that histamine caused concentration dependent contractions in rabbit aorta, and furthermore, that these contractions werc potentiated b-! endothelial denuding or b! diphenhj-dramine, H,-receptor antagonist. They suggested that stimulation of the release of E D R F in response to histamine caused suppression of phosphoinositide turnor-er and contraction. These data consequentl! support the concept that the formation of -
nitric oxide in the vascular endothelium is stimulated by histamine. I Ioweier, Nishimoto et al. (1990) also reported that in aortic strips pretreated with diphenhydramine-an HI-receptor antagonist-and precontracted with phenylephrine addition of histamine produced relasation irrespectivelq- of whether the strips \yere de-endothelialized or not. These data are in harmonj- with those presently reported, i.e. that the aortic strip can relax to histamine even in the absence of EDRF. These authors were, however, not able to demonstrate relaxation to histamine in denuded strips unless the HI-receptors had been blocked. Our data consequentlj- estend those by Nishimoto et ill. (199O), by demonstrating that the primarj- response of de-endothelialized rabbit aortic strips may be H,receptor mediated dilatation. T h e fact that some denuded strips responded with contraction to histamine-before addition of clemastine--indicates that the affinitq- balance between the Hl- and 11,receptors in the vascular smooth muscle map differ between different strips. Rabbit aortic strips or rings, precontracted with xadrenergic agonists, are frequently used in bioassay experiments for analysis of endothelium-dependent relasation to different agents. The present experiments clearly demonstrate that in such experiments the occurrence of histamine-induced relaxation cannot be taken to indicate that the vascular endothelium is patent. Supported b! the Swedish Medical Research Council (project 4311).
REFERENCES BASSESGE E. & BCSSE,R. 1988. Endothelial modulation of coronarl- tone. Prugr Crrrdiocosc. Dis 30, 319-380 FURCHGOTT, R.F. & ZAWADZKI,J.V. 1980. T h e obligator!. role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Xuture
288, 3’73-376 NISHISIOTO, T., YOKOYAMA, M. & FLKUZ-IKI, 11. 1990. Self suppression of phosphoinositide turnover and contraction by stimulating release of endogenous endothelium derived relaxing factor in vascular action of histamine, Curdiorasc Rcs 24, 364-372. P.ALSiER, R.M.J., FERRIGE, A.G. & MONCADA, S. 1987. Nitric oxide accounts for the biological activity of endothelium-derived relaxing factor. N u t i m 327, 5ZC.526. Y.IS DE YOORDE,J. & LEUSEN,I . 1984. Effect of histamine on aorta preparations of different species. .4rc/z Irit Pharmacodyn 268, 95-105.
ADONIS
00016772910015 1L
Endothelium-independent vascular relaxation by histamine in the rabbit aorta
A.W E N N M A L M and A.-S. P E T E R S S O N Department of Clinical Physiology, University of Gothenburg, Gothenburg, Sweden
The obligatory role of the endothelial cells in the relaxation of arterial smooth muscle by acetylcholine was originally described by Furchgott & Zawadzki (1980). Subsequently, a number of vasodilators have been shown to operate by stimulating the formation of the so-called endothelial-derived relaxing factor (EDRF, see e.g. Bassenge & Busse 1988), later identified as nitric oxide (Palmer et al. 1987). Among these vasodilators are histamine, which was proposed to elicit endothelium-dependent relaxation in aortic ring segments from rat and rabbit (Van de Voorde & Leusen 1984). This observation of endotheliumdependent relaxation of the rabbit aorta to histamine has been cited frequently but-to our knowledge-not confirmed by others. Here we report that strips of rabbit aorta relax to histamine, even after rubbing of the endothelial surface. Helically cut strips of thoracic aorta, taken from 13 New Zealand rabbits (2.2-2.7 kg) of mixed sexes, were used for the experiments. They were mounted in 5 ml organ baths filled with aerated Tyrode solution kept at 37 "C. Isotonic (preload 2 g) contractions and relaxations of the strips were recorded via Harvard smooth muscle transducers on a Grass polygraph. In some strips the endothelial surface was gently rubbed with a forceps before the strip was mounted in the bath. Fifty ,d additions of acetylcholine (Sigma, final conc. 0.5-50 p ~ ) ,N-monomethyl-I-arginine (1-NMMA, Sigma, 50-100 PM), cimetidine (SKF, 0.2 mM), clemastine (Sandoz, 20 p ~ ) ,histamine , noradrenaline (Sigma, 0.1(Sigma, 10-100 p ~ )and 1 ,UM) were made directly to the bath. The strips were precontracted with 0.1-1 ,UM noradrenaline. Acetylcholine elicited dose-dependent relaxations in unrubbed strips, but contraction in strips with the endothelial layer rubbed (Fig. 1). Also in unrubbed strips treated with 1-NMMA acetylcholine induced contraction. In strips responding to acetylcholine (0.5-5 ,UM) with relaxation histamine Received 27 April 1991, accepted 1 May 1991. Key words: acetylcholine, endothelium, histamine, rabbit aorta. Correspondence : Dr Ake Wennmalm, Department of Clinical Physiology, Sahlgrenska Hospital, S-413 45 Goteborg, Sweden.
(10-50 p ~ also ) elicited relaxation (Fig. l), while higher concentrations of histamine in most cases caused a contraction. In strips responding to acetylcholine (0.5-50 ,UM) with contraction histamine (l& 100 p ~in)most cases caused relaxation (Fig. I), but occasionally contraction. Relaxation responses to histamine were reversed into contraction by addition of cimetidine (0.2 mM) to the bath, both in strips responding to acetylcholine with relaxation and in those responding to acetylcholine with contraction. The contractile responses to histamine were reversed into relaxation by clemastine , in strips responding to acetylcholine (20 p ~ ) both with relaxation and to those responding to acetylcholine with contraction.
Ach
Hist.
50wM
50pM
NA 0.01 @4
Fig. 1. Representative tracings of the isotonic responses of helically cut rabbit aortic strips to different agonists. The strips were precontracted with noradrenaline, after which acetylcholine was added. In the unrubbed strip acetylcholine elicited relaxation, indicating that the endothelium was intact. In the rubbed strip acetylcholine induced contraction, indicating absence of endothelium. In either of the strips histamine elicited a pronounced relaxation.
525
11I s cc s s 10s In the present experiments histamine elicited relaxation of rabbit aortic strips, both in those responding to acetj-lcholine with relaxation and in those responding to acet?-lcholine with contraction. Gentle rubbing of the intimal surface of vascular tissue, as performed in the present experiments, removes the endothelial cells (Furchgott & Zawadzki 1980) and thereby the ahilitj- ofthe \-essel \vall to form nitric oxide and to respond to acetj-lcholine a i t h relaxation. In the present experiments the responses of the rabbit aortic strips to acetylcholine, i.e. relaxation or contraction, nere used to judge whether the strips had an intact endothelium or not. Strips responding to acetylcholine with relaxation also responded to moderate doses of histamine uith relaxation, Such a response might be taken to indicate that histamine stimulated endothelium-dependent formation of nitric oxide, therebj- causing relaxation of the strip. Howeier, strips responding to acetylcholine with contraction - i.e. strips in which the endothelial cells had been rubbed off also relased to histamine. This observation does certainl!- not disfivour the esistence of endothelium-dependent relaxation to histamine in the rabbit aorta per se; it does, hoa-e\er, strong11 indicate that endothelium-independent relaxation to histamine ma! (also) occur in the tissue. Addition of cimetidine to the organ bath reversed the relaxation response to histamine to a contraction, indicating that the tascular smooth muscle receptor responsible for the relaxation was of the €I,-type. The contractile response to histamine (100 pv) was blocked by clemastine, indicating that it was caused b!- activation of H,-receptors in the \-essel uall. The present esperiments are in contrast to those discussed above (\-an de Yoorde & Ixusen 1984) in which only contractile responses to histamine were obtained after endothelial denudation. Our results do not, however, not allow an!- conclusions as to whether histamine stimulates endothelial formation of nitric oxide or not. Recent11 Nishimoto rt ul. (1990) reported that histamine caused concentration dependent contractions in rabbit aorta, and furthermore, that these contractions werc potentiated b-! endothelial denuding or b! diphenhj-dramine, H,-receptor antagonist. They suggested that stimulation of the release of E D R F in response to histamine caused suppression of phosphoinositide turnor-er and contraction. These data consequentl! support the concept that the formation of -
nitric oxide in the vascular endothelium is stimulated by histamine. I Ioweier, Nishimoto et al. (1990) also reported that in aortic strips pretreated with diphenhydramine-an HI-receptor antagonist-and precontracted with phenylephrine addition of histamine produced relasation irrespectivelq- of whether the strips \yere de-endothelialized or not. These data are in harmonj- with those presently reported, i.e. that the aortic strip can relax to histamine even in the absence of EDRF. These authors were, however, not able to demonstrate relaxation to histamine in denuded strips unless the HI-receptors had been blocked. Our data consequentlj- estend those by Nishimoto et ill. (199O), by demonstrating that the primarj- response of de-endothelialized rabbit aortic strips may be H,receptor mediated dilatation. T h e fact that some denuded strips responded with contraction to histamine-before addition of clemastine--indicates that the affinitq- balance between the Hl- and 11,receptors in the vascular smooth muscle map differ between different strips. Rabbit aortic strips or rings, precontracted with xadrenergic agonists, are frequently used in bioassay experiments for analysis of endothelium-dependent relasation to different agents. The present experiments clearly demonstrate that in such experiments the occurrence of histamine-induced relaxation cannot be taken to indicate that the vascular endothelium is patent. Supported b! the Swedish Medical Research Council (project 4311).
REFERENCES BASSESGE E. & BCSSE,R. 1988. Endothelial modulation of coronarl- tone. Prugr Crrrdiocosc. Dis 30, 319-380 FURCHGOTT, R.F. & ZAWADZKI,J.V. 1980. T h e obligator!. role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Xuture
288, 3’73-376 NISHISIOTO, T., YOKOYAMA, M. & FLKUZ-IKI, 11. 1990. Self suppression of phosphoinositide turnover and contraction by stimulating release of endogenous endothelium derived relaxing factor in vascular action of histamine, Curdiorasc Rcs 24, 364-372. P.ALSiER, R.M.J., FERRIGE, A.G. & MONCADA, S. 1987. Nitric oxide accounts for the biological activity of endothelium-derived relaxing factor. N u t i m 327, 5ZC.526. Y.IS DE YOORDE,J. & LEUSEN,I . 1984. Effect of histamine on aorta preparations of different species. .4rc/z Irit Pharmacodyn 268, 95-105.
