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Involvement of NK receptors and β-adrenoceptors in nitric oxide-dependent relaxation of rabbit aorta rings following electrical-field stimulation
European Journal of Pharmacology, 238 (1993) 105-109
105
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 21255
Short communication
Involvement of NK receptors and/3-adrenoceptors in nitric oxide-dependent relaxation of rabbit aorta rings following electrical-field stimulation P a o l a Persico, A n t o n i o C a l i g n a n o , F r a n c a M a n c u s o and L u d o v i c o S o r r e n t i n o Department of Experimental Pharmacology, University of Naples 'Federico H', Via D. Montesano 49, 80131 Naples, Italy Received 15 April 1993, accepted 27 April 1993
Electrical-field stimulation caused an endothelium-dependent relaxation in rabbit aorta rings precontracted by phenylephrine. The relaxation was reduced in a dose-dependent manner by morphine, benzalkonium, [D-Pro2,D-TrpT'9]substance P and an fl-adrenoceptor antagonist, propranolol. The vasodilatation was enhanced by superoxide dismutase and abolished by haemoglobin and NG-monomethyl-L-arginine. The inhibitory effect of N°-monomethyl-L-arginine was reversed by L-arginine, the precursor of nitric oxide biosynthesis, but not by its enantiomer, D-arginine. These data show that the electrically induced relaxation is independent on nitric oxide released by NK receptors and fl-receptors. Moreover, morphine, by reducing substance P release, decreased the magnitude of electrically induced relaxation, suggesting an indirect role of opioids in the regulation of the peripheral circulation through the control of nitric oxide release. Furthermore our observations confirm the hypothesis that subtypes of/3-adrenoceptors releasing nitric oxide participate in the regulation of vascular tone. Substance P; Nitric oxide (NO); Morphine; Substance P receptor antagonists;/3-Adrenoceptors
1. Introduction
Substance P is a neuropeptide localized in the nerve terminals of primary sensory neurons and implicated in neurogenic inflammation and nociception (Payan, 1989). This peptide is able to cause in vivo vasodilatation and plasma extravasation through NK 1 receptors localized on the endothelium, causing an endothelium-dependent relaxation of arterial smooth muscle (Gulati et al., 1987). Endothelial cells react to mechanical or electrical stimuli as well as to various neurohumoral mediators by releasing vasodilator a n d / o r vasoconstrictor substances and, as such, contribute to the control of local vascular tone. The relaxation caused by substance P is due to a diffusible factor released by endothelial cells (Whittle et al., 1989). This diffusible factor named endothelium-derived relaxing factor ( E D R F ) (Furchgott and Zawadzki, 1980) is identical to nitric oxide (NO) derived from the terminal guanidino nitrogen atom of L-arginine (Palmer et al., 1988). It has been shown that opioids inhibit the neuro-
Correspondence to: A. Calignano, Department of Experimental Pharmacology, University of Naples 'Federico II', via D. Montesano 49, 80131 Naples, Italy. Tel. (39)-81-7486441, fax (39)-81-7486403.
genic plasma extravasation following antidromic stimulation of the saphenous nerve (Barthb and Szolcanyi, 1981; Lembeck and Gamse, 1982). Thus the existence of endogenous opioid activity controlling substance P release from the peripheral nerve endings has been postulated (Barthb and Szolcanyi, 1981). In the present study, the rabbit isolated aorta ring precontracted with phenylephrine was electrically stimulated, resulting in endothelium- and NO-dependent relaxation that was studied and characterized. Our study showed that the electrically induced relaxation is mainly mediated by substance P release and that opioids regulating this process could participate indirectly in the modulation of vascular tone through the control of NO generation.
2. Materials and methods
Albino male New Zealand rabbits (1.5-2 kg body weight) were anesthetized with pentobarbital sodium (30 m g / k g i.p.) and killed by exsanguination. The descending aorta was rapidly dissected and placed in Krebs solution gassed with 95% 02-5% CO 2 at room temperature and loose connective tissue was removed. Arterial segments were cut into rings (4-6 mm in
106 length), which w e r e s u s p e n d e d in 10-ml o r g a n baths filled with w a r m (37°C) o x y g e n a t e d Krebs solution and c o n n e c t e d to i s o m e t r ic t r a n s d u c e r s (U. Basile, Italy). A f t e r 30 min of e q u i l i b r a t i o n u n d e r a t e n s io n o f 1.5 g, the rings w e r e c o n t r a c t e d with p h e n y l e p h r i n e (10 -6 M). In s o m e e x p e r i m e n t s the e n d o t h e l i u m was rem o v e d by gently r u b b i n g the luminal surface with a c o t t o n swab m o i s t e n e d with K r e b s solution. E n d o t h e lium a b s e n c e was c o n f i r m e d by the lack of relaxation in r e s p o n s e to a c e t y i c h o l i n e (10 -6 M) a d d e d d u r i n g p h c n y l e p h r i n e - e v o k e d c o n t r a c t i o n . Rings w e r e l o c a t e d b e t w e e n p l a t i n u m e l e c t r o d e s p l a c e d at e i t h e r side of the vessel an d p e r i v a s c u l a r nerves w e r e s t i m u l a t e d by 0.5-ms pulses d e l i v e r e d at 0.4 Hz at 50 V for 1 min. N~;-Monomethyl-L-arginine and capsaicin were a d d e d to the bath 20 min b e f o r e starting stimulation, w h e r e a s all o t h e r drugs w e r e a d d e d 5 min b e f o r e e l e c t r i c a l - f i e l d stimulation. Cimetidine hydrochloride, NG-monomethyl-L-arginine, a t r o p i n e sulfate, acetylsalicylic acid, n a l o x o n e h y d r o c h l o r i d e , s u b s t a n c e P a c e t a t e salt, capsaicin, pen-
tobarbital sodium, b e n z a l k o n i u m h y d r o c h l o r i d e , Lp h e n y l c p h r i n e h y d r o c h l o r i d e , acct y l ch o l i n c hydrochloride, [D-Pro2,D-TrpT"~]substance P a c e t a t e salt, tetrodotoxin, p r o p r a n o l o l h y d r o c h l o r i d e , a l p r e n o l o l hyd r o c h l o r i d e , o x p r c n o l o l h y d r o c h l o r i d c , L - a r g i n i n e hyd r o c h l o r i d e , D - a r g i n i n c h y d r o c h l o r i d e , bovine erythrocyte s u p e r o x i d e dismutase, y o h i m b i n c h y d r o c h l o r i d e , g u a n e t h i d i n e sulfate and h a e m o g l o b i n w e r e o b t a i n e d from Sigma (St. Louis, M O , U S A ) ; m e p y r a m i n e m a l c a t e was o b t a i n e d from R h o n e Poulenc; m e t h y s e r g i d e was o b t a i n e d from Sandoz, and m o r p h i n e h y d r o c h l o r i d c was o b t a i n e d from S . A . L . A . R . S . (Italy). T h e results arc e x p r e s s e d as m e a n s + S.E.M. and w e r e analyzed for statistical significance with S t u d e n t ' s t-test. Significan ce was set at P < 0.01.
3. Results E l e c t r i c a l st i m u l at i o n o f p h e n y l e p h r i n e (10 -6 M)c o n t r a c t e d rabbit ao r t a rings r e s u l t e d in relaxation
TABLE 1 Effect of morphine (MOR), benzalkonium (BEN), [D-Pro2,D-TrpV'9]substance P (DD-SAP), propranolol (PRO), alprenolol (ALP), oxprcnolol (OXP) and capsaicin (CAP) on electrically induced relaxation of phenylephrine (10 -6 M)-contracted aorta rings. The values arc means _+S.E.M.; " P < (I.01 vs. control. Drugs
Concentration
% relaxation
Control
-
45.0 + 2.5
MOR
10 7M 7.0× 10 -7 M 1.4x 10 -6 M
31.2+2.5 ~ 23.7_+ 1.7 " 18.2_+ 1.0 ~'
28.5_+2.8 47.3_+2.4 59.6_+ 1.3
12 12 12
BEN
2.0x 10 -~' M
20.2_+2.3 "
55.0+2.7
5
DD-SAP
10 -6 M
18.2_+ 1.0 :'
59.0_+0.7
12
MOR + BEN
1.4× 10 -~' M 2.0×10 ~'M
20.1_+1.1 ~'
55.3-+1.1
12
1.4x10 6 M 10 -6 M
20.2_+ 1.3 ~'
55.2_+ 1.7
12
PRO
10 -5 M 8.0x 10 -5 M
30.1 -+ 1.2 " 22.5_+2.3 "
30.4_+ 1.3 51.0-+2.2
12 12
ALP
10 -5 M 8.0× 10 -s M
32.7_+2.4 a 22.9_+ 1.7 a
27.3_+ 1.4 49.4_+ 1.6
12 12
OXP
10 5 M 8.0×10 5 M
30.8_+ 1.5 ~ 22.5+2.3 a
32.1 _+1.8 50.6_+2.1
12 12
MOR + PRO
1.4×10 -s M 8.0x 10 -5 M
0 ~'
100
12
2.0 × 10- n M 8.0×10 5 M
0 ~'
100
12
0a
100
12
MOR+DD-SAP
BEN + PRO DD-SAP+ PRO
10 - 6 M 8.0X 10 -5 M
CAP
10 -5 M
CAP + DD-SAP
% reduction
n 24
27.4_+2.4 a
40.1 _+2.7
6
22.5_+2.8 "
50.2_+3.5
6
1(I-5 M 10 - 6 M
107
A
ES
~S
~S
L-NMMA E~
MOR
l.~b ESI
MOR +NAL
Fig. 1. (A) Effect of NC'-monomethyl-L-arginine (L-NMMA, 3 x 10 -4 M) and haemoglobin (Hb, 10 -4 M) on electrical-field stimulation (ES) of rabbit aorta rings precontracted with phenylephrine (10 -6 M) (trace representative of six experiments for L - N M M A and 10 experiments for Hb). (B) Effect of morphine (MOR, a = 1.4× 10 -6 M, b = 7 x 10 -7 M, c = 10 -7 M) and antagonism by naloxone (NAL, 5 x 10 -7 M) one electrical-field stimulation (ES) of rabbit aorta rings precontracted with phenylephrine (10 -6 M) (trace representative of 12 experiments for M O R and 5 experiments for M O R + NAL).
(45.3 + 2.5%, n = 24) observed only when the endothelium was intact. When the endothelium was removed by rubbing the luminal surface of aorta rings, no relaxation was obtained on electrical stimulation. N GMonomethyl-L-arginine (L-NMMA), added 20 min before electrical stimulation caused a concentration-dependent inhibition of the electrically induced relaxation (18 5- 1.7% by 10 -6 M, 70 + 1.5% by 10 -5 M and 100% by 3 x 10 -4 M, n = 6). Haemoglobin (10 -4 M, n = 10) addition to the Krebs solution 5 min before electrical stimulation completely abolished the electrically induced relaxation (fig. 1A). On the other hand, superoxide dismutase (70 U / m i ) addition increased the relaxant effect by 59.1 5- 3.1% (n = 6). The electrically induced vasorelaxation was markedly reduced by tetrodotoxin (10 -6 M, n = 5, 89 + 2.1%). Similar results were obtained with guanethidine (10 -5 M, n = 5, data not shown). Benzalkonium (2 x 10 -6 M) and [DPro2,D-Trp7'9]substance P (10 -6 M) completely abolished the small relaxation obtained in the presence of tetrodotoxin (10 -6 M, n = 6). Morphine addition 5 min before electrical stimulation caused a concentrationdependent inhibition of the electrically induced relaxation (table 1, fig. 1B). The morphine effect was reversed by concomitant administration of naloxone (5 x 10 -7 M, n = 5) (fig. IB). A non-significant increase of contraction was observed after naloxone administration (data not shown). The maximum morphine effect (59.6 + 1.3%, n = 12) was observed at 1.4 x 10 -6 M and a higher morphine concentration did not produce a greater inhibitory effect (data not shown). In order to further characterize the mechanisms involved in the electrically induced relaxation, the effect of several compounds were investigated in subsequent experi-
ments. Methysergide ( 1 0 - 7 - 1 0 -5 M, n = 5), mepyramine ( 1 0 - 7 - 1 0 -5 M, n = 5), cimetidine ( 1 0 - 7 - 1 0 -5 M, n = ) , atropine ( 1 0 - 9 - 1 0 -7 M, n = 5 ) , acetylsalicylic acid ( 1 0 - 7 - 1 0 -4 M, n = 5) or yohimbine (5 x 10 -7 M, n = 5) did not affect the electrically induced relaxation. Benzalkonium (2 x 10 -6 M) and [D-Pro2,D-Trp7'9]sub stance P (10 -6 M) inhibited the electrically induced relaxation by 55 5-2.7 and 59 + 0.7% respectively (table 1). Moreover, benzalkonium and [D-ProZ,D-Trp7'9]sub stance P did not significantly modify the inhibitory effect of morphine (table 1). The /3-adrenoceptor antagonists, propranolol (10 -5 and 8 x 10 -5 M), alprenolol (10 -S and 8 x 10 -5 M) and oxprenolol (10 -5 and 8 x 10 -5 M) added to the organ bath were equivalent for reducing the electrically induced relaxation in a concentration-dependent manner. A combination of propranolol (8 x 10 -5 M) with morphine (1.4 x 10 -6 M), benzalkonium (2 x 10 -6 M) or [D-Pro2,D Trp7"9]substance P (10 -6 M) completely abolished the electrically induced relaxation (table 1). Capsaicintreated (10 -5 M) aorta rings showed a reduction of 40 + 2.7% (n = 6) of electrically induced vasorelaxation, but a slight reduction, 10 5- 3% (n = 6, P < 0.05), could still be obtained with [D-Pro2,D-Trp7'9]substance P (10 -6 M) addition. A combination of capsaicin (10 -5 M) with [D-Pro2,D-Trp7"q]substance P (10 -6 M) resulted in a 50 + 3.5% reduction of electrically induced vasorelaxation (table 1).
4. D i s c u s s i o n
Considerable evidence indicates that electrical stimulation of vessels releases neurotransmitters from perivascular nerve endings (Rand and Varma, 1970). Therefore these substances may play a key role in the regulation of vascular tone and blood flow. Recently a number of vasodilators have been shown to evoke their responses by stimulating the production of endothelium-derived relaxing factor ( E D R F ) in endothelial ceils. These substances include acetylcholine, substance P, adenosine 5-triphosphate, adenosine 5-diphosphate, bradykinin, thrombin, histamine and epinephrine (Furchgott, 1983). We now report that electrical-field stimulation of rabbit aorta rings precontracted with phenylephrine leads to an endotheliumdependent and tetrodotoxin-sensitive vasorelaxation. This suggests that the nerve endings and endothelium may play an important role in the regulation of vascular tone in this experimental model. The electrically induced vasorelaxation could be enhanced by superoxide dismutase and completely prevented by haemoglobin or N°-monomethyl-L-arginine (L-NMMA), suggesting that the relaxing effect is totally due to NO release. It is well known that haemoglobin itself con-
108 tains a prosthetic heme group which binds nitric oxide, thus effectively removing nitric oxide from solution. NG-Monomethyl-L-arginine has been shown to inhibit NO synthase in endothelial cells (Moncada et al., 1986). Furthermore the data obtained in the present study using superoxide dismutase support the hypothesis that the relaxing effect is NO-dependent. Morphine addition reduced the electrically induced relaxation in a concentration-dependent manner. The maximum reduction (60%) was observed at 1.4 × 10 -6 M, and did not increase with a higher concentration of morphine. These results suggest that opioids regulate the release of NO-releasing substance(s). The electrically induced relaxation was also reduced by the substance P antagonists, benzalkonium (Piotrowski et al., 1987) or [D-Pro2,D-TrpT'9]substance P, in a concentration-dependent manner. It is of interest that the maximal reduction observed with substance P antagonists was of the same magnitude as the maximal morphineinduced reduction. Moreover the morphine inhibition was not increased by the concomitant presence of benzalkonium or [D-Pro2,D-TrpT'9]substance P. Substantial evidence indicates that opioids regulate substance P release from nerve endings (Jesel and lversen, 1977). Substance P is a ncuropeptide localised in the nerve terminals of primary sensory neurons, and is implicated in neurogenic inflammation and nociccption (Payan, 1989). This peptide is also Iocalised in the endothelial cells lining the arterial wall (Loesch and Burnstock, 1988). Substance P causes, via NK 1 receptors localized on the endothelial ceils, and endotheiium-dependent relaxation of arterial smooth muscle (Loesch and Burnstock, 1988; Gulati et al., 1987). The data obtained with tetrodotoxin indicate a neuronal origin of transmitters that cause NO release. The inhibition obtained with tetrodotoxin was extremely strong but a slight relaxation was still observed. This relaxation was abolished by substance P antagonists, thus we cannot exclude the participation of endothelial pools of substance P in addition to neuronal pools. This hypothesis was confirmed by the capsaicin data. These observations show that the endothelial pools of substancc P also could be released by electrical-field stimulation. Morphine or substance P antagonists reduced the electrically induced relaxation by only 60%. We have found that fl-adrenoceptor antagonists like propranolol, oxpranolol and alprenolol reduced the remaining 40% of electrically induced relaxation. A combination of morphine plus fl-adrenoceptor blockers or substance P antagonists plus /3-adrenoceptor blockers completely abolished the electrically induced relaxation. Recently it has been reported that, in rat thoracic aorta, isoprenaline causes an endothelium-dependent and N c,-nitro-L-arginine-sensitive vasorelaxation associated with increases of cGMP (Gray and Marshall,
1991). Our data are in agreement with this report indicating the presence of fl-adrenoceptors that release NO. We also could exclude the participation of a2-adrenoceptors that are well known to be involved in NO release, because yohimbine did not modify the electrically induced relaxation. The guanethidine data are not consistent with our hypothesis that substance P could be a major neurotransmitter released in electrically induced vasorelaxation. A possible interpretation of the data is that the concentration of guanethidine used (10 -5 M) was high enough to have a local anaesthetic action (Maxwell and Wastila, 1977) and therefore block nerve conduction in the sensory as well as adrenergic nerves. In conclusion, the electrical-field stimulation of rabbit aorta rings precontracted by phenylephrine causes a vasorelaxation that was endothelium- and NO-dependent. Our data suggest that substance P releases NO through the activation of NK-P receptors. Morphine, by reducing substance P release, decreased the magnitude of the electrically induced relaxation, suggesting an indirect role of opioids in the regulation of the peripheral circulation through the control of nitric oxide release, fl-Adrenoceptors also seem to be involved in NO release. This observatkm supports the hypothesis that in some vascular tissues such as rat thoracic aorta activation of fl-adrenoceptors could release NO.
References Barthb, L. and L. Szolcsanyi, 1981, Opiate agonists inhibit neurogenie plasma extravasation in the rat, Eur. J. Pharmacol. 73, 101. Furchgott, R.F., 1983, Role of endothelium in responses of vascular smooth muscle, Circ. Res. 53, 557. Furchgott, R.F. and J.V. Zawadzki, 1980, The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature 288, 373. Gray, D.W. and I. Marshall, 1991, Isoprenaline relaxation of rat thoracic aorta is endothelium-dependent, releases nitric oxide and raises cyclic GMP and cyclic AMP, Br. J. Pharmacol. 102, Proc. Suppl. 125P. Gulati, N., R. Mathison, H. Huggel, D. Regoli and J.L. B6ny, 1987, Effects of neurokinins on the isolated pig coronary artery, Eur. J. Pharmacol. 137, 149. Jesel, T.M. and L.L. Iversen, 1977, Opiate analgesics inhibit substance P release from rat trigeminal nucleus, Nature 268, 549. Lembeck, F. and R. Gamse, 1982, Substance P in peripheral sensory processes, in: Substance P in the Nervous System, eds. R. Porter and M. O'Connor (Pitman, London) p. 35. Loesch, A. and G. Burnstock, 1988, UItrastructural localization of serotonin and substance P in vascular endothelial cells of rat femoral and mesenteric arteries, Anat. Embriol. 178, 137. Maxwell, R.A. and W.B. Wastila, 1977, Adrenergic neuron blocking drugs, in: Hand. Exp. Pharm. XXXIX, ed. F. Gross (SpringerVerlag, Heidelberg) p. 161. Moncada, S., R.M.J. Palmer and R.J. Gryglewski, 1986, Mechanism of action of some inhibitors of endothelium-derived relaxing factor, Proc. Natl. Acad. Sci. U.S.A. 83, 9164.
109 Payan, D.G.M.D., 1989, Neuropeptides and inflammation: the role of substance P, Ann. Rev. Med. 40, 341. Palmer, R.M.J., D.S. Ashton and S. Moncada, 1988, Vascular endothelial cells synthesize nitric oxide from L-arginine, Nature 333, 664. Piotrowski, W., M. Mead and J.C. Foreman, 1987, Action of the SAP2_11 and SAP3.11 fragments of substance P on rat peritoneal mast cells, Agents Actions 20, 178.
Rand, M.J. and B. Varma, 1970, The effects of cholinomimetic drugs on responses to sympathetic nerve stimulation and noradrenaline in the rabbit ear artery, Br. J. Pharmacol. 38, 758. Whittle, B.J.R., J. Lopez-Belmonte and D.D. Rees, 1989, Modulation of the vasodepressor actions of acetylcholine, bradykinin, substance P and endothelin in the rat by a specific inhibitor of nitric oxide formation, Br. J. Pharmacol. 98, 646.
105
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 21255
Short communication
Involvement of NK receptors and/3-adrenoceptors in nitric oxide-dependent relaxation of rabbit aorta rings following electrical-field stimulation P a o l a Persico, A n t o n i o C a l i g n a n o , F r a n c a M a n c u s o and L u d o v i c o S o r r e n t i n o Department of Experimental Pharmacology, University of Naples 'Federico H', Via D. Montesano 49, 80131 Naples, Italy Received 15 April 1993, accepted 27 April 1993
Electrical-field stimulation caused an endothelium-dependent relaxation in rabbit aorta rings precontracted by phenylephrine. The relaxation was reduced in a dose-dependent manner by morphine, benzalkonium, [D-Pro2,D-TrpT'9]substance P and an fl-adrenoceptor antagonist, propranolol. The vasodilatation was enhanced by superoxide dismutase and abolished by haemoglobin and NG-monomethyl-L-arginine. The inhibitory effect of N°-monomethyl-L-arginine was reversed by L-arginine, the precursor of nitric oxide biosynthesis, but not by its enantiomer, D-arginine. These data show that the electrically induced relaxation is independent on nitric oxide released by NK receptors and fl-receptors. Moreover, morphine, by reducing substance P release, decreased the magnitude of electrically induced relaxation, suggesting an indirect role of opioids in the regulation of the peripheral circulation through the control of nitric oxide release. Furthermore our observations confirm the hypothesis that subtypes of/3-adrenoceptors releasing nitric oxide participate in the regulation of vascular tone. Substance P; Nitric oxide (NO); Morphine; Substance P receptor antagonists;/3-Adrenoceptors
1. Introduction
Substance P is a neuropeptide localized in the nerve terminals of primary sensory neurons and implicated in neurogenic inflammation and nociception (Payan, 1989). This peptide is able to cause in vivo vasodilatation and plasma extravasation through NK 1 receptors localized on the endothelium, causing an endothelium-dependent relaxation of arterial smooth muscle (Gulati et al., 1987). Endothelial cells react to mechanical or electrical stimuli as well as to various neurohumoral mediators by releasing vasodilator a n d / o r vasoconstrictor substances and, as such, contribute to the control of local vascular tone. The relaxation caused by substance P is due to a diffusible factor released by endothelial cells (Whittle et al., 1989). This diffusible factor named endothelium-derived relaxing factor ( E D R F ) (Furchgott and Zawadzki, 1980) is identical to nitric oxide (NO) derived from the terminal guanidino nitrogen atom of L-arginine (Palmer et al., 1988). It has been shown that opioids inhibit the neuro-
Correspondence to: A. Calignano, Department of Experimental Pharmacology, University of Naples 'Federico II', via D. Montesano 49, 80131 Naples, Italy. Tel. (39)-81-7486441, fax (39)-81-7486403.
genic plasma extravasation following antidromic stimulation of the saphenous nerve (Barthb and Szolcanyi, 1981; Lembeck and Gamse, 1982). Thus the existence of endogenous opioid activity controlling substance P release from the peripheral nerve endings has been postulated (Barthb and Szolcanyi, 1981). In the present study, the rabbit isolated aorta ring precontracted with phenylephrine was electrically stimulated, resulting in endothelium- and NO-dependent relaxation that was studied and characterized. Our study showed that the electrically induced relaxation is mainly mediated by substance P release and that opioids regulating this process could participate indirectly in the modulation of vascular tone through the control of NO generation.
2. Materials and methods
Albino male New Zealand rabbits (1.5-2 kg body weight) were anesthetized with pentobarbital sodium (30 m g / k g i.p.) and killed by exsanguination. The descending aorta was rapidly dissected and placed in Krebs solution gassed with 95% 02-5% CO 2 at room temperature and loose connective tissue was removed. Arterial segments were cut into rings (4-6 mm in
106 length), which w e r e s u s p e n d e d in 10-ml o r g a n baths filled with w a r m (37°C) o x y g e n a t e d Krebs solution and c o n n e c t e d to i s o m e t r ic t r a n s d u c e r s (U. Basile, Italy). A f t e r 30 min of e q u i l i b r a t i o n u n d e r a t e n s io n o f 1.5 g, the rings w e r e c o n t r a c t e d with p h e n y l e p h r i n e (10 -6 M). In s o m e e x p e r i m e n t s the e n d o t h e l i u m was rem o v e d by gently r u b b i n g the luminal surface with a c o t t o n swab m o i s t e n e d with K r e b s solution. E n d o t h e lium a b s e n c e was c o n f i r m e d by the lack of relaxation in r e s p o n s e to a c e t y i c h o l i n e (10 -6 M) a d d e d d u r i n g p h c n y l e p h r i n e - e v o k e d c o n t r a c t i o n . Rings w e r e l o c a t e d b e t w e e n p l a t i n u m e l e c t r o d e s p l a c e d at e i t h e r side of the vessel an d p e r i v a s c u l a r nerves w e r e s t i m u l a t e d by 0.5-ms pulses d e l i v e r e d at 0.4 Hz at 50 V for 1 min. N~;-Monomethyl-L-arginine and capsaicin were a d d e d to the bath 20 min b e f o r e starting stimulation, w h e r e a s all o t h e r drugs w e r e a d d e d 5 min b e f o r e e l e c t r i c a l - f i e l d stimulation. Cimetidine hydrochloride, NG-monomethyl-L-arginine, a t r o p i n e sulfate, acetylsalicylic acid, n a l o x o n e h y d r o c h l o r i d e , s u b s t a n c e P a c e t a t e salt, capsaicin, pen-
tobarbital sodium, b e n z a l k o n i u m h y d r o c h l o r i d e , Lp h e n y l c p h r i n e h y d r o c h l o r i d e , acct y l ch o l i n c hydrochloride, [D-Pro2,D-TrpT"~]substance P a c e t a t e salt, tetrodotoxin, p r o p r a n o l o l h y d r o c h l o r i d e , a l p r e n o l o l hyd r o c h l o r i d e , o x p r c n o l o l h y d r o c h l o r i d c , L - a r g i n i n e hyd r o c h l o r i d e , D - a r g i n i n c h y d r o c h l o r i d e , bovine erythrocyte s u p e r o x i d e dismutase, y o h i m b i n c h y d r o c h l o r i d e , g u a n e t h i d i n e sulfate and h a e m o g l o b i n w e r e o b t a i n e d from Sigma (St. Louis, M O , U S A ) ; m e p y r a m i n e m a l c a t e was o b t a i n e d from R h o n e Poulenc; m e t h y s e r g i d e was o b t a i n e d from Sandoz, and m o r p h i n e h y d r o c h l o r i d c was o b t a i n e d from S . A . L . A . R . S . (Italy). T h e results arc e x p r e s s e d as m e a n s + S.E.M. and w e r e analyzed for statistical significance with S t u d e n t ' s t-test. Significan ce was set at P < 0.01.
3. Results E l e c t r i c a l st i m u l at i o n o f p h e n y l e p h r i n e (10 -6 M)c o n t r a c t e d rabbit ao r t a rings r e s u l t e d in relaxation
TABLE 1 Effect of morphine (MOR), benzalkonium (BEN), [D-Pro2,D-TrpV'9]substance P (DD-SAP), propranolol (PRO), alprenolol (ALP), oxprcnolol (OXP) and capsaicin (CAP) on electrically induced relaxation of phenylephrine (10 -6 M)-contracted aorta rings. The values arc means _+S.E.M.; " P < (I.01 vs. control. Drugs
Concentration
% relaxation
Control
-
45.0 + 2.5
MOR
10 7M 7.0× 10 -7 M 1.4x 10 -6 M
31.2+2.5 ~ 23.7_+ 1.7 " 18.2_+ 1.0 ~'
28.5_+2.8 47.3_+2.4 59.6_+ 1.3
12 12 12
BEN
2.0x 10 -~' M
20.2_+2.3 "
55.0+2.7
5
DD-SAP
10 -6 M
18.2_+ 1.0 :'
59.0_+0.7
12
MOR + BEN
1.4× 10 -~' M 2.0×10 ~'M
20.1_+1.1 ~'
55.3-+1.1
12
1.4x10 6 M 10 -6 M
20.2_+ 1.3 ~'
55.2_+ 1.7
12
PRO
10 -5 M 8.0x 10 -5 M
30.1 -+ 1.2 " 22.5_+2.3 "
30.4_+ 1.3 51.0-+2.2
12 12
ALP
10 -5 M 8.0× 10 -s M
32.7_+2.4 a 22.9_+ 1.7 a
27.3_+ 1.4 49.4_+ 1.6
12 12
OXP
10 5 M 8.0×10 5 M
30.8_+ 1.5 ~ 22.5+2.3 a
32.1 _+1.8 50.6_+2.1
12 12
MOR + PRO
1.4×10 -s M 8.0x 10 -5 M
0 ~'
100
12
2.0 × 10- n M 8.0×10 5 M
0 ~'
100
12
0a
100
12
MOR+DD-SAP
BEN + PRO DD-SAP+ PRO
10 - 6 M 8.0X 10 -5 M
CAP
10 -5 M
CAP + DD-SAP
% reduction
n 24
27.4_+2.4 a
40.1 _+2.7
6
22.5_+2.8 "
50.2_+3.5
6
1(I-5 M 10 - 6 M
107
A
ES
~S
~S
L-NMMA E~
MOR
l.~b ESI
MOR +NAL
Fig. 1. (A) Effect of NC'-monomethyl-L-arginine (L-NMMA, 3 x 10 -4 M) and haemoglobin (Hb, 10 -4 M) on electrical-field stimulation (ES) of rabbit aorta rings precontracted with phenylephrine (10 -6 M) (trace representative of six experiments for L - N M M A and 10 experiments for Hb). (B) Effect of morphine (MOR, a = 1.4× 10 -6 M, b = 7 x 10 -7 M, c = 10 -7 M) and antagonism by naloxone (NAL, 5 x 10 -7 M) one electrical-field stimulation (ES) of rabbit aorta rings precontracted with phenylephrine (10 -6 M) (trace representative of 12 experiments for M O R and 5 experiments for M O R + NAL).
(45.3 + 2.5%, n = 24) observed only when the endothelium was intact. When the endothelium was removed by rubbing the luminal surface of aorta rings, no relaxation was obtained on electrical stimulation. N GMonomethyl-L-arginine (L-NMMA), added 20 min before electrical stimulation caused a concentration-dependent inhibition of the electrically induced relaxation (18 5- 1.7% by 10 -6 M, 70 + 1.5% by 10 -5 M and 100% by 3 x 10 -4 M, n = 6). Haemoglobin (10 -4 M, n = 10) addition to the Krebs solution 5 min before electrical stimulation completely abolished the electrically induced relaxation (fig. 1A). On the other hand, superoxide dismutase (70 U / m i ) addition increased the relaxant effect by 59.1 5- 3.1% (n = 6). The electrically induced vasorelaxation was markedly reduced by tetrodotoxin (10 -6 M, n = 5, 89 + 2.1%). Similar results were obtained with guanethidine (10 -5 M, n = 5, data not shown). Benzalkonium (2 x 10 -6 M) and [DPro2,D-Trp7'9]substance P (10 -6 M) completely abolished the small relaxation obtained in the presence of tetrodotoxin (10 -6 M, n = 6). Morphine addition 5 min before electrical stimulation caused a concentrationdependent inhibition of the electrically induced relaxation (table 1, fig. 1B). The morphine effect was reversed by concomitant administration of naloxone (5 x 10 -7 M, n = 5) (fig. IB). A non-significant increase of contraction was observed after naloxone administration (data not shown). The maximum morphine effect (59.6 + 1.3%, n = 12) was observed at 1.4 x 10 -6 M and a higher morphine concentration did not produce a greater inhibitory effect (data not shown). In order to further characterize the mechanisms involved in the electrically induced relaxation, the effect of several compounds were investigated in subsequent experi-
ments. Methysergide ( 1 0 - 7 - 1 0 -5 M, n = 5), mepyramine ( 1 0 - 7 - 1 0 -5 M, n = 5), cimetidine ( 1 0 - 7 - 1 0 -5 M, n = ) , atropine ( 1 0 - 9 - 1 0 -7 M, n = 5 ) , acetylsalicylic acid ( 1 0 - 7 - 1 0 -4 M, n = 5) or yohimbine (5 x 10 -7 M, n = 5) did not affect the electrically induced relaxation. Benzalkonium (2 x 10 -6 M) and [D-Pro2,D-Trp7'9]sub stance P (10 -6 M) inhibited the electrically induced relaxation by 55 5-2.7 and 59 + 0.7% respectively (table 1). Moreover, benzalkonium and [D-ProZ,D-Trp7'9]sub stance P did not significantly modify the inhibitory effect of morphine (table 1). The /3-adrenoceptor antagonists, propranolol (10 -5 and 8 x 10 -5 M), alprenolol (10 -S and 8 x 10 -5 M) and oxprenolol (10 -5 and 8 x 10 -5 M) added to the organ bath were equivalent for reducing the electrically induced relaxation in a concentration-dependent manner. A combination of propranolol (8 x 10 -5 M) with morphine (1.4 x 10 -6 M), benzalkonium (2 x 10 -6 M) or [D-Pro2,D Trp7"9]substance P (10 -6 M) completely abolished the electrically induced relaxation (table 1). Capsaicintreated (10 -5 M) aorta rings showed a reduction of 40 + 2.7% (n = 6) of electrically induced vasorelaxation, but a slight reduction, 10 5- 3% (n = 6, P < 0.05), could still be obtained with [D-Pro2,D-Trp7'9]substance P (10 -6 M) addition. A combination of capsaicin (10 -5 M) with [D-Pro2,D-Trp7"q]substance P (10 -6 M) resulted in a 50 + 3.5% reduction of electrically induced vasorelaxation (table 1).
4. D i s c u s s i o n
Considerable evidence indicates that electrical stimulation of vessels releases neurotransmitters from perivascular nerve endings (Rand and Varma, 1970). Therefore these substances may play a key role in the regulation of vascular tone and blood flow. Recently a number of vasodilators have been shown to evoke their responses by stimulating the production of endothelium-derived relaxing factor ( E D R F ) in endothelial ceils. These substances include acetylcholine, substance P, adenosine 5-triphosphate, adenosine 5-diphosphate, bradykinin, thrombin, histamine and epinephrine (Furchgott, 1983). We now report that electrical-field stimulation of rabbit aorta rings precontracted with phenylephrine leads to an endotheliumdependent and tetrodotoxin-sensitive vasorelaxation. This suggests that the nerve endings and endothelium may play an important role in the regulation of vascular tone in this experimental model. The electrically induced vasorelaxation could be enhanced by superoxide dismutase and completely prevented by haemoglobin or N°-monomethyl-L-arginine (L-NMMA), suggesting that the relaxing effect is totally due to NO release. It is well known that haemoglobin itself con-
108 tains a prosthetic heme group which binds nitric oxide, thus effectively removing nitric oxide from solution. NG-Monomethyl-L-arginine has been shown to inhibit NO synthase in endothelial cells (Moncada et al., 1986). Furthermore the data obtained in the present study using superoxide dismutase support the hypothesis that the relaxing effect is NO-dependent. Morphine addition reduced the electrically induced relaxation in a concentration-dependent manner. The maximum reduction (60%) was observed at 1.4 × 10 -6 M, and did not increase with a higher concentration of morphine. These results suggest that opioids regulate the release of NO-releasing substance(s). The electrically induced relaxation was also reduced by the substance P antagonists, benzalkonium (Piotrowski et al., 1987) or [D-Pro2,D-TrpT'9]substance P, in a concentration-dependent manner. It is of interest that the maximal reduction observed with substance P antagonists was of the same magnitude as the maximal morphineinduced reduction. Moreover the morphine inhibition was not increased by the concomitant presence of benzalkonium or [D-Pro2,D-TrpT'9]substance P. Substantial evidence indicates that opioids regulate substance P release from nerve endings (Jesel and lversen, 1977). Substance P is a ncuropeptide localised in the nerve terminals of primary sensory neurons, and is implicated in neurogenic inflammation and nociccption (Payan, 1989). This peptide is also Iocalised in the endothelial cells lining the arterial wall (Loesch and Burnstock, 1988). Substance P causes, via NK 1 receptors localized on the endothelial ceils, and endotheiium-dependent relaxation of arterial smooth muscle (Loesch and Burnstock, 1988; Gulati et al., 1987). The data obtained with tetrodotoxin indicate a neuronal origin of transmitters that cause NO release. The inhibition obtained with tetrodotoxin was extremely strong but a slight relaxation was still observed. This relaxation was abolished by substance P antagonists, thus we cannot exclude the participation of endothelial pools of substance P in addition to neuronal pools. This hypothesis was confirmed by the capsaicin data. These observations show that the endothelial pools of substancc P also could be released by electrical-field stimulation. Morphine or substance P antagonists reduced the electrically induced relaxation by only 60%. We have found that fl-adrenoceptor antagonists like propranolol, oxpranolol and alprenolol reduced the remaining 40% of electrically induced relaxation. A combination of morphine plus fl-adrenoceptor blockers or substance P antagonists plus /3-adrenoceptor blockers completely abolished the electrically induced relaxation. Recently it has been reported that, in rat thoracic aorta, isoprenaline causes an endothelium-dependent and N c,-nitro-L-arginine-sensitive vasorelaxation associated with increases of cGMP (Gray and Marshall,
1991). Our data are in agreement with this report indicating the presence of fl-adrenoceptors that release NO. We also could exclude the participation of a2-adrenoceptors that are well known to be involved in NO release, because yohimbine did not modify the electrically induced relaxation. The guanethidine data are not consistent with our hypothesis that substance P could be a major neurotransmitter released in electrically induced vasorelaxation. A possible interpretation of the data is that the concentration of guanethidine used (10 -5 M) was high enough to have a local anaesthetic action (Maxwell and Wastila, 1977) and therefore block nerve conduction in the sensory as well as adrenergic nerves. In conclusion, the electrical-field stimulation of rabbit aorta rings precontracted by phenylephrine causes a vasorelaxation that was endothelium- and NO-dependent. Our data suggest that substance P releases NO through the activation of NK-P receptors. Morphine, by reducing substance P release, decreased the magnitude of the electrically induced relaxation, suggesting an indirect role of opioids in the regulation of the peripheral circulation through the control of nitric oxide release, fl-Adrenoceptors also seem to be involved in NO release. This observatkm supports the hypothesis that in some vascular tissues such as rat thoracic aorta activation of fl-adrenoceptors could release NO.
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