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Inhibition by calmodulin antagonists of the neurogenic relaxation in cerebral arteries
ELSEVIER
European Journal of Pharmacology 256 (1994) 79-83
Inhibition by calmodulin antagonists of the neurogenic relaxation in cerebral arteries Tomio Okamura, Noboru Toda
*
Department of Pharmacology, Shiga Universityof Medical Science, Seta, Ohtsu 520-21, Japan (Received 30 August 1993; revised MS received 14 January 1994; accepted 18 January 1994)
Abstract
The present study was aimed to determine the effect of calmodulin inhibitors on the relaxant response of isolated dog and monkey cerebral arteries to vasodilator nerve stimulation, which is hypothesized to be mediated by nitric oxide (NO) from nerve endings. The relaxations caused by nerve stimulation by electrical pulses in endothelium-denuded arteries were attenuated by treatment with caimidazolium and W-7 (N-(6-aminohexyl)-5-chloro-l-naphthalene sulfonamide hydrochloride) and were abolished by NG-nitro-L-arginine, an inhibitor of nitric oxide synthase, and tetrodotoxin. The calmodulin inhibitors also attenuated the relaxations caused by nicotine and substance P, which were endothelium-independent and -dependent, respectively, but did not influence the relaxant response to NO. It is concluded that calmodulin is required for activation of the NO synthase present in the vasodilator nerve as well as that in the endothelium. Key words: Nitric oxide (NO); Vasodilator nerve; Cerebral artery; Calmodulin; Endothelium
1. Introduction
Endogenous nitric oxide (NO) synthesized from Larginine plays an important role in the regulation of vascular smooth muscle tone not only as an endothelium-derived relaxing factor ( E D R F ; Furchgott and Zawadzki, 1980) but also as a neurotransmitter of vasodilator nerves (Toda and Okamura, 1992a). At least two distinct types of N O synthase have been identified (Nathan and Stuehr, 1990); the constitutive type is present in endothelial ceils and neural tissues, and its activity is calcium- and calmodulin-dependent, whereas the inducible type is induced by cytokines such as interleukin-1 or tumor necrosis factor and is present in endothelial and smooth muscle cells, macrophages and chondrocytes, and its activity is calcium- and calmodulin-independent, except in chondrocytes (Palmer et al., 1992). The E D R F - m e d i a t e d relaxation is markedly suppressed by removal of extracellular calcium (Griffith et al., 1986) and by treatment with non-selective, but not dihydropyridine, calcium channel antagonists (Toda and Okamura, 1992b) and calmod-
* Corresponding author. Tel. 81-775-48-2181, fax 81-775-48-2183. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0014-2999(94)00065-F
ulin antagonists (Weinheimer and Osswald, 1986; Schini and Vanhoutte, 1992), indicating that intracellular free calcium supplied from extracellular fluids through nonL-type calcium channel activates N O synthase by a calmodulin-dependent mechanism in endothelial cells. However, whether or not nitroxidergic, vasodilator nerve function is dependent on calmodulin has not been determined. The present study was therefore undertaken to determine whether calmodulin inhibitors selectively inhibit the neurally induced relaxation in isolated monkey and dog cerebral arteries.
2. Materials and methods
2.1. Preparation Mongrel dogs of either sex, weighing 8-13 kg, were anesthetized with intravenous injections of sodium thiopental (30 m g / k g ) . Japanese monkeys (Macaca fuscata) of either sex, weighing 6 - 1 0 kg, were anesthetized with intramuscular injections of ketamine (40 m g / k g ) and sodium thiopental (20 m g / k g ) . The animals were killed by bleeding from the carotid arteries.
80
T Okamura, A~ Toda / European Journal of Pharmacology 256 (1994) 79-83
The brain was rapidly removed, and basilar and middle cerebral arteries (0.5-0.7 mm outside diameter) were isolated. The arteries were helically cut into strips, approximately 20 mm long. The specimens were vertically fixed between hooks in a muscle bath (20 ml capacity) containing modified Ringer-Locke solution (in mM, NaC1 120, KC1 5.4, CaC12 2.2, MgC12 1.0, NaHCO 3 25.0 and dextrose 5.6), which was aerated with a mixture of 95% 0 2 and 5% CO 2 and maintained at 37 __+0.3°C. The pH of the solution was 7.357.43. The hook anchoring the upper end of the strips was connected to the lever of a force-displacement transducer. The resting tension was adjusted to 1.5 g (for dog artery) or 1.0 g (for monkey artery), which is optimal for inducing maximal contraction. Before the start of experiments, all of the strips were allowed to equilibrate in the bathing media for 60-90 min., during which time the solution was replaced every 10-15 rain. Some of the arterial strips were placed between stimulating electrodes. The gaps between the strip and the electrodes were wide enough to allow undisturbed contraction and relaxation, yet sufficiently narrow to stimulate intramural nerve terminals effectively. A train of 0.2 ms square pulses of supramaximal intensity (10 V) were transmurally applied at a frequency of 5 Hz for 40 s. The stimulus pulses were delivered by an electronic stimulator (Nihonkohden Kogyo, Tokyo, Japan).
or marked suppression of the relaxation caused by substance P (10 - 7 M) in the arteries treated with 10 -6 M indomethacin and also by the silver staining procedure. 2.3. Statistics and drugs used The results shown in the text, tables and figures are expressed as means_+ S.E. Statistical analyses were done using Student's paired or unpaired t-test for two groups and Tukey's method after one-way analysis of variance for more than three groups. The drugs used were NG-nitro-L-arginine, N%nitro-D-arginine, L-arginine, D-arginine, substance P (Peptide Institute Inc., Minoh, Japan), nicotine (Nacalai Tesque, Kyoto, Japan), tetrodotoxin (Sankyo, Tokyo), prostaglandin F2~ (Ono, Osaka, Japan), calmidazolium (Boehringer Mannheim, Mannheim, Germany), W-7 (N-(6-aminohexyl)-5-chloro-l-naphthalene sulfonamide hydrochloride, Sigma, St Louis, USA) and papaverine hydrochloride (Dainippon, Osaka). Responses to NO were obtained by adding NaNO e solution adjusted at pH 2 (Furchgott, 1988).
3. Results
3.1. Modification by calmodulin inhibitors of the response to transmural electrical stimulation
2.2. Recording Isometric contractions and relaxations were displayed on an ink-writing recorder. The contractile response to 30 mM K + was first obtained, and then the strips were washed three times with the fresh medium and equilibrated for 30-40 min. The strips were contracted partially with prostaglandin Fza (2-10 X 10 -7 M); the contractions were in a range between 25 and 40% of the contraction induced by 30 mM K +. Transmural electrical stimulation was applied repeatedly at intervals of 10 min until steady responses were obtained, and then blocking agents were applied. At the end of each series, tetrodotoxin (3 × 10 -7 M) was applied to confirm the neurally induced response. Nicotine and NO at submaximal concentrations (10 -4 M and 10 -7 M, respectively) were applied directly to the bathing medium, and the strips were repeatedly washed. After the responses to the agents had been stabilized, preparations were treated for about 20 min with blocking agents. Substance P at 10 -8 M was also used in some experiments. At the end of each series of experiments, papaverine (10 -4 M ) w a s added to obtain maximal relaxation. Relaxations induced by transmural electrical stimulation, nicotine or other vasodilators relative to those induced by papaverine are presented. Removal of the endothelium was verified by abolition
In dog cerebral artery strips denuded of the endothelium and partially precontracted with prostaglandin F2~, transmural electrical stimulation at 5 Hz produced a relaxation, which was abolished by 3 × 10-7 M tetrodotoxin or 10 -6 M N%nitro-L-arginine (n = 5). The inhibition elicited by NG-nitro-L-arginine was reversed by 3 × 10 -4 M L-, but not i>, arginine. 10 6 M NG-nitro-D-arginine was without effect (n = 5). The relaxation caused by transmural stimulation was attenuated by treatment with calmidazolium (3 × 10 -6 M and 10 .5 M) and W-7 (3 × 10 -6 M and 10 5 M) in a concentration-related manner (Fig. 1). Calmidazolium, but not W-7, elicited a contraction which was endothelium-independent; the contractions induced by calmidazolium (10 .5 M) in the endothelium-intact and -denuded strips obtained from the same dogs were 30.5 _+ 7.9% and 14.5 + 5.0% (n = 5, statistically insignificant), respectively, at 3 × 10 .6 M, and 44.6_+ 12.4% and 42.0 _+ 8.6% (n = 5, statistically insignificant), respectively, at 10 .5 M, of the contraction induced by 30 mM K +. On the other hand, relaxations induced by 10 -7 M substance P in the endothelium-intact and -denuded strips were 75.6 _+ 1.6% and 26.7 _+ 3.8%, respectively (n = 5, P < 0.01). Dimethylsulfoxide, the vehicle for calmidazolium, at the same concentration as that included in the 10 .5 M calmidazolium solution caused
T. Okamura, N. Toda / European Journal of Pharmacology 256 (1994) 79-83 DOG CEREBRAL A R T E R Y - - T r a n s m u r a l 0
MONKEY CEREBRAL A R T E R Y -
stimulation 5Hz
81 Transmural stimulation 5Hz
O_
-
!!iiiiiii!!l iiiiiiiiii!l ilililililiiii!iii!ili~
!i!!ii!ii!i!i!i!i!i!i!i 10 -
10 -
J
x rr
20
+
-
o
30 -
2 0 -
30 C
CLM 3X 10-6
CLM 10-5M
C
W-7 3Xl0-S
-
W-7 10-5M
Fig. 1. Modification by calmidazolium (CLM, left panel) and W-7 (right) of the relaxant response to transmural electrical stimulation (5 Hz) of dog cerebral artery strips d e n u d e d of the endothelium and contracted with prostaglandin F2,~. Relaxations induced by papaverine were taken as 100%. Significantly different from control (C), a p < 0.01 (Tukey's method). Vertical bars represent S.E.M.
no contraction and did not influence the neurally induced relaxation. The addition of 2.2 m M calcium chloride did not reverse the inhibition caused by calmidazolium. A typical recording is shown in Fig. 2. In monkey artery strips denuded of the endothelium and precontracted with prostaglandin F2,, relaxations caused by transmural electrical stimulation at 5 Hz were abolished by tetrodotoxin or N°-nitro-L-arginine and suppressed by calmidazolium and W-7 in a concentration-related manner; the suppression elicited by calmidazolium was greater than that elicited by W-7 (Fig. 3).
C
CLM 10-SM
C
W-7 3X10-6
W-7 10-5M
Fig. 3. Modification by calmidazolium (CLM, left panel) and W-7 (right) of the relaxant response to transmural electrical stimulation (5 Hz) of monkey cerebral artery strips d e n u d e d of the endothelium and contracted with prostaglandin F2,~. Relaxations induced by papaverine were taken as 100%. Significantly different from control (C), a p < 0.01; b p < 0.05 (Tukey's method). Vertical bars represent S.E.M.
response to substance P, but did not affect the responses to nicotine and N O (n = 8). Treatment with calmidazolium significantly inhibited the relaxant responses to nicotine and substance P in a concentration-
DOG CEREBRAL ARTERY Nicotine 10 - 4 M 0 20
rr• 40
3.2. Modification by calmodulin inhibitors of the responses to nicotine, substance P and nitric oxide D o g cerebral artery strips with intact endothelium responded to nicotine (10 -4 M), substance P (10 -8 M) and N O (10 -7 M) with relaxations to a similar extent. Removal of the endothelium abolished the relaxant
CLM 3 X 1 0 -s
i
I
Substance P I o - s M
~
0
8
20
:=
40
I NO 10 -7 M
DOG CEREBRAL A R T E R Y - Transmural electrical stimulation, 5Hz Calmidazolium 3X10 -e M 5Hz
/
0
8
=
10-5 M [ ~'~k
Ca 2+
TTX
PA
1020
20
4o
C
CLM CLM 3X10 -6 IO-SM
C
W-7 3X10-6M
lOmin
Fig. 2. Typical tracings of the response to transmural electrical stimulation (5 Hz) of a dog middle cerebral artery strip d e n u d e d of the endothelium and partially contracted with prostaglandin Fed (5 × 10 - 7 M), before and after treatment with calmidazolium. Ca 2+ = 2.2 m M calcium chloride; P A = 10 - 4 M papaverine; T T X = 3 × 10 -7 M tetrodotoxin.
Fig. 4. Modification by calmidazolium (CLM, left panel) and W-7 (right) of the relaxant response to nicotine (top), substance P (middle) and nitric oxide (NO, bottom) of dog cerebral artery strips with an intact endothelium and contracted with prostaglandin F2~. Relaxations induced by papaverine were taken as 100%. Significantly different from control (C), a p < 0.01 (Tukey's method). Vertical bars represent S.E.M.
82
T. Okamura, N. Toda / European Journal of Pharmacology 256 (1994) 79-83
related manner, whereas the response to NO was not influenced (Fig. 4). W-7 markedly inhibited the nicotine-induced relaxations, but did not affect the response to NO (Fig. 4). Neither calmodulin antagonist at the concentrations used significantly affected the contractions evoked by K + and prostaglandin F2~ (data not shown). The relaxations caused by nicotine and substance P, but not by NO, were markedly suppressed by NG-nitro-L-arginine (n = 5).
4. Discussion The present study demonstrated that calmodulin inhibitors impair the neurogenic vasodilatation elicited by stimulation of nitroxidergic nerves as well as the endothelium-dependent relaxation caused by substance P in cerebral arteries. In dog and monkey cerebral artery strips, transmural electrical stimulation and nicotine produced relaxation, possibly by stimulating nitroxidergic, vasodilator nerves, since the relaxation is abolished by treatment with NO synthase inhibitors such as NG-monomethylL-arginine (Toda and Okamura, 1990a,b,c) and N Gnitro-L-arginine (present study, Toda and Okamura, 1991a), a NO scavenger such as oxyhemoglobin and a soluble guanylate cyclase inhibitor such as methylene blue (Toda, 1988). The inhibition produced by the NO synthase inhibitors was reversed by L-, but not D-, arginine. This hypothesis is strongly supported by the findings that stimulation of vasodilator nerves releases nitroxy compounds from superfused endothelium-denuded cerebral artery strips (Toda and Okamura, 1990b) and increases the production of cGMP in these strips (Toda and Okamura, 1991a). Our recent immunohistochemical studies demonstrated the innervation of NO synthase-containing nerves in dog cerebral arteries (Yoshida et al., 1993) and showed that the nerve fibers arise from the ipsilateral pterygopalatine ganglion (Toda et al., 1993b). NO synthase in the brain has been purified (Bredt and Snyder, 1990), cloned (Bredt et al., 1991) and identified as a constitutive type, the enzyme requires NADPH, calcium and calmodulin for activity (Bredt and Snyder, 1990), as does the enzyme derived from the endothelial ceils (Palmer and Moncada, 1989; F6rstermann et al., 1991; Busse and Miilsch, 1990). However, the NO synthase localized in the terminal of peripheral nerves innervating blood vessels (Toda and Okamura, 1991b; 1992c), intestine (Bult et al., 1990; Toda et al., 1991), penis (Burnett et al., 1992), etc., has not been purified nor cloned. Therefore, it is not yet known whether or not the NO synthase in the peripheral nerves is biochemically identical to that in the brain. Bredt et al. (1990) have demonstrated that the nerves innervating peripheral tissues are stained by an
antibody raised against the NO synthase purified from cerebellum, suggesting that the NO synthase derived from peripheral nerves is also a constitutive type, but the functional properties of the enzyme are not known. In the present study, the relaxant responses to stimulation of nitroxidergic vasodilator nerves by electrical pulses and nicotine were significantly suppressed by treatment with calmodulin antagonists, such as calmidazolium and W-7, in a concentration-dependent manner. The inhibitors at the concentrations used in this study did not significantly affect the prostaglandin F2,~induced contractions, but the concentrations used were sufficient to bind the hydrophobic domains on calmodulin (Johnson and Wittenauer, 1983) and to inhibit the EDRF-mediated relaxations caused by acetylcholine, ATP and calcium ionophore A23187 in the rat aorta (Schini and Vanhoutte, 1992). Therefore, it is suggested that the calmodulin inhibitors are likely to impair neurogenic relaxations by inhibition of calmodulin. The possibility that calmidazolium and W-7 attenuate the relaxation by inhibiting the soluble guanylate cyclase-cGMP effector pathway is unlikely, as the calmodulin inhibitors did not affect the relaxations induced by exogenously applied NO. The mechanism of the contraction caused by calmidazolium is unknown, but it seems to be unrelated to the inhibitory action of calmodulin, as W-7 did not cause the significant contraction. Calmidazolium markedly inhibited the relaxations caused by substance P in dog cerebral arteries. The peptide-induced relaxation is primarily endotheliumdependent (present study, Onoue et al., 1988), is markedly suppressed by treatment with N%nitro-Larginine (present study, Toda et al., 1993a) and is associated with the production of cGMP (Toda et al., 1993a), suggesting the involvement of endothelium-derived NO in the relaxation. Therefore, it appears that calmodulin inhibitors impair the action of the NO synthase present in the endothelium of dog cerebral arteries. Similar data have been reported for the rat aorta (Schini and Vanhoutte, 1992) and the guinea-pig pulmonary artery (Weinheimer and Osswald, 1986). Our recent study has demonstrated that calcium is a prerequisite for the synthesis of NO in the vasodilator nerve and the endothelium of dog cerebral arteries (Toda and Okamura, 1992b). In addition, the present findings suggest the necessity of calmodulin for activation of the NO synthase present in the terminal of vasodilator nerve as well as that in the endothelium of dog and monkey cerebral arteries.
5. Acknowledgements The work was supported in part by Grant-in-Aid for Scientific Research in priority areas: 'Vascular Endothelium-Smooth Muscle
T. Okamura, N. Toda / European Journal of Pharmacology 256 (1994) 79-83 Coupling', from the Ministry of Education, Science and Culture, Japan.
6. References Bredt, D.S. and S.H. Snyder, 1990, Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme, Proc. Natl. Acad. Sci. USA 87, 682. Bredt, D.S., P.M. Hwang and S.H. Snyder, 1990, Localization of nitric oxide synthase indicating a neural role for nitric oxide, Nature 347, 768. Bredt, D.S., P.M. Hwang, C.E. Glatt, C.J. Lowenstein, R.R. Reed and S.H. Snyder, 1991, Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase, Nature 351, 714. Bult, H., G.E. Boeckxstaens, P.A. Pelckmans, F.H. Jordaens, Y.M. Van Maercke and A.G. Herman, 1990, Nitric oxide as an inhibitory non-adrenergic non-cholinergic ncurotransmitter, Nature 345, 346. Burnett, A.L., C.J. Lowenstein, D.S. Bredt, T.S.K. Chang and S.H. Snyder, 1992, Nitric oxide: a physiologic mediator of penile erection, Science 257, 401. Busse, R. and A. Miilsch, 1990, Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin, FEBS Lett. 265, 133. F6rstermann, U., J.S. Pollock, H.H.H.W. Schmidt, M. Heller and F. Murad, 1991, Calmodulin-dependent endothelium-derived relaxing factor/nitric oxide synthase activity is present in the particulate and cytosolic fractions of bovine aortic endothelial cells, Proc. Natl. Acad. Sci. USA 88, 1788. Furchgott, R.F., 1988, Studies on relaxation of rabbit aorta by sodium nitrite: the basis for the proposal that the acid activatable inhibitory factor from bovine retractor penis is inorganic nitrite and the endothelium-derived relaxing factor is nitric oxide, in: Vasodilatation, Vol. IV, ed. P.M. Vanhoutte (Raven Press, New York) p. 401. 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. Griffith, T.M., D.H. Edwards, A.C. Newby, M.J. Lewis and A.H. Henderson, 1986, Production of endothelium-derived relaxaing factor is dependent on oxydative phosphorylation and extracellular calcium, Cardiovasc. Res. 20, 7. Johnson, J.D. and L.A. Wittenauer, 1983, A fluorescent calmodulin that reports the binding of hydrophobic inhibitory ligands, Biochem. J. 211, 473. Nathan, C.F. and D.J. Stuehr, 1990, Does endothelium-derived nitric oxide have a role in cytokine-induced hypotension?, J. Natl. Cancer Inst. 82, 726. Onoue, H., N. Nakamura and N. Toda, 1988, Endothelium-dependent and -independent responses to vasodilators of isolated dog cerebral arteries, Stroke 19, 1388.
83
Palmer, R.M.J. and S. Moncada, 1989, A novel citrulline-forming enzyme implicated in the formation of nitric oxide by vascular endothelial cells, Biochem. Biophys. Res. Commun. 158, 348. Palmer, R.M.J., T. Andrews, N.A. Foxwell and S. Moncada, 1992, Glucocorticoids do not affect the induction of a novel calcium-dependent nitric oxide synthase in rabbit chondrocytes, Biochem. Biophys. Res. Commun. 188, 209. Schini, V.B. and P.M. Vanhoutte, 1992, Inhibitors of calmodulip impair the constitutive but not the inducible nitric oxide synthase activity in the rat aorta, J. Pharmacol. Exp. Ther. 261,553. Toda, N., 1988, Hemolysate inhibits cerebral artery relaxation, J. Cereb. Blood Flow Metab. 8, 46. Toda, N. and T. Okamura, 1990a, Modification by L-N6-monomethyl arginine (L-NMMA) of the response to nerve stimulation in isolated dog mesenteric and cerebral arteries, Jpn. J. Pharmacol. 52, 170. Toda, N. and T. Okamura, 1990b, Possible role of nitric oxide in transmitting information from vasodilator nerve to cerebroarterial muscle, Biochem. Biophys. Res. Commun. 170, 308. Toda, N. and T. Okamura, 1990c, Mechanism underlying the response to vasodilator nerve stimulation in isolated dog and monkey cerebral arteries, Am. J. Physiol. 259, H1511. Toda, N. and T. Okamura, 1991a, Role of nitric oxide in neurally induced cerebroarterial relaxation, J. Pharmacol. Exp. Ther. 258, 1027. Toda, N. and T. Okamura, 1991b, Reciprocal regulation by putatively nitroxidergic and adrenergic nerves of monkey and dog temporal arterial tone, Am. J. Physiol. 261, H1740. Toda, N. and T. Okamura, 1992a, Regulation by nitroxidergic nerve of arterial tone, News Physiol. Sci. 7, 148. Toda, N. and T. Okamura, 1992b, Different susceptibility of vasodilator nerve, endothelium and smooth muscle functions to Ca ++ antagonists in cerebral arteries, J. Pharmacol. Exp. Ther. 261,234. Toda, N. and T. Okamura, 1992c, Mechanism of neurally induced monkey mesenteric artery relaxation and contraction, Hypertension 19, 161. Toda, N., Y. Tanobe and H. Baba, 1991, Suppression by N6-nitro L-arginine of relaxations induced by non-adrenergic, noncholinergic nerve stimualtion in dog duodenal longitudinal muscle, Jpn. J. Pharmacol. 57, 527. Toda, N., K. Ayajiki and T. Okamura, 1993a, Cerebroarterial relaxations mediated by nitric oxide derived from endothelium and vasodilator nerve, J. Vasc. Res. 30, 61. Toda, N., K. Ayajiki, K. Yoshida, H. Kimura and T. Okamura, 1993b, Impairment by damage of the pterygopalatine ganglion of nitroxidergic vasodilator nerve function in canine cerebral and retinal arteries, Circ. Res. 72, 206. Weinheimer, G. and H. Osswald, 1986, Inhibition of endotheliumdependent smooth muscle relaxation by calmodulin antagonists, Naunyn-Schmied. Arch. Pharmacol. 332, 391. Yoshida, K., T. Okamura, H. Kimura, D.S. Bredt, S.H. Snyder and N. Toda, 1993, Nitric oxide synthase-immunoreactive nerve fibers in dog cerebral and peripheral arteries. Brain Res. 629, 67.
European Journal of Pharmacology 256 (1994) 79-83
Inhibition by calmodulin antagonists of the neurogenic relaxation in cerebral arteries Tomio Okamura, Noboru Toda
*
Department of Pharmacology, Shiga Universityof Medical Science, Seta, Ohtsu 520-21, Japan (Received 30 August 1993; revised MS received 14 January 1994; accepted 18 January 1994)
Abstract
The present study was aimed to determine the effect of calmodulin inhibitors on the relaxant response of isolated dog and monkey cerebral arteries to vasodilator nerve stimulation, which is hypothesized to be mediated by nitric oxide (NO) from nerve endings. The relaxations caused by nerve stimulation by electrical pulses in endothelium-denuded arteries were attenuated by treatment with caimidazolium and W-7 (N-(6-aminohexyl)-5-chloro-l-naphthalene sulfonamide hydrochloride) and were abolished by NG-nitro-L-arginine, an inhibitor of nitric oxide synthase, and tetrodotoxin. The calmodulin inhibitors also attenuated the relaxations caused by nicotine and substance P, which were endothelium-independent and -dependent, respectively, but did not influence the relaxant response to NO. It is concluded that calmodulin is required for activation of the NO synthase present in the vasodilator nerve as well as that in the endothelium. Key words: Nitric oxide (NO); Vasodilator nerve; Cerebral artery; Calmodulin; Endothelium
1. Introduction
Endogenous nitric oxide (NO) synthesized from Larginine plays an important role in the regulation of vascular smooth muscle tone not only as an endothelium-derived relaxing factor ( E D R F ; Furchgott and Zawadzki, 1980) but also as a neurotransmitter of vasodilator nerves (Toda and Okamura, 1992a). At least two distinct types of N O synthase have been identified (Nathan and Stuehr, 1990); the constitutive type is present in endothelial ceils and neural tissues, and its activity is calcium- and calmodulin-dependent, whereas the inducible type is induced by cytokines such as interleukin-1 or tumor necrosis factor and is present in endothelial and smooth muscle cells, macrophages and chondrocytes, and its activity is calcium- and calmodulin-independent, except in chondrocytes (Palmer et al., 1992). The E D R F - m e d i a t e d relaxation is markedly suppressed by removal of extracellular calcium (Griffith et al., 1986) and by treatment with non-selective, but not dihydropyridine, calcium channel antagonists (Toda and Okamura, 1992b) and calmod-
* Corresponding author. Tel. 81-775-48-2181, fax 81-775-48-2183. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0014-2999(94)00065-F
ulin antagonists (Weinheimer and Osswald, 1986; Schini and Vanhoutte, 1992), indicating that intracellular free calcium supplied from extracellular fluids through nonL-type calcium channel activates N O synthase by a calmodulin-dependent mechanism in endothelial cells. However, whether or not nitroxidergic, vasodilator nerve function is dependent on calmodulin has not been determined. The present study was therefore undertaken to determine whether calmodulin inhibitors selectively inhibit the neurally induced relaxation in isolated monkey and dog cerebral arteries.
2. Materials and methods
2.1. Preparation Mongrel dogs of either sex, weighing 8-13 kg, were anesthetized with intravenous injections of sodium thiopental (30 m g / k g ) . Japanese monkeys (Macaca fuscata) of either sex, weighing 6 - 1 0 kg, were anesthetized with intramuscular injections of ketamine (40 m g / k g ) and sodium thiopental (20 m g / k g ) . The animals were killed by bleeding from the carotid arteries.
80
T Okamura, A~ Toda / European Journal of Pharmacology 256 (1994) 79-83
The brain was rapidly removed, and basilar and middle cerebral arteries (0.5-0.7 mm outside diameter) were isolated. The arteries were helically cut into strips, approximately 20 mm long. The specimens were vertically fixed between hooks in a muscle bath (20 ml capacity) containing modified Ringer-Locke solution (in mM, NaC1 120, KC1 5.4, CaC12 2.2, MgC12 1.0, NaHCO 3 25.0 and dextrose 5.6), which was aerated with a mixture of 95% 0 2 and 5% CO 2 and maintained at 37 __+0.3°C. The pH of the solution was 7.357.43. The hook anchoring the upper end of the strips was connected to the lever of a force-displacement transducer. The resting tension was adjusted to 1.5 g (for dog artery) or 1.0 g (for monkey artery), which is optimal for inducing maximal contraction. Before the start of experiments, all of the strips were allowed to equilibrate in the bathing media for 60-90 min., during which time the solution was replaced every 10-15 rain. Some of the arterial strips were placed between stimulating electrodes. The gaps between the strip and the electrodes were wide enough to allow undisturbed contraction and relaxation, yet sufficiently narrow to stimulate intramural nerve terminals effectively. A train of 0.2 ms square pulses of supramaximal intensity (10 V) were transmurally applied at a frequency of 5 Hz for 40 s. The stimulus pulses were delivered by an electronic stimulator (Nihonkohden Kogyo, Tokyo, Japan).
or marked suppression of the relaxation caused by substance P (10 - 7 M) in the arteries treated with 10 -6 M indomethacin and also by the silver staining procedure. 2.3. Statistics and drugs used The results shown in the text, tables and figures are expressed as means_+ S.E. Statistical analyses were done using Student's paired or unpaired t-test for two groups and Tukey's method after one-way analysis of variance for more than three groups. The drugs used were NG-nitro-L-arginine, N%nitro-D-arginine, L-arginine, D-arginine, substance P (Peptide Institute Inc., Minoh, Japan), nicotine (Nacalai Tesque, Kyoto, Japan), tetrodotoxin (Sankyo, Tokyo), prostaglandin F2~ (Ono, Osaka, Japan), calmidazolium (Boehringer Mannheim, Mannheim, Germany), W-7 (N-(6-aminohexyl)-5-chloro-l-naphthalene sulfonamide hydrochloride, Sigma, St Louis, USA) and papaverine hydrochloride (Dainippon, Osaka). Responses to NO were obtained by adding NaNO e solution adjusted at pH 2 (Furchgott, 1988).
3. Results
3.1. Modification by calmodulin inhibitors of the response to transmural electrical stimulation
2.2. Recording Isometric contractions and relaxations were displayed on an ink-writing recorder. The contractile response to 30 mM K + was first obtained, and then the strips were washed three times with the fresh medium and equilibrated for 30-40 min. The strips were contracted partially with prostaglandin Fza (2-10 X 10 -7 M); the contractions were in a range between 25 and 40% of the contraction induced by 30 mM K +. Transmural electrical stimulation was applied repeatedly at intervals of 10 min until steady responses were obtained, and then blocking agents were applied. At the end of each series, tetrodotoxin (3 × 10 -7 M) was applied to confirm the neurally induced response. Nicotine and NO at submaximal concentrations (10 -4 M and 10 -7 M, respectively) were applied directly to the bathing medium, and the strips were repeatedly washed. After the responses to the agents had been stabilized, preparations were treated for about 20 min with blocking agents. Substance P at 10 -8 M was also used in some experiments. At the end of each series of experiments, papaverine (10 -4 M ) w a s added to obtain maximal relaxation. Relaxations induced by transmural electrical stimulation, nicotine or other vasodilators relative to those induced by papaverine are presented. Removal of the endothelium was verified by abolition
In dog cerebral artery strips denuded of the endothelium and partially precontracted with prostaglandin F2~, transmural electrical stimulation at 5 Hz produced a relaxation, which was abolished by 3 × 10-7 M tetrodotoxin or 10 -6 M N%nitro-L-arginine (n = 5). The inhibition elicited by NG-nitro-L-arginine was reversed by 3 × 10 -4 M L-, but not i>, arginine. 10 6 M NG-nitro-D-arginine was without effect (n = 5). The relaxation caused by transmural stimulation was attenuated by treatment with calmidazolium (3 × 10 -6 M and 10 .5 M) and W-7 (3 × 10 -6 M and 10 5 M) in a concentration-related manner (Fig. 1). Calmidazolium, but not W-7, elicited a contraction which was endothelium-independent; the contractions induced by calmidazolium (10 .5 M) in the endothelium-intact and -denuded strips obtained from the same dogs were 30.5 _+ 7.9% and 14.5 + 5.0% (n = 5, statistically insignificant), respectively, at 3 × 10 .6 M, and 44.6_+ 12.4% and 42.0 _+ 8.6% (n = 5, statistically insignificant), respectively, at 10 .5 M, of the contraction induced by 30 mM K +. On the other hand, relaxations induced by 10 -7 M substance P in the endothelium-intact and -denuded strips were 75.6 _+ 1.6% and 26.7 _+ 3.8%, respectively (n = 5, P < 0.01). Dimethylsulfoxide, the vehicle for calmidazolium, at the same concentration as that included in the 10 .5 M calmidazolium solution caused
T. Okamura, N. Toda / European Journal of Pharmacology 256 (1994) 79-83 DOG CEREBRAL A R T E R Y - - T r a n s m u r a l 0
MONKEY CEREBRAL A R T E R Y -
stimulation 5Hz
81 Transmural stimulation 5Hz
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-
o
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2 0 -
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CLM 3X 10-6
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C
W-7 3Xl0-S
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Fig. 1. Modification by calmidazolium (CLM, left panel) and W-7 (right) of the relaxant response to transmural electrical stimulation (5 Hz) of dog cerebral artery strips d e n u d e d of the endothelium and contracted with prostaglandin F2,~. Relaxations induced by papaverine were taken as 100%. Significantly different from control (C), a p < 0.01 (Tukey's method). Vertical bars represent S.E.M.
no contraction and did not influence the neurally induced relaxation. The addition of 2.2 m M calcium chloride did not reverse the inhibition caused by calmidazolium. A typical recording is shown in Fig. 2. In monkey artery strips denuded of the endothelium and precontracted with prostaglandin F2,, relaxations caused by transmural electrical stimulation at 5 Hz were abolished by tetrodotoxin or N°-nitro-L-arginine and suppressed by calmidazolium and W-7 in a concentration-related manner; the suppression elicited by calmidazolium was greater than that elicited by W-7 (Fig. 3).
C
CLM 10-SM
C
W-7 3X10-6
W-7 10-5M
Fig. 3. Modification by calmidazolium (CLM, left panel) and W-7 (right) of the relaxant response to transmural electrical stimulation (5 Hz) of monkey cerebral artery strips d e n u d e d of the endothelium and contracted with prostaglandin F2,~. Relaxations induced by papaverine were taken as 100%. Significantly different from control (C), a p < 0.01; b p < 0.05 (Tukey's method). Vertical bars represent S.E.M.
response to substance P, but did not affect the responses to nicotine and N O (n = 8). Treatment with calmidazolium significantly inhibited the relaxant responses to nicotine and substance P in a concentration-
DOG CEREBRAL ARTERY Nicotine 10 - 4 M 0 20
rr• 40
3.2. Modification by calmodulin inhibitors of the responses to nicotine, substance P and nitric oxide D o g cerebral artery strips with intact endothelium responded to nicotine (10 -4 M), substance P (10 -8 M) and N O (10 -7 M) with relaxations to a similar extent. Removal of the endothelium abolished the relaxant
CLM 3 X 1 0 -s
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Ca 2+
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PA
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20
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Fig. 2. Typical tracings of the response to transmural electrical stimulation (5 Hz) of a dog middle cerebral artery strip d e n u d e d of the endothelium and partially contracted with prostaglandin Fed (5 × 10 - 7 M), before and after treatment with calmidazolium. Ca 2+ = 2.2 m M calcium chloride; P A = 10 - 4 M papaverine; T T X = 3 × 10 -7 M tetrodotoxin.
Fig. 4. Modification by calmidazolium (CLM, left panel) and W-7 (right) of the relaxant response to nicotine (top), substance P (middle) and nitric oxide (NO, bottom) of dog cerebral artery strips with an intact endothelium and contracted with prostaglandin F2~. Relaxations induced by papaverine were taken as 100%. Significantly different from control (C), a p < 0.01 (Tukey's method). Vertical bars represent S.E.M.
82
T. Okamura, N. Toda / European Journal of Pharmacology 256 (1994) 79-83
related manner, whereas the response to NO was not influenced (Fig. 4). W-7 markedly inhibited the nicotine-induced relaxations, but did not affect the response to NO (Fig. 4). Neither calmodulin antagonist at the concentrations used significantly affected the contractions evoked by K + and prostaglandin F2~ (data not shown). The relaxations caused by nicotine and substance P, but not by NO, were markedly suppressed by NG-nitro-L-arginine (n = 5).
4. Discussion The present study demonstrated that calmodulin inhibitors impair the neurogenic vasodilatation elicited by stimulation of nitroxidergic nerves as well as the endothelium-dependent relaxation caused by substance P in cerebral arteries. In dog and monkey cerebral artery strips, transmural electrical stimulation and nicotine produced relaxation, possibly by stimulating nitroxidergic, vasodilator nerves, since the relaxation is abolished by treatment with NO synthase inhibitors such as NG-monomethylL-arginine (Toda and Okamura, 1990a,b,c) and N Gnitro-L-arginine (present study, Toda and Okamura, 1991a), a NO scavenger such as oxyhemoglobin and a soluble guanylate cyclase inhibitor such as methylene blue (Toda, 1988). The inhibition produced by the NO synthase inhibitors was reversed by L-, but not D-, arginine. This hypothesis is strongly supported by the findings that stimulation of vasodilator nerves releases nitroxy compounds from superfused endothelium-denuded cerebral artery strips (Toda and Okamura, 1990b) and increases the production of cGMP in these strips (Toda and Okamura, 1991a). Our recent immunohistochemical studies demonstrated the innervation of NO synthase-containing nerves in dog cerebral arteries (Yoshida et al., 1993) and showed that the nerve fibers arise from the ipsilateral pterygopalatine ganglion (Toda et al., 1993b). NO synthase in the brain has been purified (Bredt and Snyder, 1990), cloned (Bredt et al., 1991) and identified as a constitutive type, the enzyme requires NADPH, calcium and calmodulin for activity (Bredt and Snyder, 1990), as does the enzyme derived from the endothelial ceils (Palmer and Moncada, 1989; F6rstermann et al., 1991; Busse and Miilsch, 1990). However, the NO synthase localized in the terminal of peripheral nerves innervating blood vessels (Toda and Okamura, 1991b; 1992c), intestine (Bult et al., 1990; Toda et al., 1991), penis (Burnett et al., 1992), etc., has not been purified nor cloned. Therefore, it is not yet known whether or not the NO synthase in the peripheral nerves is biochemically identical to that in the brain. Bredt et al. (1990) have demonstrated that the nerves innervating peripheral tissues are stained by an
antibody raised against the NO synthase purified from cerebellum, suggesting that the NO synthase derived from peripheral nerves is also a constitutive type, but the functional properties of the enzyme are not known. In the present study, the relaxant responses to stimulation of nitroxidergic vasodilator nerves by electrical pulses and nicotine were significantly suppressed by treatment with calmodulin antagonists, such as calmidazolium and W-7, in a concentration-dependent manner. The inhibitors at the concentrations used in this study did not significantly affect the prostaglandin F2,~induced contractions, but the concentrations used were sufficient to bind the hydrophobic domains on calmodulin (Johnson and Wittenauer, 1983) and to inhibit the EDRF-mediated relaxations caused by acetylcholine, ATP and calcium ionophore A23187 in the rat aorta (Schini and Vanhoutte, 1992). Therefore, it is suggested that the calmodulin inhibitors are likely to impair neurogenic relaxations by inhibition of calmodulin. The possibility that calmidazolium and W-7 attenuate the relaxation by inhibiting the soluble guanylate cyclase-cGMP effector pathway is unlikely, as the calmodulin inhibitors did not affect the relaxations induced by exogenously applied NO. The mechanism of the contraction caused by calmidazolium is unknown, but it seems to be unrelated to the inhibitory action of calmodulin, as W-7 did not cause the significant contraction. Calmidazolium markedly inhibited the relaxations caused by substance P in dog cerebral arteries. The peptide-induced relaxation is primarily endotheliumdependent (present study, Onoue et al., 1988), is markedly suppressed by treatment with N%nitro-Larginine (present study, Toda et al., 1993a) and is associated with the production of cGMP (Toda et al., 1993a), suggesting the involvement of endothelium-derived NO in the relaxation. Therefore, it appears that calmodulin inhibitors impair the action of the NO synthase present in the endothelium of dog cerebral arteries. Similar data have been reported for the rat aorta (Schini and Vanhoutte, 1992) and the guinea-pig pulmonary artery (Weinheimer and Osswald, 1986). Our recent study has demonstrated that calcium is a prerequisite for the synthesis of NO in the vasodilator nerve and the endothelium of dog cerebral arteries (Toda and Okamura, 1992b). In addition, the present findings suggest the necessity of calmodulin for activation of the NO synthase present in the terminal of vasodilator nerve as well as that in the endothelium of dog and monkey cerebral arteries.
5. Acknowledgements The work was supported in part by Grant-in-Aid for Scientific Research in priority areas: 'Vascular Endothelium-Smooth Muscle
T. Okamura, N. Toda / European Journal of Pharmacology 256 (1994) 79-83 Coupling', from the Ministry of Education, Science and Culture, Japan.
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Palmer, R.M.J. and S. Moncada, 1989, A novel citrulline-forming enzyme implicated in the formation of nitric oxide by vascular endothelial cells, Biochem. Biophys. Res. Commun. 158, 348. Palmer, R.M.J., T. Andrews, N.A. Foxwell and S. Moncada, 1992, Glucocorticoids do not affect the induction of a novel calcium-dependent nitric oxide synthase in rabbit chondrocytes, Biochem. Biophys. Res. Commun. 188, 209. Schini, V.B. and P.M. Vanhoutte, 1992, Inhibitors of calmodulip impair the constitutive but not the inducible nitric oxide synthase activity in the rat aorta, J. Pharmacol. Exp. Ther. 261,553. Toda, N., 1988, Hemolysate inhibits cerebral artery relaxation, J. Cereb. Blood Flow Metab. 8, 46. Toda, N. and T. Okamura, 1990a, Modification by L-N6-monomethyl arginine (L-NMMA) of the response to nerve stimulation in isolated dog mesenteric and cerebral arteries, Jpn. J. Pharmacol. 52, 170. Toda, N. and T. Okamura, 1990b, Possible role of nitric oxide in transmitting information from vasodilator nerve to cerebroarterial muscle, Biochem. Biophys. Res. Commun. 170, 308. Toda, N. and T. Okamura, 1990c, Mechanism underlying the response to vasodilator nerve stimulation in isolated dog and monkey cerebral arteries, Am. J. Physiol. 259, H1511. Toda, N. and T. Okamura, 1991a, Role of nitric oxide in neurally induced cerebroarterial relaxation, J. Pharmacol. Exp. Ther. 258, 1027. Toda, N. and T. Okamura, 1991b, Reciprocal regulation by putatively nitroxidergic and adrenergic nerves of monkey and dog temporal arterial tone, Am. J. Physiol. 261, H1740. Toda, N. and T. Okamura, 1992a, Regulation by nitroxidergic nerve of arterial tone, News Physiol. Sci. 7, 148. Toda, N. and T. Okamura, 1992b, Different susceptibility of vasodilator nerve, endothelium and smooth muscle functions to Ca ++ antagonists in cerebral arteries, J. Pharmacol. Exp. Ther. 261,234. Toda, N. and T. Okamura, 1992c, Mechanism of neurally induced monkey mesenteric artery relaxation and contraction, Hypertension 19, 161. Toda, N., Y. Tanobe and H. Baba, 1991, Suppression by N6-nitro L-arginine of relaxations induced by non-adrenergic, noncholinergic nerve stimualtion in dog duodenal longitudinal muscle, Jpn. J. Pharmacol. 57, 527. Toda, N., K. Ayajiki and T. Okamura, 1993a, Cerebroarterial relaxations mediated by nitric oxide derived from endothelium and vasodilator nerve, J. Vasc. Res. 30, 61. Toda, N., K. Ayajiki, K. Yoshida, H. Kimura and T. Okamura, 1993b, Impairment by damage of the pterygopalatine ganglion of nitroxidergic vasodilator nerve function in canine cerebral and retinal arteries, Circ. Res. 72, 206. Weinheimer, G. and H. Osswald, 1986, Inhibition of endotheliumdependent smooth muscle relaxation by calmodulin antagonists, Naunyn-Schmied. Arch. Pharmacol. 332, 391. Yoshida, K., T. Okamura, H. Kimura, D.S. Bredt, S.H. Snyder and N. Toda, 1993, Nitric oxide synthase-immunoreactive nerve fibers in dog cerebral and peripheral arteries. Brain Res. 629, 67.