Journal of Cerebral Blood Flow and Metabolism 11:366-370 © 1991 The International Society of Cerebral Blood Flow and Metabolism Published by Raven Press, Ltd., New York
Nitric Oxide Mediates the Neurogenic Vasodilation of Bovine Cerebral Arteries Carmen Gonzalez and Carmen Estrada Department of Physiology, School of Medicine, Universidad Autonoma de Madrid, Madrid, Spain
Summary:
Nitric oxide (NO) is a mediator of the vasodi lation induced by a variety of physiological and pharma cological stimuli. The possible role of NO in the relax ation elicited in cerebral arteries by perivascular nerve stimulation has been investigated. Electrical field stimu lation of precontracted bovine cerebral arteries induced a relaxation that was blocked by tetrodotoxin, but not by adrenergic or muscarinic receptor antagonists, suggesting the existence of nonadrenergic, noncholinergic dilator nerves, as has been shown in other species. The relax ation was significantly reduced by the inhibitors of NO
synthesis, NG-monomethyl-L-arginine and nitro-L arginine methyl ester, but not by the enantiomer, � monomethyl-D-arginine. Such a reduction was reversed by L-arginine. In addition, transmural nerve stimulation (TNS)-induced relaxation was potentiated by superoxide dismutase. No response to TNS was observed in arteries without endothelium. These results suggested that neuro genic relaxation of bovine cerebral arteries is mediated by endothelium-derived NO. Key Words: Nitric oxide Neurogenic vasodilation-Transmural nerve stimula tion-Endothelium-Cerebral blood vessels.
Cerebral arteries are endowed with nonadrener gic, noncholinergic nerves whose stimulation in duces a relaxation of arterial segments in vitro (Lee et aI., 1978; Duckles, 1979) and an increase in cor tical blood flow in vivo (Seylaz et aI., 1988; Susuki et aI., 1990). The neurotransmitter(s) involved as well as the mechanism(s) underlying such neuro genic relaxation remain undetermined. Several authors have recently demonstrated that hemolysate or hemoglobin abolished the relaxation induced by transmural nerve stimulation (TNS) of isolated cerebral arteries (Lee et aI., 1984; Toda, 1988; Linnik and Lee, 1989; Gaw et aI., 1990). In other vascular territories, hemoglobin has been shown to prevent endothelium-dependent vascular relaxation as well as the relaxation produced by ni trodilators (Martin et aI., 1985). In both cases, the
inhibitory effect can be explained by the capacity of hemoglobin to bind nitric oxide (NO) (Martin et aI., 1985), which is the final mediator that interacts with the smooth muscle cells (Ignarro et aI., 1987; Palmer et aI., 1987). The ability to synthesize NO has been demon strated not only in endothelial cells (Palmer et aI., 1988), but also in neutrophils (McCall et aI., 1989), macrophages (Marietta et aI., 1988; Stuehr and Nathan, 1989), and in several organs including the adrenal gland (Palacios et aI., 1989) and the central nervous system (Garthwaite et aI., 1989; Knowles et aI., 1989). In all cases, NO is synthesized from the terminal guanidino nitrogen atom(s) of L arginine (Moncada et aI., 1989), and the synthesis can be inhibited by the L-arginine analogs, N'l monomethyl-L-arginine (L-NMM A) and nitro L-arginine methyl ester (L-NAME). In this work, the possible involvement of NO in the neurogenic relaxation of bovine pial arteries has been investi gated using specific inhibitors of the synthesis of NO.
Received June 19, 1990; revised September II, 1990; accepted September 14, 1990. Address correspondence and reprint requests to Dr. C. Es trada at Departamento de Fisiologfa, Facultad de Medicina, UAM, Arzobispo Morcillo I, 28029 Madrid, Spain. Ab b reviations used: ACh, acetylcholine; DAMP, 4-diphenyl acetoxy-N-methylpiperidine; 5-HT, 5-hydroxytryptamine; L-NAME, nitro-L-arginine methyl ester; L-NMMA, �-mono methyl-L-arginine; SNP, sodium nitroprussiate; SOD, superox ide dismutase; TNS, transmural nerve stimulation; TTX, tetro dotoxin; UTP, uridine triphosphate.
METHODS Bovine brains were obtained from a local slaughter house (GIRESA, Madrid, Spain) and transported to the laboratory in a phosphate-buffered saline s ol ution at 4°C.
366
NITRIC OXIDE MEDIATES NEUROGENIC VASODILATION A first-order branch of the anterior cerebral artery of ap proximately I mm in diameter was dissected out and cut into cylindrical segments, 4 mm in length. The rings were suspended on two intraluminal parallel wires, introduced into an organ bath containing a physiological solution, and connected to a Statham strain gauge (Gould Inc., Cleveland, OH, U. S. A. ) for isometric tension recording. The composition of the physiological solution was as fol lows On mM): NaCl, 115; KC1, 4.6; CaCI2, 1. 25; NaHC03, 25; KH2P04, 1. 2; MgS04, 1.2; EDTA, 0. 01; and glucose, II. All experiments were performed in the presence of 10-6 M phentolamine, 10-6 M propranolol, and 5 x 10-6 M indomethacin to avoid adrenergic or prostaglandin-mediated effects. In some segments, the endothelium was removed by gentle rubbing of the intima surface with a small pipe cleaner. Prior to the beginning of each experiment, arteries were precontracted with uri dine triphosphate (UTP) or 5-hydroxytryptamine (5-HT). TNS (200 rnA, 0. 2 ms) was carried out using two par allel silver electrodes, one on each side of the arterial segment, connected to a CS-20 stimulator (Cibertec, Madrid, Spain). The interval between consecutive stimuli was 8-10 min, and two or three reproducible responses were obtained before the addition of the drugs. The effect of acetylcholine (ACh) was tested in each vessel at the beginning and at the end of the experiments to assess the functional integrity of the endothelial layer. Results are expressed as mean ± SD. Data were ana lyzed using Student's t test for paired data. A p value less than 0.05 was considered significant. L-NMMA and ,vG-monomethyl-o-arginine (o-NMMA) were a generous gift from Dr. Moncada (Wellcome, U.K.). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, U. S. A. ).
RESULTS Pial arteries with an active tone (743 ± 375 mg) induced by UTP or 5-HT relaxed when subjected to TNS ( 1-8 Hz). Thirteen of 18 arteries from six an imals relaxed 29 ± 18% of their initial active tension when stimulated at 1 Hz for 40 s. Two arteries tested from one animal contracted, and three seg ments did not respond. The relaxation to TNS was blocked in the presence of 10- 6 M tetrodotoxin (TTX); this drug did not affect the dilation induced by ACh. Addition to the organ bath of the musca rinic antagonists, atropine ( 10 6 M) or 4-diphenyl acetoxy-N-methylpiperidine (DAMP, 10-7 M), in hibited the response to ACh, but not to TNS (not shown). The TNS-elicited relaxation was significantly re duced by the L-arginine:NO synthase inhibitor L NMMA (Figs. 1A,lB, and 2). A complete reversal of the L-NMMA effect was achieved by adding L arginine to the bathing medium (Fig. 2). The enan tiomer o-NMMA was without effect (Fig. 1A and 2). The more potent inhibitor of the synthesis of NO, L-NAME, also significantly reduced the relax ation to TNS; this effect was reversed only in part by L-arginine (Fig. 2). -
367
Both L-NMMA and L-NAME, but not o-NMMA, also had a direct effect on cerebral blood vessels, by causing additional dose-dependent contractions (30 fl-M L-NMMA, 576 ± 3 12 mg, n = 10) of arteries that were previously contracted with UTP or 5-HT (Fig. 1A and B). Superoxide dis mutase (SOD), which has been shown to increase the half-life of NO (Ignarro et aI., 1987; Palmer et aI. , 1987), elicited a relaxation of the cerebral arteries (70 ± 9. 5% of the initial ten sion, n = 6). After recovery of the active tone with additional UTP, a significant enhancement of the dilatory response to TNS was observed (Figs. lC and 2). Six de-endothelized arterial segments, which did not relax in response to ACh, showed no response to TNS (Fig. 1D), and no additional contraction in the presence of L-NMMA (not shown). The integ rity of the muscle layer was confirmed by the relax ation to sodium nitroprussiate ( 10-5 M SNP, 73.5 ± 23% of the initial tension, Fig. 1D) and the contrac tile response to 40 mM K + (2 ± 0.6 g, not shown). DISCUSSION Transmural electrical stimulation induced a relax ation of precontracted bovine pial arteries that was prevented by TTX but not by phentolamine, pro pranolol, atropine, or DAMP, indicating that, as has been reported in other species (Lee et aI., 1978; Duckles, 1979), these arteries are supplied with a nonadrenergic, noncholinergic vasorelaxant inner vation. Although the nature of the neurotransmit teres) involved remains unknown, the present work is an attempt to provide new insights into the mech anism by which such vasodilation takes place. The significant reduction in TNS-induced relax ation in the presence of the L-arginine:NO synthase inhibitors, L-NMMA and L-NAME, and its reversal by L-arginine revealed that the synthesis of NO is involved in the neurogenic dilation of bovine cere bral arteries. The implication of NO production in such vascular response was further confirmed by the enhanced relaxation observed when NO inacti vation was prevented by SOD (Ignarro et aI., 1987; Palmer et aI., 1987). A role of NO in the neurogenic dilation of pial vessels may explain previous results in which the relaxant response to TNS was blocked by hemolysates (Lee et aI., 1984; Toda, 1988) or hemoglobin (Linnik and Lee, 1989). Hemoglobin binds NO and therefore prevents the arterial re sponse to nitrovasodilators as well as the endothe lium-dependent relaxation induced by a variety of stimuli (Martin et aI., 1985). Because hemoglobin reacts with NO in less than 100 ms (KeIrn et aI.,
J Cereb Blood Flow Metab, Vol. 11, No. 3, 1991
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FIG. 1. Representative recordings of the relaxation induced by trans mural nerve stimulation (TNS, S) at 1 Hz (A,C,O) or 8 Hz (B) in bovine cerebral arteries with (A,B,C) or without (D) endothelium. Arteries were precontracted with uridine triphosphate (UTP) and tested for acetylcholine (ACh) response as a control for the functional integrity of the endothelial layer. (A,B) NG_ monomethyl-L-arginine (L-NMMA), but not D-NMMA, produced a dose dependent increase in the resting tone and inhibited the relaxation induced by TN S. This inhibition was reversed when a three times higher concentration of L-arginine was added. (C) Superoxide dismu tase (SO�) produced a decrease in the resting tone of the artery and a potentiation of the TNS-induced re laxation. (D) Arteries without endo thelium did not relax to TNS or ACh, but their response to sodium nitroprussiate (NP) was preserved.
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vided with fast sodium channels. In support of this assumption is the observation that precontracted denervated rat tail arteries with intact endothelium do not relax to electrical field stimulation with pa rameters similar to those used in the present study (Hynes et al. , 1988). Another possibility is that the neurotransmitter(s) released by TNS interacted with endothelial cell re ceptors and induced NO release. Several experi mental data are in accordance with this hypothesis. It has been shown that the endothelium inhibits ar terial contractions induced by stimulation of peri vascular adrenergic nerves (Tesfamarian and Co hen, 1988; Hynes et al. , 1988; Gonzalez et al. , 1990), and that this inhibitory effect is blocked by hemoglobin, suggesting that endothelial NO coun teracts the direct contractile effect of endogenous noradrenaline on smooth muscle (Hynes et al. , 1988; Gonzalez et al. , 1990). The saturation kinetics of such endothelium-mediated inhibition was com patible with a receptor-mediated mechanism (Gonzalez et al., 1990). It could be argued that the
NITRIC OXIDE MEDIATES NEUROGENIC VASODILATION
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thickness of the arterial wall may prevent neuro transmitters from reaching the endothelial cell layer. However, in perfused rabbit ear arteries, it has been reported that a few seconds after perivas cular administration of e H]ACh, a considerable amount of the labeled amine was recovered in the vessel lumen, and radioautography revealed an ac cumulation in the endothelial layer (Gonzalez et aI., 1990). Furthermore, Cohen and Weisbrod ( 1988), using rabbit carotid arteries, demonstrated that, af ter stimulation of adrenergic nerve terminals la beled with e Hlnoradrenaline, 20% of the released radioactivity overflowed into the luminal side of the vessel. Lee ( 1982) and Lee et al. ( 1982) have reported an endothelium-independent neurogenic relaxation in cat and pig cerebral vessels. In these cases, a direct release of NO-or a NO precursor-from the nerve terminals should be considered. NO can be synthe sized in brain synaptosomal cytosol (Knowles et aI., 1989), and NO release has been shown in cere bellar cells when stimulated via N-methyl-D aspartate (NMDA) receptors (Garthwaite et aI., 1989). The influx of calcium induced by nerve de polarization might be the triggering signal for the synthesis of NO, which is strictly dependent on the free calcium concentration within the physiological range (Knowles et aI., 1989). In this regard, it has recently been demonstrated that NO is released by electrical stimulation of the isolated canine ileoco lonic junction (Bult et aI., 1990). Whatever the origin of NO, its involvement in the neurogenic relaxation of cerebral vessels has impor tant physiopathological implications. Cerebrovas-
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cular dilator nerves have been demonstrated to have a functional role in vivo (Seylaz et aI., 1988; Susuki et aI., 1990) and therefore NO may mediate the neurogenic regulation of cerebral blood flow. During subarachnoid hemorrhage (SAH), the re leased NO, which readily diffuses through cell membranes, could be avidly bound by perivascular hemoglobin and thus the effect of both humoral and neurogenic dilators would be substantially reduced. Such a mechanism may explain in part the cerebral vasospasm that follows SAH. Moreover, the re lease of endothelial NO induced by nerve stimula tion suggests that endothelial cell integrity may be an essential factor in maintaining the cerebral blood flow within physiological limits, both in resting con ditions and when an additional blood supply is pro vided by an increased activity of vasodilator nerves.
Acknowledgment: This work was supported by a grant from Fondo de Investigaciones Sanitarias, Spain. The au thors wish to thank Dr. O. Dieguez for providing part of the technical equipment, and Dr. S. Moncada for his help ful advice. REFERENCES Bult H, Boeckxstaens GE, Pelckmans PA, Iordaens FH, Van Maercke YM, Herman AG (1990) Nitric oxide as an inhibi tory non-adrenergic non-cholinergic neurotransmitter. Na
ture (Lond) 345:34&-347 Cannell MB, Sage SO (1989) Bradykinin-evoked changes in cy tosolic calcium and membrane currents in cultured bovine pulmonary artery endothelial cells. J Physiol (Lond)
419:555-568 Cohen RA, Weisbrod RM (1988) Endothelium inhibits norepi nephrine release from adrenergic nerves of rabbit carotid artery. AmJ PhysioI254:H871-H878
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Duckles SP ( 1979) Neurogenic dilator and constrictor responses of pial arteries in vitro. Differences between dogs and sheep.
Circ Res44:482-490 Garthwaite J, Garthwaite G, Palmer RMJ, Moncada S ( 1989) NMDA receptor activation induces nitric oxide synthesis from arginine in rat brain slices. EurJ Pharmacoll72:413-
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4 16 Gaw AJ, Wadsworth RM, Humphrey PPA ( 1990) Neurotrans mission in the sheep middle cerebral artery: modulation of responses by 5-HT and haemolysate. J Cereb Blood Flow
Metab 10:409-4 16 Gonzalez MC, Martin C, Hamel E, Galea E, Gomez B, Lluch S, Estrada C ( 1990) Endothelial cells inhibit the vascular re sponse to adrenergic nerve stimulation by a receptor mediated mechanism. CanJ PhysiolPharmacol68: 104-109 Hynes MR, Dang H, Duckles SP ( 1988) Contractile responses to adrenergic nerve stimulation are enhanced with removal of endothelium in rat caudal artery. Lf i eSci42:357-365 Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G (1987) Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. ProcNatl Acad Sci USA
84:9265-9269 Kelm M, Feelisch M, Spahr R, Piper HM, Noack E, Schrader J ( 1988) Quantitative and kinetic characterization of nitric ox ide and EDRF released from cultured endothelial cells. Bio
chern BiophysRes Commun154:236-244 Knowles RG, Palacios M, Palmer RMJ, Moncada S (1989) For mation of nitric oxide from L-arginine in the central nervous system: A transduction mechanism for stimulation of the soluble guanylate cyclase. Proc Natl Acad Sci USA 86:
5 159-5162 Lee TJ-F ( 1982) Cholinergic mechanism in the large cat cerebral artery. CircRes50:870-879 Lee TJ-F, Hume WR, Su C, Bevan JA ( 1978) Neurogenic va sodilation of cat cerebral arteries. CirRes 42:535-542 Lee TJ-F, Kinkead LR, Sarwinski S ( 1982) Norepinephrine and acetylcholine transmitter mechanisms in large cerebral ar teries of the pig. J Cereb Blood Flow Metab 2:439-450 Lee TJ-F, McIlhany MP, Sarwinski S ( 1984) Erythrocyte ex tracts enhance neurogenic vasoconstriction of dog cerebral arteries in vitro. J Cereb Blood Flow Metab 4:474-476 Linnik MD, Lee TJ-F ( 1989) Effect of hemoglobin on neurogenic responses and cholinergic parameters in porcine cerebral ar teries. J Cereb Blood Flow Metab 9:219-225 Marietta MA, Yoon PS, Iyengar R, Leaf CD, Wishnok JS (1988)
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Macrophage oxidation of L-arginine to nitrite and nitrate: Nitric oxide is an intermediate. Biochemistry 27:8706-87 1 1 Martin W , Villani GM, Jothianandan D , Furchgott R F ( 1985) Selective blockade of endothelium-dependent and giyceryl trinitrate-induced relaxation by hemoglobin and by methyl ene blue in the rabbit aorta. J PharmacolExp Ther232:708-
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McCall TB, Boughton-Smith NK, Palmer RMJ, Whittle BJR, Moncada S ( 1989) Synthesis of nitric oxide from L-arginine by neutrophils. BiochemJ 26 1:293-296 Moncada S, Palmer RM, Higgs A ( 1989) Biosynthesis of nitric oxide from L-arginine. A pathway for the regulation of cell function and communication. BiochemPharmacoI38:1709-
1715 Palacios M, Knowles RG, Palmer RMJ, Moncada S ( 1989) Nitric oxide from L-arginine stimulates the soluble guanylate cy clase in adrenal glands. BiochemBiophysResCommun 165:
802-809 Palmer RMJ, Ferrige AG, Moncada S ( 1987) Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature (Lond) 327:524-526 Palmer RMJ, Ashton DS, Moncada S ( 1988) Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature(Lond)
333 :664-666 Rees DD, Palmer RMJ, Hodson HF, Moncada S ( 1989) A spe cific inhibitor of nitric oxide formation from L-arginine at tenuates endothelium-dependent relaxation. BrJ Pharmacol
96:418-424 Seylaz J, Hara H, Pinard E, Mraovitch S, MacKenzie ET, Ed vinsson L ( 1988) Effect of stimulation of the sphenopalatine ganglion on cortical blood flow in the rat. J Cereb Blood
Flow Metab 8:875-878 Stuehr DJ, Nathan CF ( 1989) Nitric oxide. A macrophage prod uct responsible for cytostasis and respiratory inhibition in tumor target cells. J ExpMed 169:1543-1555 Susuki N, Hardebo JE, Kahrstrom J, Owman CH ( 1990) Selec tive electrical stimulation of postganglionic cerebrovascular parasympathetic nerve fibers originating from the spheno palatine ganglion enhances cortical blood flow in the rat. J
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720-725 Toda N ( 1988) Hemolysate inhibits cerebral artery relaxation. J
Cereb Blood Flow Metab 8:46-53
Nitric Oxide Mediates the Neurogenic Vasodilation of Bovine Cerebral Arteries Carmen Gonzalez and Carmen Estrada Department of Physiology, School of Medicine, Universidad Autonoma de Madrid, Madrid, Spain
Summary:
Nitric oxide (NO) is a mediator of the vasodi lation induced by a variety of physiological and pharma cological stimuli. The possible role of NO in the relax ation elicited in cerebral arteries by perivascular nerve stimulation has been investigated. Electrical field stimu lation of precontracted bovine cerebral arteries induced a relaxation that was blocked by tetrodotoxin, but not by adrenergic or muscarinic receptor antagonists, suggesting the existence of nonadrenergic, noncholinergic dilator nerves, as has been shown in other species. The relax ation was significantly reduced by the inhibitors of NO
synthesis, NG-monomethyl-L-arginine and nitro-L arginine methyl ester, but not by the enantiomer, � monomethyl-D-arginine. Such a reduction was reversed by L-arginine. In addition, transmural nerve stimulation (TNS)-induced relaxation was potentiated by superoxide dismutase. No response to TNS was observed in arteries without endothelium. These results suggested that neuro genic relaxation of bovine cerebral arteries is mediated by endothelium-derived NO. Key Words: Nitric oxide Neurogenic vasodilation-Transmural nerve stimula tion-Endothelium-Cerebral blood vessels.
Cerebral arteries are endowed with nonadrener gic, noncholinergic nerves whose stimulation in duces a relaxation of arterial segments in vitro (Lee et aI., 1978; Duckles, 1979) and an increase in cor tical blood flow in vivo (Seylaz et aI., 1988; Susuki et aI., 1990). The neurotransmitter(s) involved as well as the mechanism(s) underlying such neuro genic relaxation remain undetermined. Several authors have recently demonstrated that hemolysate or hemoglobin abolished the relaxation induced by transmural nerve stimulation (TNS) of isolated cerebral arteries (Lee et aI., 1984; Toda, 1988; Linnik and Lee, 1989; Gaw et aI., 1990). In other vascular territories, hemoglobin has been shown to prevent endothelium-dependent vascular relaxation as well as the relaxation produced by ni trodilators (Martin et aI., 1985). In both cases, the
inhibitory effect can be explained by the capacity of hemoglobin to bind nitric oxide (NO) (Martin et aI., 1985), which is the final mediator that interacts with the smooth muscle cells (Ignarro et aI., 1987; Palmer et aI., 1987). The ability to synthesize NO has been demon strated not only in endothelial cells (Palmer et aI., 1988), but also in neutrophils (McCall et aI., 1989), macrophages (Marietta et aI., 1988; Stuehr and Nathan, 1989), and in several organs including the adrenal gland (Palacios et aI., 1989) and the central nervous system (Garthwaite et aI., 1989; Knowles et aI., 1989). In all cases, NO is synthesized from the terminal guanidino nitrogen atom(s) of L arginine (Moncada et aI., 1989), and the synthesis can be inhibited by the L-arginine analogs, N'l monomethyl-L-arginine (L-NMM A) and nitro L-arginine methyl ester (L-NAME). In this work, the possible involvement of NO in the neurogenic relaxation of bovine pial arteries has been investi gated using specific inhibitors of the synthesis of NO.
Received June 19, 1990; revised September II, 1990; accepted September 14, 1990. Address correspondence and reprint requests to Dr. C. Es trada at Departamento de Fisiologfa, Facultad de Medicina, UAM, Arzobispo Morcillo I, 28029 Madrid, Spain. Ab b reviations used: ACh, acetylcholine; DAMP, 4-diphenyl acetoxy-N-methylpiperidine; 5-HT, 5-hydroxytryptamine; L-NAME, nitro-L-arginine methyl ester; L-NMMA, �-mono methyl-L-arginine; SNP, sodium nitroprussiate; SOD, superox ide dismutase; TNS, transmural nerve stimulation; TTX, tetro dotoxin; UTP, uridine triphosphate.
METHODS Bovine brains were obtained from a local slaughter house (GIRESA, Madrid, Spain) and transported to the laboratory in a phosphate-buffered saline s ol ution at 4°C.
366
NITRIC OXIDE MEDIATES NEUROGENIC VASODILATION A first-order branch of the anterior cerebral artery of ap proximately I mm in diameter was dissected out and cut into cylindrical segments, 4 mm in length. The rings were suspended on two intraluminal parallel wires, introduced into an organ bath containing a physiological solution, and connected to a Statham strain gauge (Gould Inc., Cleveland, OH, U. S. A. ) for isometric tension recording. The composition of the physiological solution was as fol lows On mM): NaCl, 115; KC1, 4.6; CaCI2, 1. 25; NaHC03, 25; KH2P04, 1. 2; MgS04, 1.2; EDTA, 0. 01; and glucose, II. All experiments were performed in the presence of 10-6 M phentolamine, 10-6 M propranolol, and 5 x 10-6 M indomethacin to avoid adrenergic or prostaglandin-mediated effects. In some segments, the endothelium was removed by gentle rubbing of the intima surface with a small pipe cleaner. Prior to the beginning of each experiment, arteries were precontracted with uri dine triphosphate (UTP) or 5-hydroxytryptamine (5-HT). TNS (200 rnA, 0. 2 ms) was carried out using two par allel silver electrodes, one on each side of the arterial segment, connected to a CS-20 stimulator (Cibertec, Madrid, Spain). The interval between consecutive stimuli was 8-10 min, and two or three reproducible responses were obtained before the addition of the drugs. The effect of acetylcholine (ACh) was tested in each vessel at the beginning and at the end of the experiments to assess the functional integrity of the endothelial layer. Results are expressed as mean ± SD. Data were ana lyzed using Student's t test for paired data. A p value less than 0.05 was considered significant. L-NMMA and ,vG-monomethyl-o-arginine (o-NMMA) were a generous gift from Dr. Moncada (Wellcome, U.K.). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, U. S. A. ).
RESULTS Pial arteries with an active tone (743 ± 375 mg) induced by UTP or 5-HT relaxed when subjected to TNS ( 1-8 Hz). Thirteen of 18 arteries from six an imals relaxed 29 ± 18% of their initial active tension when stimulated at 1 Hz for 40 s. Two arteries tested from one animal contracted, and three seg ments did not respond. The relaxation to TNS was blocked in the presence of 10- 6 M tetrodotoxin (TTX); this drug did not affect the dilation induced by ACh. Addition to the organ bath of the musca rinic antagonists, atropine ( 10 6 M) or 4-diphenyl acetoxy-N-methylpiperidine (DAMP, 10-7 M), in hibited the response to ACh, but not to TNS (not shown). The TNS-elicited relaxation was significantly re duced by the L-arginine:NO synthase inhibitor L NMMA (Figs. 1A,lB, and 2). A complete reversal of the L-NMMA effect was achieved by adding L arginine to the bathing medium (Fig. 2). The enan tiomer o-NMMA was without effect (Fig. 1A and 2). The more potent inhibitor of the synthesis of NO, L-NAME, also significantly reduced the relax ation to TNS; this effect was reversed only in part by L-arginine (Fig. 2). -
367
Both L-NMMA and L-NAME, but not o-NMMA, also had a direct effect on cerebral blood vessels, by causing additional dose-dependent contractions (30 fl-M L-NMMA, 576 ± 3 12 mg, n = 10) of arteries that were previously contracted with UTP or 5-HT (Fig. 1A and B). Superoxide dis mutase (SOD), which has been shown to increase the half-life of NO (Ignarro et aI., 1987; Palmer et aI. , 1987), elicited a relaxation of the cerebral arteries (70 ± 9. 5% of the initial ten sion, n = 6). After recovery of the active tone with additional UTP, a significant enhancement of the dilatory response to TNS was observed (Figs. lC and 2). Six de-endothelized arterial segments, which did not relax in response to ACh, showed no response to TNS (Fig. 1D), and no additional contraction in the presence of L-NMMA (not shown). The integ rity of the muscle layer was confirmed by the relax ation to sodium nitroprussiate ( 10-5 M SNP, 73.5 ± 23% of the initial tension, Fig. 1D) and the contrac tile response to 40 mM K + (2 ± 0.6 g, not shown). DISCUSSION Transmural electrical stimulation induced a relax ation of precontracted bovine pial arteries that was prevented by TTX but not by phentolamine, pro pranolol, atropine, or DAMP, indicating that, as has been reported in other species (Lee et aI., 1978; Duckles, 1979), these arteries are supplied with a nonadrenergic, noncholinergic vasorelaxant inner vation. Although the nature of the neurotransmit teres) involved remains unknown, the present work is an attempt to provide new insights into the mech anism by which such vasodilation takes place. The significant reduction in TNS-induced relax ation in the presence of the L-arginine:NO synthase inhibitors, L-NMMA and L-NAME, and its reversal by L-arginine revealed that the synthesis of NO is involved in the neurogenic dilation of bovine cere bral arteries. The implication of NO production in such vascular response was further confirmed by the enhanced relaxation observed when NO inacti vation was prevented by SOD (Ignarro et aI., 1987; Palmer et aI., 1987). A role of NO in the neurogenic dilation of pial vessels may explain previous results in which the relaxant response to TNS was blocked by hemolysates (Lee et aI., 1984; Toda, 1988) or hemoglobin (Linnik and Lee, 1989). Hemoglobin binds NO and therefore prevents the arterial re sponse to nitrovasodilators as well as the endothe lium-dependent relaxation induced by a variety of stimuli (Martin et aI., 1985). Because hemoglobin reacts with NO in less than 100 ms (KeIrn et aI.,
J Cereb Blood Flow Metab, Vol. 11, No. 3, 1991
C. GONZALEZ AND C. ESTRADA
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1988), it should be expected that its effect is similar to that of agents that prevent the generation of NO. The inhibitors of the synthesis of NO (Rees et al. , 1989, and present results) as well as hemoglobin (Martin et al., 1985; Linnik and Lee, 1989) also pro duced a direct contraction in vessels with an active tone elicited by other drugs. This direct effect could be explained if NO were tonically released, either spontaneously or due to the stimulation of endothe lial cells by the vasoconstrictors used to provide the active tone. No TNS-induced relaxation was observed in bo vine arteries in which the endothelium had been mechanically removed, indicating that, in our ex perimental conditions, NO was released from endo thelial cells. The NO release could be due to a direct depolarization of these cells. However, such a pos sibility is unlikely, because endothelial cells are de void of voltage-dependent calcium channels (Can nell and Sage, 1989). Also, the blockade of the re laxing effect by TTX suggests rather that the depolarization occurs in a nerve membrane pro-
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FIG. 1. Representative recordings of the relaxation induced by trans mural nerve stimulation (TNS, S) at 1 Hz (A,C,O) or 8 Hz (B) in bovine cerebral arteries with (A,B,C) or without (D) endothelium. Arteries were precontracted with uridine triphosphate (UTP) and tested for acetylcholine (ACh) response as a control for the functional integrity of the endothelial layer. (A,B) NG_ monomethyl-L-arginine (L-NMMA), but not D-NMMA, produced a dose dependent increase in the resting tone and inhibited the relaxation induced by TN S. This inhibition was reversed when a three times higher concentration of L-arginine was added. (C) Superoxide dismu tase (SO�) produced a decrease in the resting tone of the artery and a potentiation of the TNS-induced re laxation. (D) Arteries without endo thelium did not relax to TNS or ACh, but their response to sodium nitroprussiate (NP) was preserved.
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vided with fast sodium channels. In support of this assumption is the observation that precontracted denervated rat tail arteries with intact endothelium do not relax to electrical field stimulation with pa rameters similar to those used in the present study (Hynes et al. , 1988). Another possibility is that the neurotransmitter(s) released by TNS interacted with endothelial cell re ceptors and induced NO release. Several experi mental data are in accordance with this hypothesis. It has been shown that the endothelium inhibits ar terial contractions induced by stimulation of peri vascular adrenergic nerves (Tesfamarian and Co hen, 1988; Hynes et al. , 1988; Gonzalez et al. , 1990), and that this inhibitory effect is blocked by hemoglobin, suggesting that endothelial NO coun teracts the direct contractile effect of endogenous noradrenaline on smooth muscle (Hynes et al. , 1988; Gonzalez et al. , 1990). The saturation kinetics of such endothelium-mediated inhibition was com patible with a receptor-mediated mechanism (Gonzalez et al., 1990). It could be argued that the
NITRIC OXIDE MEDIATES NEUROGENIC VASODILATION
369
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methyl-D-arginine (D-NMMA), NG-monomethyl L-arginine (L-NMMA), nitro-L-arginine methyl es ter (L-NAME), and superoxide dismutase (SOD) on the relaxation induced by transmural nerve stimulation of bovine cerebral arteries. The re versal of the inhibitory effects by L-arginine is also shown. Results are expressed as percent of the control relaxation measured in each artery when stimulated with 1 Hz. Values are the mean ± SO of the data obtained in four to six arteries. *p < 0.05; **p < 0.0 1 , when compared with the control.
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thickness of the arterial wall may prevent neuro transmitters from reaching the endothelial cell layer. However, in perfused rabbit ear arteries, it has been reported that a few seconds after perivas cular administration of e H]ACh, a considerable amount of the labeled amine was recovered in the vessel lumen, and radioautography revealed an ac cumulation in the endothelial layer (Gonzalez et aI., 1990). Furthermore, Cohen and Weisbrod ( 1988), using rabbit carotid arteries, demonstrated that, af ter stimulation of adrenergic nerve terminals la beled with e Hlnoradrenaline, 20% of the released radioactivity overflowed into the luminal side of the vessel. Lee ( 1982) and Lee et al. ( 1982) have reported an endothelium-independent neurogenic relaxation in cat and pig cerebral vessels. In these cases, a direct release of NO-or a NO precursor-from the nerve terminals should be considered. NO can be synthe sized in brain synaptosomal cytosol (Knowles et aI., 1989), and NO release has been shown in cere bellar cells when stimulated via N-methyl-D aspartate (NMDA) receptors (Garthwaite et aI., 1989). The influx of calcium induced by nerve de polarization might be the triggering signal for the synthesis of NO, which is strictly dependent on the free calcium concentration within the physiological range (Knowles et aI., 1989). In this regard, it has recently been demonstrated that NO is released by electrical stimulation of the isolated canine ileoco lonic junction (Bult et aI., 1990). Whatever the origin of NO, its involvement in the neurogenic relaxation of cerebral vessels has impor tant physiopathological implications. Cerebrovas-
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cular dilator nerves have been demonstrated to have a functional role in vivo (Seylaz et aI., 1988; Susuki et aI., 1990) and therefore NO may mediate the neurogenic regulation of cerebral blood flow. During subarachnoid hemorrhage (SAH), the re leased NO, which readily diffuses through cell membranes, could be avidly bound by perivascular hemoglobin and thus the effect of both humoral and neurogenic dilators would be substantially reduced. Such a mechanism may explain in part the cerebral vasospasm that follows SAH. Moreover, the re lease of endothelial NO induced by nerve stimula tion suggests that endothelial cell integrity may be an essential factor in maintaining the cerebral blood flow within physiological limits, both in resting con ditions and when an additional blood supply is pro vided by an increased activity of vasodilator nerves.
Acknowledgment: This work was supported by a grant from Fondo de Investigaciones Sanitarias, Spain. The au thors wish to thank Dr. O. Dieguez for providing part of the technical equipment, and Dr. S. Moncada for his help ful advice. REFERENCES Bult H, Boeckxstaens GE, Pelckmans PA, Iordaens FH, Van Maercke YM, Herman AG (1990) Nitric oxide as an inhibi tory non-adrenergic non-cholinergic neurotransmitter. Na
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