Phylogenesis of constitutively formed nitric oxide in non-mammals.

It is widely recognized that nitric oxide (NO) in mammalian tissues is produced from L-arginine via catalysis by NO synthase (NOS) isoforms such as neuronal NOS (nNOS) and endothelial NOS (eNOS) that are constitutively expressed mainly in the central and peripheral nervous system and vascular endothelial cells, respectively. This review concentrates only on these constitutive NOS (cNOS) isoforms while excluding information about iNOS, which is induced mainly in macrophages upon stimulation by cytokines and polysaccharides. The NO signaling pathway plays a crucial role in the functional regulation of mammalian tissues and organs. Evidence has also been accumulated for the role of NO in invertebrates and non-mammalian vertebrates. Expression of nNOS in the brain and peripheral nervous system is widely determined by staining with NADPH (reduced nicotinamide adenine dinucleotide phosphate) diaphorase or NOS immunoreactivity, and functional roles of NO formed by nNOS are evidenced in the early phylogenetic stages (invertebrates and fishes). On the other hand, the endothelium mainly produces vasodilating prostanoids rather than NO or does not liberate endothelium-derived relaxing factor (EDRF) (fishes), and the ability of endothelial cells to liberate NO is observed later in phylogenetic stages (amphibians). This review article summarizes various types of interesting information obtained from lower organisms (invertebrates, fishes, amphibians, reptiles, and birds) about the properties and distribution of nNOS and eNOS and also the roles of NO produced by the cNOS as an important intercellular signaling molecule.

Relatively selective neuronal nitric oxide synthase inhibition by 7-nitroindazole in monkey isolated cerebral arteries.

The selectivity of 7-nitroindazole in inhibiting endothelial and neuronal nitric oxide synthases (eNOS and nNOS) was investigated by comparing its inhibitory action on relaxations mediated by nitric oxide (NO) in response to stimulation of perivascular nerves and in response to histamine in monkey cerebral artery strips. 7-Nitroindazole at 2 x 10(-5) M moderately attenuated the response to transmural electrical stimulation and to nicotine, but did to alter the endothelium-dependent relaxation in response to histamine in cimetidine-treated strips. Raising the concentration of 7-nitroindazole to 10(-4) M abolished the neurogenic response, partially inhibited the histamine-induced relaxation, but did not affect the response to NO. It is concluded that 7-nitroindazole is a relatively selective nNOS inhibitor; however, at high concentrations, it inhibits eNOS in monkey cerebral arteries.

Modifications by sumatriptan and acetylcholine of nitric oxide-mediated neurogenic dilatation in dog cerebral arteries.

Canine cerebral arterial strips denuded of endothelium responded to nicotine and transmural electrical stimulation with relaxations, which were abolished by NG-nitro-L-arginine and methylene blue. Magnitudes of relaxation did not differ in the arteries contracted with prostaglandin F2alpha and sumatriptan, an effective therapeutic of migraine. Sumatriptan concentration-dependently contracted the arteries responding to 2 Hz stimulation with persistent relaxations, and the concentration of this 5-HT1B/1D/1F receptor agonist to overcome the relaxation averaged 1.06 x 10(-7) M. Acetylcholine inhibited the response to nerve stimulation due possibly to its action on prejunctional nitroxidergic nerves; the inhibition did not differ in the arteries contracted with prostaglandin F2alpha and K+. It appears that sumatriptan does not interfere with the release of nitric oxide from nerves but counteracts the neurogenic relaxation by functional antagonistic action on smooth muscle. Prejunctional inhibition by muscarinic receptor activation is unlikely associated with opening of neuronal K+ channels.

Ginsenoside potentiates NO-mediated neurogenic vasodilatation of monkey cerebral arteries.

The aqueous extract of the Panax ginseng (GE) potentiated the relaxation induced by transmural electrical stimulation or nicotine in monkey cerebral arterial strips denuded of the endothelium and partially contracted with prostaglandin F(2 alpha). The response to electrical stimulation was abolished by tetrodotoxin, whereas that to nicotine was suppressed by hexamethonium. N(G)-nitro-L-arginine abolished both of the neurogenic relaxation. Atropine did not alter the potentiating effect of GE. Relaxations induced by exogenous NO were unaffected by GE. The enhancement by GE, of the neurogenic response, appears to be associated with increment in the synthesis or release of NO from the perivascular nerve. Blockade of muscarinic prejunctional inhibition, superoxide scavenging action and phosphodiesterase inhibition are not involved.

Hypothermia on NO-mediated neurogenic relaxation and on hypoxic inhibition in the response of canine cerebral arteries.

Cerebral arteries are innervated by nitric oxide (NO)-mediated vasodilator nerves, and hypoxia has been shown to attenuate neurogenic vasorelaxation. The present study examines the effects of hypothermia on neurogenic vasorelaxation and on the hypoxia-induced inhibition of the neurogenic vasorelaxation response. In isolated canine cerebral arteries, relaxant responses to transmural electrical stimulation (5 Hz for 40 s), mediated via NO synthesized from L-arginine, were not influenced by lowering the bathing media temperature from 37 degrees C to 30 degrees C but were attenuated at 25 degrees C. On the other hand, relaxations caused by nicotine and exogenous NO were not significantly attenuated but were prolonged by cooling to 25 degrees C. The responses associated with nerve stimulation by electrical pulses or nicotine were depressed by hypoxia (from about 500 mmHg of partial O2 pressure to about 45 mmHg) under normothermia. However, hypothermia at 25 degrees C prevented the inhibition by hypoxia of the neurogenic relaxation. It is concluded that the hypothermia-induced inhibition in the response to electrical nerve stimulation is not associated with a decreased synthesis and release of NO in vasodilator nerves nor with a reduced ability of smooth muscle to relax in response to NO. Interference with the propagation of action potentials might be involved in the inhibition via a fall of temperature. The fact that the hypoxia-induced impairment of vasodilator nerve function was prevented by cooling may partially explain the efficacy of hypothermia in protecting against ischemic neuronal injury in the brain.

Effects of calcium antagonists on the nitrergic nerve function in canine corpus cavernosum.

Effects of calcium antagonists on nitrergic nerve function were examined in the isolated canine corpus cavernosum. In the cavernous strips precontracted with phenylephrine, transmural electrical stimulation elicited frequency-dependent (2 - 5 Hz) relaxations that were abolished by N(G)-nitro-L-arginine (10(-5) M), a nitric oxide (NO) synthase inhibitor; 1H[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ, 10(-6) M), a soluble guanylate cyclase inhibitor; and tetrodotoxin (3 x 10(-7) M). The relaxations were not affected by treatment with nifedipine or nicardipine (10(-8) - 10(-6) M), L-type specific calcium channel inhibitors, but were significantly inhibited by amlodipine or cilnidipine, inhibitors of L- plus N-type calcium channels, in a concentration-related manner (10(-7) - 10(-6) M). All of the inhibitors used did not affect the relaxations induced by exogenous NO (acidifed NaNO2). These findings suggest that N-type, but not L-type, calcium channels are responsible for increasing cytosolic free calcium, a prerequisite for the synthesis of NO, in the nitrergic dilator nerves innervating the corpus cavernosum.

Mechanisms underlying endothelium-dependent, nitric oxide/prostacyclin-independent, acetylcholine-induced relaxation in canine corpus cavernosum.

The mechanisms underlying endothelium-dependent and nitric oxide (NO)/prostacyclin-independent, acetylcholine-induced relaxation in isolated canine corpus cavernosum were investigated. In isolated canine corpus cavernous strips treated with indomethacin (10(-6) M) and N(G)-nitro-L-arginine (10(-4) M), acetylcholine produced relaxations in a concentration-dependent manner. The relaxations were not affected by treatment with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide, sodium salt (carboxy PTIO, 3 x 10(-4) M), glibenclamide (10(-6) M), iberiotoxin (10(-7) M) or charybdotoxin (10(-7) M), but were abolished or reversed to contractions by treatment with apamin (10(-8) M) or scyllatoxin (10(-8) M). Levcromakalim (10(-7)-10(-6) M) induced a concentration-dependent relaxation which was abolished by treatment with glibenclamide (10(-6) M), but was not affected by treatment with apamin (10(-8) M) or scyllatoxin (10(-8) M). These findings indicate that endothelial cells of canine corpus cavernosum have an ability to produce a relaxing substance(s) other than NO or prostacyclin in response to acetylcholine. The substance(s) may open solely small conductance Ca2+-dependent K+ channels.

Comparison of endothelium-dependent relaxation in carotid arteries from Japanese white and Watanabe heritable hyperlipidemic rabbits.

Modifications by atherosclerosis of endothelium-dependent and -independent relaxations were evaluated in carotid arteries isolated from Watanabe heritable hyperlipidemic (WHHL; age 20-29 months) and age-matched Japanese white (JW) rabbits. Marked, patchy atherosclerotic lesions were observed in all WHHL rabbit arteries. Endothelium-dependent relaxations induced by acetylcholine, partly depressed by N(G)-nitro-L-arginine (L-NA), were significantly inhibited in the WHHL rabbit arteries with atherosclerosis, compared with those in the arteries without atherosclerotic lesions from JW and WHHL rabbits. No difference was observed in the relaxation caused by superoxide dismutase in these arteries. Conversely, endothelium-dependent relaxations by substance P were greater in the arteries with and without atherosclerosis from WHHL rabbits than in the arteries from JW rabbits. Endothelium-independent relaxations elicited by sodium nitroprusside and 2,2-(hydroxynitrosohydrazino)bis-ethanamine (NOC18) did not differ in the arteries from JW and WHHL rabbits. The responses to acetylcholine and substance P of JW rabbit arteries with the endothelium were not attenuated by treatment with pertussis toxin. L-NA-resistant, endothelium-dependent relaxations by substance P were almost abolished by charybdotoxin, and atherosclerosis did not alter the response. It is concluded that endothelial functions, evaluated by substance P, in rabbit carotid arteries are not impaired by atherosclerosis and by long exposure to hyperlipidemia in vivo. Dysfunction of muscarinic receptors may be involved in the depressed response to acetylcholine. As far as the arteries used in the present study are concerned, responses mediated possibly by endothelium-derived hyperpolarizing factor (EDHF) are unlikely to be modulated by atherosclerosis.

Mechanisms underlying contraction and relaxation induced by nerve stimulation in monkey uterine arteries.

We investigated the mechanisms of contractile and relaxant responses to nerve stimulation by electrical pulses and nicotine in isolated monkey uterine artery strips denuded of the endothelium. In the strips contracted with prostaglandin F(2alpha), transmural electrical stimulation (5 Hz, 40 s) produced a contraction which was partially attenuated by prazosin and abolished or reversed to a relaxation by additional treatment with alpha,beta-methylene ATP. The relaxation was abolished by N(G)-nitro-L-arginine (L-NA) and restored by L-arginine but not by D-arginine. Atropine, D-NA, aminophylline and suramin, an inhibitor of P(2Y) purinoceptors, were without effect. The neurogenic relaxation was abolished by 1H-(1,2, 4)oxadiazolo(4,3)quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase. Nicotine (10(-4) mol/l) elicited contraction or relaxation of uterine arteries; the contraction was reversed by combined treatment with prazosin and alpha,beta-methylene ATP. Nicotine-induced relaxations were abolished by L-NA and restored by L-arginine. The relaxation induced by exogenously applied NO (acidified NaNO(2) solution) was not influenced by L-NA but abolished by ODQ. It is concluded that contractions induced by nerve stimulation are mediated by norepinephrine and ATP liberated from sympathetic nerves that stimulate alpha(1)-adrenoceptors and P(2x) purinoceptors, respectively. The neurogenic relaxation seems to be mediated exclusively by nitric oxide synthesized from L-arginine in perivascular nerves that activates guanylate cyclase and produces cyclic GMP in smooth muscle.

Evidence for nitroxidergic innervation in monkey ophthalmic arteries in vivo and in vitro.

In anesthetized monkeys, electrical stimulation (ES) of the pterygopalatine or geniculate ganglion dilated the ipsilateral ophthalmic artery (OA). The induced vasodilatation was unaffected by phentolamine but potentiated by atropine. Intravenous N(G)-nitro-L-arginine (L-NNA) abolished the response, which was restored by L-arginine. Hexamethonium-abolished vasodilator responses induced solely by geniculate ganglionic stimulation. The L-NNA constricted OA; L-arginine reversed the effect. Destruction of the pterygopalatine ganglion constricted the ipsilateral artery. Helical strips of OA isolated under deep anesthesia from monkeys, denuded of endothelium, responded to transmural ES with relaxations, which were abolished by tetrodotoxin and L-NNA but were potentiated by atropine. It is concluded that neurogenic vasodilatation of monkey OA is mediated by nerve-derived nitric oxide (NO), and the nerve is originated from the ipsilateral pterygopalatine ganglion that is innervated by cholinergic neurons from the brain stem via the geniculate ganglion. The OA appears to be dilated by mediation of NO continuously liberated from nerves that receive tonic discharges from the vasomotor center. Acetylcholine liberated from postganglionic cholinergic nerves would impair the release of neurogenic NO.

Preganglionic and postganglionic neurons responsible for cerebral vasodilation mediated by nitric oxide in anesthetized dogs.

The authors performed investigations to functionally determine the route of efferent innervation in vivo responsible for cerebral vasodilation mediated by nitric oxide (NO). In anesthetized beagles, electrical stimulation of the pterygopalatine ganglion vasodilated ipsilateral cerebral arteries such as the middle cerebral and posterior communicating arteries. Intravenous injections of NG-nitro-L-arginine (L-NA) markedly inhibited the response to nerve stimulation, and the effect was reversed by L-arginine. Stimulation of the proximal portion of the greater superficial petrosal nerve, upstream of the pterygopalatine ganglion, also produced cerebral vasodilation, which was abolished by L-NA and restored by L-arginine. Treatment with hexamethonium abolished the response to stimulation of the petrosal nerve but did not affect the response to pterygopalatine ganglion stimulation. Destruction of the pterygopalatine ganglion by cauterization constricted the cerebral arteries. Postganglionic denervation abolished the vasodilation, lacrimation, and nasal secretion induced on the ipsilateral side by stimulation of the pterygopalatine ganglion and petrosal nerve. The vasodilator response was suppressed by L-NA but unaffected by atropine, whereas lacrimation and nasal secretion were abolished solely by atropine. It is concluded that postganglionic neurons from the pterygopalatine ganglion play crucial roles in cerebral vasodilation mediated by NO from the nerve, and preganglionic neurons, possibly from the superior salivatory nucleus through the greater superficial petrosal nerve, innervate the pterygopalatine ganglion. Tonic discharges from the vasomotor center participate significantly in the maintenance of cerebral vasodilation.

Cerebral vasodilatation induced by stimulation of the pterygopalatine ganglion and greater petrosal nerve in anesthetized monkeys.

Although brain cell viability depends largely on cerebral circulation, mechanisms of blood flow control, such as autoregulation, or of the pathogenesis of functionally impaired blood supply to brain regions, such as in cerebral vasospasm after subarachnoid hemorrhage, have not been clearly defined. Our recent studies support the hypothesis that nitric oxide, released from nitrergic nerves, plays a crucial role as a neurotransmitter in vasodilating cerebral arteries from primate and subprimate mammals. In the present study, we demonstrated, by using arterial angiography, that electrical stimulation of the pterygopalatine ganglion produced vasodilatation of ipsilateral cerebral arteries of anesthetized Japanese monkeys. The response was abolished by intravenous injections of N(G)-nitro-L-arginine, a nitric oxide synthase inhibitor. Denervation of the ganglion elicited cerebral vasoconstriction, indicating that vasodilator nerves from the vasomotor center were tonically active. Stimulation of the greater petrosal nerve, upstream of the pterygopalatine ganglion, also elicited cerebral vasodilatation, which was abolished by treatment with the nitric oxide synthase inhibitor and with hexamethonium, indicating that the nerve is in connection via synapses with the nitrergic nerve innervating cerebral arteries. Endogenous nitric oxide released from the nerve may contribute to the maintenance of blood flow in major cerebral arteries necessary to supply blood to the different brain regions. Without this influence, cerebral arteries might be constricted to the extent that blood flow is impeded. This is the first direct evidence indicating an important role of nitric oxide liberated by pre- and postganglionic nerve stimulation in the control of cerebral arterial tone in primates.

Involvement of CYP3A-derived arachidonic acid metabolite(s) in responses to endothelium-derived K+ channel opening substance in monkey lingual artery.

1. In monkey lingual artery strips partially contracted with prostaglandin F2alpha, acetylcholine-induced, concentration-related relaxations were abolished by removal of the endothelium. The response was not significantly influenced by indomethacin but attenuated by NG-nitro-L-arginine (L-NOARG); the effect of the nitric oxide (NO) synthase inhibitor was reversed by L-arginine. 2. The response to acetylcholine resistant to L-NOARG was suppressed in the strips exposed to high K+ media. Charybdotoxin partially inhibited the relaxation, and the remaining relaxation was abolished by additional treatment with apamin, whereas glibenclamide, iberiotoxin or apamin alone was without effect. Relaxations induced by sodium nitroprusside were not influenced by charybdotoxin. 3. The L-NOARG-resistant acetylcholine-induced relaxation was inhibited by metyrapone, proadifen and 17-octadecynoic acid, non-selective cytochrome P450 mono-oxygenase (CYP) inhibitors, and progesterone and ketoconazole, inhibitors selective to CYP3A. The inhibitors did not affect the nitroprusside-induced relaxation. Selective inhibitors of other CYP isoforms, such as debrisoquine and lauric acid, did not reduce the response to acetylcholine. 4. Reaction mixture containing human liver microsome rich in CYPs, arachidonic acid and NADPH incubated at 37 degrees C and filtrated relaxed endothelium-denuded monkey lingual artery strips, used as bioassay tissues. This response was abolished in the strips exposed to high K+ media. The response was also suppressed by combined treatment of the assay tissue with charybdotoxin plus apamin, but was not affected by treatment with iberiotoxin. The reaction mixture co-incubated with ketoconazole failed to relax the strips. 5. It is concluded that the monkey lingual arterial relaxation dependent on the endothelium is mediated by NO and also by a charybdotoxin plus apamin-sensitive but iberiotoxin-insensitive Ca2+-activated K+ channel opening substance(s) that may be a CYP3A-derived arachidonic acid metabolite(s).

Neurogenic vasoconstriction as affected by cholinergic and nitroxidergic nerves in dog ciliary and ophthalmic arteries.

PURPOSE: To determine the involvement of noradrenergic and other vasoconstrictor nerves in the contraction of ocular arteries and the modification by cholinergic and nitroxidergic nerves of vasoconstrictor nerve function. METHODS: Changes in isometric tension were recorded in helical strips of the canine posterior ciliary and external ophthalmic arteries denuded of the endothelium, which were stimulated by transmurally applied electrical pulses (5 Hz). Vasoconstrictor mediators were analyzed by pharmacological antagonists, such as prazosin, alpha,beta-methylene ATP, a P2alpha-purinoceptor antagonist, and BIBP3226, a neuropeptide Y receptor antagonist. RESULTS: Transmural electrical stimulation produced contractions that were potentiated by N(G)-nitro-L-arginine (L-NA), a nitric oxide (NO) synthase inhibitor. The contraction was partially inhibited by prazosin and abolished by combined treatment with alpha,beta-methylene ATP but was not influenced by BIBP3226. Stimulation-induced contraction was attenuated by physostigmine and potentiated by atropine. Contractions induced by exogenous ATP were reversed to relaxations by alpha,beta-methylene ATP. In the strips treated with L-NA, prazosin, and alpha,beta-methylene ATP, the addition of L-arginine elicited relaxations by nerve stimulation. The ATP-induced relaxation was attenuated by aminophylline, whereas neurogenic relaxation was unaffected. CONCLUSIONS: Ciliary and ophthalmic arterial contractions by nerve stimulation are mediated by norepinephrine and ATP, which stimulate alpha1-adrenoceptor and P2X purinoceptor, respectively. ATP from the nerve is unlikely involved in vasodilatation. Acetylcholine derived from the nerve impairs the neurogenic contraction, possibly by interfering with the release of vasoconstrictor transmitters, and neurogenic NO also inhibits the contraction postjunctionally by physiological antagonism.

Effects of endothelial impairment by saponin on the responses to vasodilators and nitrergic nerve stimulation in isolated canine corpus cavernosum.

1. Responsiveness to EDRF-releasing substances and inhibitory nerve stimulation of canine isolated penile corpus cavernosum with and without saponin treatment were investigated. 2. Histological studies demonstrated that saponin did not detach endothelial cells from underlying tissues, but induced degenerative changes in the endothelial cells selectively. 3. In the cavernous strips contracted with phenylephrine, addition of acetylcholine, sodium nitroprusside, ATP and Ca2+ ionophore A23187 induced relaxations, but substance P and bradykinin did not change the muscle tone. 4. Acetylcholine-induced relaxation was significantly attenuated but not abolished by NG-nitro-L-arginine (L-NOARG). L-arginine restored the response inhibited by L-NOARG. The L-NOARG resistant relaxation was not influenced by 1H[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) but was suppressed in the strips contracted with K+. Treatment with saponin abolished the relaxation elicited by acetylcholine and A23187 but did not influence the response to nitroprusside and ATP. The ATP-induced relaxation was attenuated by aminophylline. 5. Transmural electrical stimulation at 2-20 Hz produced endothelium-independent relaxations which were abolished by tetrodotoxin and L-NOARG but unaffected by treatment with saponin. In saponin-treated cavernous strips, the neurogenic relaxation was not affected by acetylcholine, physostigmine, atropine and vasoactive intestinal peptide (VIP) but was abolished by ODQ. 6. It is concluded that acetylcholine-induced relaxations are endothelium-dependent and mediated partly by NO and also by other substances from the endothelium. The endothelium-independent relaxation to ATP is likely to be mediated by P1 purinoceptors. The function of nitrergic nerve does not seem to be prejunctionally modulated by acetylcholine and VIP.

Mechanisms underlying arginine vasopressin-induced relaxation in monkey isolated coronary arteries.

OBJECTIVE: The present study was undertaken to examine whether arginine vasopressin (AVP) relaxes primate coronary artery and to analyse the mechanisms of its action in reference to endothelial nitric oxide and AVP receptor subtype. METHODS: Isometrical tension responses to AVP and desmopressin were recorded in isolated monkey coronary arteries. RESULTS: AVP (10(-9) to 10(-7) mol/l) induced a concentration-related relaxation; endothelium-denudation abolished the response. Treatment with N(G)-nitro-L-arginine, but not the D-enantiomer, abolished the endothelium-dependent relaxation, which was restored by L-arginine. Treatment with SR49059 and [Pmp1,Tyr(Me)2]-Arg8-vasopressin, selective inhibitors of V1 receptor subtype, attenuated the relaxant response to AVP, whereas the relaxation induced by sodium nitroprusside was not affected by SR49059. Desmopressin, a V2 receptor agonist, up to 10(-8) mol/l did not elicit relaxation. CONCLUSIONS: It is concluded that AVP-induced monkey coronary arterial relaxation is mediated via nitric oxide synthesized from L-arginine in association with stimulation of V1 receptor subtypes in the endothelium.

Influence of denervation on neurogenic inhibitory response of corpus cavernosum and nitric oxide synthase histochemistry.

Aims of this study were to functionally and histologically determine the localization of ganglia that distribute inhibitory nerves to the penile corpus cavernosum in dogs. In isolated corpus cavernosa from seven control dogs contracted with endothelin-1, transmural electrical stimulation (5 Hz for 40 s) elicited contractions which were reversed to relaxations by prazosin. The relaxation was abolished by NG-nitro-l-arginine (l-NNA), a nitric oxide (NO) synthase inhibitor, and restored by l-arginine. Parts of bilateral pelvic nerve plexuses running to the penis were surgically denervated in anesthetized three dogs, or the bilateral neuronal tissues close to the corpus cavernosum were removed for denervation in seven dogs. One week after the operation, the dogs were sacrificed. Denervation of pelvic plexus did not attenuate neurogenic relaxations, whereas denervation of the distal portion abolished the responses. In the tissues close to the corpus cavernosum excised for denervation, ganglia containing abundant nerve cells and fibers stained by nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase method were histochemically detected. One week after the denervation, there were no NADPH diaphorase-positive nerve fibers in the trabecula of corpus cavernosum. It is concluded that neurogenic relaxations of canine corpus cavernosum are mediated by NO synthesized from l-arginine in nerve terminals, and this nerve is originated from ganglia located close to the corpus cavernosum but not directly from the pelvic nerve plexus.

Neurogenic vasodilatation of canine isolated small labial arteries.

Mechanisms underlying vasodilatation to nerve stimulation by electrical pulses and nicotine were analyzed in isolated canine small labial arteries. Transmural electrical stimulation (5 and 20 Hz) produced a contraction followed by a relaxation in labial arterial strips denuded of the endothelium, partially contracted with prostaglandin F2alpha. The contraction was abolished by prazosin or combined treatment with alpha, beta-methylene ATP. In the treated strips, neurogenic relaxation was abolished by NG-nitro-L-arginine (L-NA), a nitric oxide (NO) synthase inhibitor, and restored by L-arginine. The D-enantiomers were without effect. Nicotine (10(-4) M) also relaxed the arteries, in which the contractile response was abolished by prazosin and alpha, beta-methylene ATP. The relaxant response was attenuated but not abolished by L-NA; the inhibition was reversed by L-arginine. The remaining relaxation by nicotine was abolished by calcitonin gene-related peptide (CGRP)-[8 to 37], a CGRP1 receptor antagonist. Relaxations elicited by a lower concentration of nicotine (2 x 10(-5) M) sufficient to produce similar magnitudes of response to those induced by 5-Hz electrical nerve stimulation were also inhibited partially by L-NA. Histochemical study with the NADPH-diaphorase method demonstrated positively stained nerve fibers and bundles in the arterial wall, suggesting the presence of neuronal NO synthase. It is concluded that the relaxation induced by electrical nerve stimulation of small labial arteries is mediated exclusively by NO synthesized from L-arginine in nerve terminals, whereas nicotine in the concentrations used evokes relaxations by a mediation of nerve-derived NO and also CGRP, possibly from sensory nerves. The reason why nicotine but not electrical pulses stimulates sensory nerves and elicits vasorelaxation remains unsolved.

Mechanism of neurogenic relaxation and modification of the response by enteric substances in isolated dog colon.

The mechanisms of neurogenic relaxation in the longitudinal muscle of the isolated canine colon and its modification by enteric substances were investigated. Relaxations induced by transmural electrical stimulation with electrical pulses, nicotine or K+ in the muscle strips contracted with bradykinin and treated with atropine were attenuated but not abolished by NG-nitro-L-arginine (L-NA), and the inhibition was reversed by L-arginine. Oxyhemoglobin and ouabain inhibited the response, whereas K+ channel inhibitors, such as glibenclamide, tetraethylammonium, apamin and charybdotoxin, were without effect. In L-NA-treated strips, stimulation-induced relaxations were reduced by ouabain but not by oxyhemoglobin. Among substances tested, only norepinephrine, ATP, vasoactive intestinal peptide (VIP) and galanin produced relaxations. However, alpha- and beta-adrenoceptor antagonists and aminophylline did not alter the response to nerve stimulation. In the strips made unresponsive to VIP and galanin, stimulation-induced relaxations were not influenced. Indomethacin, calcitonin gene-related peptide, cholecystokinin, peptide YY, substance P and serotonin did not modulate the neurogenic response. It is concluded that the relaxation associated with nerve stimulation is mediated by nitric oxide (NO) synthesized from L-arginine and also by substance(s) activating the electrogenic Na+ pump but not that opening K+ channels. Norepinephrine, ATP, VIP and galanin can be excluded as candidate inhibitory neurotransmitters, and the substances used so far are unlikely to modulate inhibitory nerve function.

Functional studies on blockade by neosurugatoxin of nicotinic receptors in nitroxidergic and sensory nerve terminals and intramural ganglionic cells.

In isolated canine cerebral and cutaneous arteries and duodenum, effects of neosurugatoxin (NSTX) on the response to nicotine were compared. Nicotine-induced relaxations are mediated by nitric oxide (NO) from vasodilator nerves in cerebral arteries and by calcitonin gene-related peptide (CGRP) in cutaneous arteries treated with NO synthase inhibitors. Duodenal relaxation to nicotine is mediated by NO from inhibitory nerves. Cerebral arterial strips without endothelium responded to nicotine with relaxations that were inhibited by NSTX (3 x 10(-10) to 3 x 10(-9) M) concentration-dependently. Relaxations to nicotine of duodenal strips were attenuated by NSTX and abolished by tetrodotoxin, whereas those induced by K+ and electrical stimulation were not influenced. In cutaneous arteries treated with N(G)-nitro-L-arginine, nicotine-induced relaxations were attenuated by NSTX as low as 10(-10) M, but unaffected by tetrodotoxin. The inhibitory potency of NSTX was in the order of cutaneous artery > duodenum > cerebral artery. It is concluded that NSTX selectively antagonizes actions on nicotinic receptors in nitroxidergic nerves of cerebral arteries, CGRP-mediated sensory nerves of cutaneous artery and ganglionic cells of the duodenum, but the affinity of this toxin to nicotinic receptors in sensory nerves is the highest.