Impaired nitric oxide function in the basilar artery of the obese Zucker rat.

The effect of insulin-resistance syndrome on vascular function has been examined in isolated basilar arteries using the obese Zucker rat (OZR) and age-matched lean littermate controls (lean Zucker rat; LZR) at 36 weeks of age. The OZR showed significantly reduced oral glucose tolerance and increased body weight, blood pressure, proteinuria, plasma levels of triglycerides, cholesterol, and insulin compared with the LZR. The contractile response to serotonin was significantly increased in the OZR. Furthermore, contractions to serotonin in LZR but not OZR were enhanced in the presence of the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (NAME). Relaxations to acetylcholine (ACh), histamine, and A23187 were significantly reduced in precontracted arteries from the OZR. In the presence of NAME, histamine responses were significantly reduced whereas ACh and A23187 responses were almost abolished. Relaxations to free-radical nitric oxide (NO) and papaverine were not different in arteries from the OZR, even though responses to sodium nitroprusside were reduced in the OZR. Western blot and immunofluorescent quantitative analyses of eNOS content in cerebral microvessel fractions and basilar artery preparations, respectively, were not significantly different between OZR and LZR. The results suggest impairment in endothelial function resulting in reduced NO function in the basilar artery from the OZR.

Effects of agents that inactivate free radical NO (NO*) on nitroxyl anion-mediated relaxations, and on the detection of NO* released from the nitroxyl anion donor Angeli's salt.

1. The effects of agents that inactivate free radical nitric oxide (carboxy-PTIO, hydroxocobalamin and pyrogallol) were tested on relaxations produced by the nitroxyl anion (NO(-)) donor Angeli's salt in rat aortic rings and anococcygeus muscles. The amount of NO(*) generated from Angeli's salt in the presence of these agents was measured using a NO(*)-selective electrode sensor. 2. Carboxy-PTIO (100, 300 microM), hydroxocobalamin (30, 100 microM) and pyrogallol (10, 30 microM) significantly reduced relaxations produced by Angeli's salt (0.3 microM) in aortic rings but not in anococcygeus muscles. 3. NO(*) generated from Angeli's salt (0.1 - 10 microM), as detected by the sensor electrode, was less than 0.5% of the amount of Angeli's salt added. Carboxy-PTIO (100 microM) and hydroxocobalamin (30 microM), but not pyrogallol significantly increased the amount of NO(*) detected. 4. In the presence of an oxidizing agent copper [II] (as CuSO(4) 100 microM), the amount of NO(*) detected from 0.3 microM of Angeli's salt increased from an undetectable level of 142.7+/-15.7 nM (equivalent to 47.6% of Angeli's salt added). Under these conditions, carboxy-PTIO, hydroxocobalamin and pyrogallol significantly reduced the amount of NO(*) detected from Angeli's salt as well as the signal generated by an equivalent amount of authentic NO (0.33 microM). 5. The difference in effects of these agents on relaxations to Angeli's salt in the aorta and the anococcygeus muscle may be explained by the ready conversion of NO(-) to NO(*) in the aorta through an unidentified mechanism, which makes NO(-) susceptible to inactivation by these agents. Furthermore, in addition to inactivating NO(*), carboxy-PTIO and hydroxocobalamin may themselves oxidize NO(-) to NO(*), albeit slightly.

A morpholinocatechol compound (UK42620) with clonidine- and tyramine-like actions.

1. The actions of a morpholinocatechol (UK42620) were studied in rat isolated atria preparations consisting of spontaneously beating left and right atrial pairs. 2. UK42620 produced positive inotropic and chronotropic responses and, in atria that were incubated with [(3)H]-noradrenaline, it also produced a massive increase in the release of radioactivity. 3. These actions of UK42620 were similar to those of tyramine and were blocked by the beta-adrenoceptor antagonist propranolol (0.3 microM) and by the neuronal uptake blocker desipramine (1 microM). 4. In the presence of desipramine, UK42620 but not tyramine produced a decrease in the stimulation-induced efflux of radioactivity that was antagonized by idazoxan. 5. Thus, UK42620 had prejunctional alpha(2)-adrenoceptor activity like that of clonidine- and tyramine-like activity releasing large amounts of noradrenaline.

Mechanisms of nitric oxide-independent relaxations induced by carbachol and acetylcholine in rat isolated renal arteries.

1. In rat isolated renal artery segments contracted with 0.1 microM phenylephrine and in the presence of the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME), carbachol and acetylcholine produced endothelium-dependent relaxations. The mechanisms underlying these relaxations were studied. 2. These relaxations were not affected by ODQ (1H-[1,2,4]oxadiazolo[4,3, -a]quinoxalin-1-one) or indomethacin. In arteries contracted with 20 - 30 mM K(+), L-NAME-resistant relaxations induced by carbachol and acetylcholine were virtually absent. 3. The Na(+)-K(+) ATPase inhibitor ouabain reduced these relaxations in a concentration-dependent manner. 4. In K(+)-free media, addition of K(+) (5 mM) produced 90. 5+/-3.9% (n=3) relaxation of phenylephrine-induced tone. This relaxation was endothelium-independent and ouabain-sensitive. 5. Tetraethylammonium (TEA), charybdotoxin (ChTX) and iberiotoxin (IbTX) reduced the sensitivity of carbachol-induced relaxations, but did not change the maximal response. These relaxations were not altered by 4-aminopyridine (4-AP), glibenclamide or apamin. Acetylcholine (1 microM)-induced relaxation was reduced by ChTX, but not by TEA or IbTX. 6. The cytochrome P450 inhibitor miconazole, but not 17-octadecynoic acid, reduced the sensitivity of carbachol-induced relaxations, without changing the maximal response. 7. In conclusion, in rat isolated renal arteries, acetylcholine and carbachol produced a non-NO/non-PGI(2) relaxation which is mediated by an endothelium-derived hyperpolarizing factor (EDHF). This factor does not appear to be a cytochrome P450 metabolite. The inhibition by ouabain of these relaxations suggests the possible involvement of Na(+)-K(+) ATPase activation in EDHF responses, although other mechanisms cannot be totally ruled out.

Differential actions of L-cysteine on responses to nitric oxide, nitroxyl anions and EDRF in the rat aorta.

1. The effects of L-cysteine were tested in rat aortic rings on responses to nitric oxide free radical (NO(*)), nitroxyl (NO(-)) derived from Angeli's salt and endothelium-derived relaxing factor (EDRF) activated by acetylcholine, ATP and the calcium ionophore A23187. Concentrations of 300 microM or less of L-cysteine had no effect on responses. 2. Relaxations produced by exogenous NO(*) (0.25 - 2.5 microM) were markedly prolonged and relaxations produced by sodium nitroprusside (0.001 - 0.3 microM) were enhanced by 1 and 3 mM L-cysteine. The enhancements by L-cysteine of responses to NO(*) and sodium nitroprusside may be attributed to the formation of S-nitrosocysteine. 3. Relaxations mediated by the nitroxyl anion (0.3 microM) donated from Angeli's salt were more prolonged than those produced by NO(*), and nitroxyl-induced relaxations were reduced by L-cysteine (1 and 3 mM). 4. EDRF-mediated relaxations produced by acetylcholine (0.01 - 10 microM), ATP (3 - 100 microM) and the calcium ionophore A23187 (0.1 microM) were significantly reduced by 3 mM L-cysteine. 5. The similarity between the inhibitory effects of L-cystei on responses to EDRF and on those to nitroxyl suggests that a component of the response to EDRF may be mediated by nitroxyl anion.

Cholinergic prejunctional inhibition of nitrergic neurotransmission in the guinea-pig isolated basilar artery.

1. The effects of endogenous and exogenous acetylcholine (ACh) on nitrergic relaxations elicited by electrical field stimulation (EFS) were studied in guinea-pig endothelium-denuded basilar artery preparations precontracted with 1 micromol/L prostaglandin F(2 alpha) and a possible role of K+ channels in mediating the effects was investigated. 2. Acetylcholine (3 micromol/L) and physostigmine (10 micromol/L) produced small, yet statistically significant, inhibitions of EFS-induced nitrergic relaxations, while atropine (1 micromol/L) slightly enhanced the nitrergic response. The ACh-induced inhibition was atropine sensitive. Acetylcholine or atropine did not affect relaxations induced by sodium nitroprusside. 3. The inhibition of nitrergic relaxations by 3 micromol/L ACh was prevented by the K+ channel blockers tetraethylammonium and 4-aminopyridine, but was not changed by iberiotoxin, apamin or glibenclamide. 4. Neither vasoactive intestinal polypeptide nor the alpha2-adrenoceptor agonists noradrenaline and clonidine modulated nitrergic neurotransmission in the guinea-pig basilar artery. 5. The findings show that ACh acts on prejunctional muscarinic receptors of nitrergic nerves to inhibit nitrergic neurotransmission. It is suggested that endogenous ACh may have this effect; however, the physiological significance of this prejunctional modulation is not clear due to the relatively small effect produced. The prejunctional inhibitory action of ACh may involve opening of neuronal K+ channels.

Effects of some imidazolidine alpha2-adrenoceptor agonists in rat isolated atria.

1. The prejunctional alpha2-adrenoceptor agonist activity of four imidazolidines (UK14819, UK14304, UK15121 and UK11957) were compared to that of clonidine in rat isolated atrial preparations. 2. The preparations consisted of spontaneously beating left and right atrial pairs that were incubated with [3H]-noradrenaline. The efflux of radioactivity induced by electrical field stimulation of intramural sympathetic nerves was used as an index of neurotransmitter release and inotropic and chronotropic responses were recorded. 3. Two quinoxalinyl imidazolidines (UK14819 and UK14304 which have chloride and bromide substitution, respectively, in the phenyl moiety) caused decreases in the efflux of radioactivity with stimulation at 2 Hz and 10 Hz but not at 20 Hz. These effects were antagonised by the alpha2-adrenoceptor antagonist idazoxan (0.3 microM) but they were not affected by the alpha1-adrenoceptor antagonist prazosin (0.1 microM). 4. The third halogenated quinoxalinyl imidazolidine analogue (UK15121, which has an iodide substitution in the phenyl ring) and a quinolinyl imidazolidine (UK11957) have actions similar to clonidine. They decreased the efflux of radioactivity with stimulation at 1 Hz (for UK11957) or 2 Hz (for UK15121) and enhanced it at higher frequencies of stimulation. Both the inhibitory and enhancing effects were antagonised by idazoxan but they were not affected by prazosin. 5. Unlike the other three imidazolidines in the present study, the quinolinyl imidazolidine (UK11957), caused a decrease in resting release of radioactivity and this effect was prevented by the monoamine oxidase inhibitor pargyline (30 microM). 6. These findings suggest that the 5-chloro-or 5-bromo substituted quinoxalinyl imidazolidines (UK14819 and UK14304) are full agonists at prejunctional alpha2-adrenoceptors, but the 5-iodo-substituted quinoxalinyl imidazolidine (UK15121) and the quinolinyl analogue of UK14304 (UK11957), like clonidine, appear to be partial agonists at these receptors.

Comparison of the redox forms of nitrogen monoxide with the nitrergic transmitter in the rat anococcygeus muscle.

1. A sustained tone was produced in rat isolated anococcygeus muscles with guanethidine and clonidine and relaxant responses were elicited by electrical stimulation of its nitrergic nerves and by the three redox forms of nitrogen monoxide. 2. The nitroxyl anion (NO ) was donated by dissociation of Angeli's salt; the free radical (NO*) was from an aqueous solution of nitric oxide gas; the nitrosonium cation (NO+) was donated by dissociation of nitrosonium tetrafluoroborate. 3. The concentrations producing approximately 50% relaxations of the anococcygeus muscle were 0.3 microM for Angeli's salt (nitroxyl), 0.5 microM for NO* and 100 microM for nitrosonium tetrafluoroborate. Nitrergic nerve stimulation at 1 Hz for 10 s produced equivalent relaxant responses. 4. The superoxide generator pyrogallol (100 microM) had no effect on responses to nitrergic nerve stimulation or Angeli's salt but significantly reduced responses to NO* and nitrosonium tetrafluoroborate. 5. The NO* scavenger carboxy-PTIO (100 microM) had no effect on responses to nitrergic nerve stimulation or Angeli's salt but significantly reduced responses to NO* and nitrosonium tetrafluoroborate. 6. Hydroxocobalamin (30 microM) had no significant effect on responses to the nitrergic transmitter, enhanced the response to Angeli's salt, and significantly reduced responses to NO* and nitrosonium tetrafluoroborate. 7. The findings suggest that the nitroxyl anion donated by Angeli's salt is a better candidate than NO* to serve as the nitrergic transmitter in the rat anococcygeus muscle, although it still does not behave exactly like the transmitter.

Nitrergic and purinergic mechanisms and their interactions for relaxation of the rat internal anal sphincter.

1. The NANC neuronal mechanisms for relaxations of the rat internal anal sphincter in response to electrical field stimulation (EFS) were studied in isolated preparations in the presence of atropine (1 microM), propranolol (3 microM) and phentolamine (3 microM). 2. EFS-induced relaxations were abolished by tetrodotoxin (1 microM) and reduced to 64% of control by the guanylate cyclase inhibitor ODQ (1 microM), but were not significantly reduced by the nitric oxide synthase inhibitor L-NAME (100 microM) or oxyhaemoglobin (10 microM). However, in the presence of tubocurarine (10 microM) or apamin (0.1 microM), L-NAME or oxyhaemoglobin greatly reduced or abolished EFS-induced relaxations. 3. The EFS-induced relaxations were mimicked by NO (10-100 microM) and by ATP (3-10 mM). The relaxations elicited by these agents were not affected by tetrodotoxin, L-NAME, tubocurarine or apamin. However, ATP-induced relaxations were reduced by the combination of L-NAME with tubocurarine or apamin. 4. Nicotine (10-100 microM) produced concentration-dependent relaxations that were abolished by tubocurarine (10 microM) or hexamethonium (200 microM). After desensitisation to nicotine (100 microM) and in its continued presence, the addition of L-NAME (100 microM) resulted in almost complete abolition of EFS-induced relaxations. 5. It is suggested that tubocurarine, hexamethonium and desensitisation to nicotine have an apamin-like action in the rat internal anal sphincter, the main effect being blockade of a purinergic component of the relaxant transmission process. 6. The findings suggest that both nitrergic and purinergic transmissions are involved in EFS-induced NANC relaxations of the rat internal anal sphincter, and there appears to be a complex interaction between these two pathways of transmission.

Effects of hydroxocobalamin and carboxy-PTIO on nitrergic transmission in porcine anococcygeus and retractor penis muscles.

The effects of carboxy-PTIO and hydroxocobalamin were studied on nitrergic transmission in anococcygeus and retractor penis muscles taken during post mortem examination from young male pigs. In both muscles under resting conditions, electrical field stimulation (EFS) caused contractions that were sensitive to tetrodotoxin (1 microM) and were greatly inhibited by prazosin (1 microM) and guanethidine (10-30 microM), but were not significantly affected by atropine (1 microM). In the anococcygeus muscle, but not in the retractor penis muscle, guanethidine produced a prolonged contraction. After tone was raised by guanethidine in the anococcygeus or by phenylephrine (1 microM) in the presence of guanethidine in the retractor penis, EFS caused tetrodotoxin-sensitive relaxations. The EFS-induced relaxations were abolished by the NO synthase inhibitor N(G)-L-nitro-arginine methyl ester (L-NAME; 100 microM) and its effect was partly overcome by L-arginine (1 mM), indicating it was mediated by nitrergic nerves. Carboxy-PTIO (0.1-1 mM) had no significant effect in reducing stimulation-induced nitrergic relaxations in either muscle. However, hydroxocobalamin (0.1-1 mM) caused concentration-dependent reductions of nitrergic relaxations in both muscles. Relaxations to exogenous nitric oxide (1 microM) in both muscles were abolished by carboxy-PTIO (0.3 mM) and hydroxocobalamin (0.1 mM). There were no differences in reactivity to carboxy-PTIO or hydroxocobalamin between anococcygeus and retractor penis muscles from the same species (pig). The finding also confirms earlier observations that the nitrergic transmitter is generally resistant to the NO-scavenger carboxy-PTIO.

Human placental nitric oxide synthase activity is not altered in diabetes.

Endothelial nitric oxide synthase (NOS) protein and mRNA have been identified and calcium-dependent NOS activity has been measured in human placentae during normal pregnancy. Recently, mRNA and protein for the inducible isoform of NOS have been detected in placentae of women with gestational diabetes. The aim of this study was to determine whether calcium-independent (ciNOS) and/or total (tNOS) NOS activities were increased in placentae obtained after vaginal delivery or Caesarean section from women assigned to the following groups according to standard obstetric criteria: gestational diabetes, diabetes before pregnancy and non-diabetic controls. tNOS and ciNOS were assessed by measuring the conversion of [3H]L-arginine to [3H]L-citrulline in the three groups. Michaelis-Menten constants (Km) and maximum velocities of reaction (Vmax) were calculated using Lineweaver-Burk analysis for tNOS. There were no significant differences in either ciNOS, Vmax or Km values between any of the three groups (normal, ciNOS 12.7+/-1.6%, Vmax 16.6+/-3.3 pmol.min-1.mg-1 protein, Km 15.30+/-2.6 micromol/l; gestational diabetes, ciNOS 15.4+/-1.4%, Vmax 14.8+/-5.2 pmol.min-1. mg-1 protein, Km 10.5+/-1.7 micromol/l; diabetes before pregnancy, ciNOS 13.4+/-1.1%, Vmax 14.9+/-3.4 pmol.min-1.mg-1 protein, Km 17. 7+/-2.2 micromol/l). The presence of macrosomia did not affect tNOS activity in those with diabetes before pregnancy, and glycosylated haemoglobin levels measured between weeks 27 and 39 were not correlated with ciNOS activity. The results from the present study do not provide evidence for increased placental tNOS or ciNOS activities in pregnancies complicated by gestational diabetes or diabetes present before pregnancy.

Cholinergic prejunctional inhibition of nitrergic neurotransmission in the guinea-pig isolated basilar artery.

1. The effects of endogenous and exogenous acetylcholine (ACh) on nitrergic relaxations elicited by electrical field stimulation (EFS) were studied in guinea-pig endothelium-denuded basilar artery preparations precontracted with 1 mumol/L prostaglandin F2 alpha and a possible role of K+ channels in mediating the effects was investigated. 2. Acetylcholine (3 mmol/L) and physostigmine (10 mumol/L) produced small, yet statistically significant, inhibitions of EFS-induced nitrergic relaxations, while atropine (1 mumol/L) slightly enhanced the nitrergic response. The ACh-induced inhibition was atropine sensitive. Acetylcholine or atropine did not affect relaxations induced by sodium nitroprusside. 3. The inhibition of nitrergic relaxations by 3 mumol/L ACh was prevented by the K+ channel blockers tetraethylammonium and 4-aminopyridine, but was not changed by iberiotoxin, apamin or glibenclamide. 4. Neither vasoactive intestinal polypeptide nor the alpha 2-adrenoceptor agonists noradrenaline and clonidine modulated nitrergic neurotransmission in the guinea-pig basilar artery. 5. The findings show that ACh acts on prejunctional muscarinic receptors of nitrergic nerves to inhibit nitrergic neurotransmission. It is suggested that endogenous ACh may have this effect; however, the physiological significance of this prejunctional modulation is not clear due to the relatively small effect produced. The prejunctional inhibitory action of ACh may involve opening of neuronal K+ channels.

Effects of pyrogallol, hydroquinone and duroquinone on responses to nitrergic nerve stimulation and NO in the rat anococcygeus muscle.

1. The hypothesis that endogenous superoxide dismutase (SOD) protects the nitrergic transmitter from inactivation by superoxide and that this explains the lack of sensitivity of the transmitter to superoxide generators was tested in the rat isolated anococcygeus muscle. 2. Responses to nitrergic nerve stimulation or to NO were not significantly affected by exogenous SOD or by the Cu/Zn SOD inhibitor diethyldithiocarbamic acid (DETCA). 3. Hydroquinone produced a concentration-dependent reduction of responses to NO with an IC50 of 27 microM, and higher concentrations reduced relaxant responses to nitrergic nerve stimulation with an IC50 of 612 microM. The effects of hydroquinone were only slightly reversed by SOD, so it does not appear to be acting as a superoxide generator. 4. Pyrogallol produced a concentration-dependent reduction in responses to NO with an IC50 value of 39 microM and this effect was reversed by SOD (100-1000 u ml(-1)). Pyrogallol did not affect responses to nitrergic nerve stimulation. Treatment with DETCA did not alter the differentiating action of pyrogallol. 5. Duroquinone produced a concentration-dependent reduction of relaxations to NO with an IC50 value of 240 microM and 100 microM slightly decreased nitrergic relaxations. After treatment with DETCA, duroquinone produced greater reductions of relaxant responses to NO and to nitrergic stimulation, the IC50 values being 8.5 microM for NO and 40 microM for nitrergic nerve stimulation: these reductions were reversed by SOD. 6. The findings do not support the hypothesis that the presence of Cu/Zn SOD explains the greater susceptibility of NO than the nitrergic transmitter to the superoxide generator pyrogallol, but suggest that it may play a role in the effects of duroquinone.

Effect of xanthine oxidase inhibition on endothelium-dependent and nitrergic relaxations.

The effects of inhibition of xanthine oxidase on responses mediated by nitric oxide (NO) were examined using the selective xanthine oxidase inhibitors allopurinol and 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine (AHPP). In rat aortic rings precontracted with phenylephrine (1 microM), allopurinol (300 microM) and AHPP (100, 300 microM) significantly reduced tone, an effect not seen after inhibition of NO synthase with Nomega-nitro-L-arginine (NOLA 100 microM). Relaxations produced by acetylcholine (0.01-10 microM) were significantly enhanced by AHPP (100, 300 microM) but not by allopurinol. Nitrergic relaxations in the rat anococcygeus muscle (field stimulation 1 ms pulses; 1 Hz: 10 s) were not affected by either allopurinol or AHPP. However, relaxations produced by exogenous NO (0.25 microM) were significantly enhanced by AHPP, allopurinol (100 microM) and superoxide dismutase (100 U/ml). Xanthine (500 microM) partially, but significantly, reversed the enhancement produced by AHPP. These findings suggest that superoxide generated by xanthine oxidase modulates the activity of basal and stimulated NO derived from the rat aortic endothelium, but does not affect the activity of the nitrergic transmitter in the rat anococcygeus muscle, despite its ability to modulate responses to exogenous NO.

Effects of P2-receptor agonists on sympathetic neuroeffector transmission in the rat isolated anococcygeus muscle.

The effects of nicotinamide adenine dinucleotide phosphate (NADPH), alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-MeATP), L-beta,gamma-methylene adenosine 5'-triphosphate (L-beta,gamma-MeATP), 2-methylthio-adenosine 5'-triphosphate (MeSATP) and adenosine-5-O-(2'-thiodiphosphate) (ADPbetaS) were investigated on the contractions to electrical field stimulation in the rat anococcygeus muscle. Stimulation-induced contractions were not affected by L-beta,gamma-MeATP (3-100 microM) or MeSATP (3-100 microM), but were enhanced by NADPH (10-100 microM), alpha,beta-MeATP (3-30 microM) and ADPbetaS (3-10 microM) in a concentration-dependent manner, and the enhancements were antagonised by the P2-receptor antagonist suramin (100 microM). The enhancement produced by alpha,beta-MeATP (10 microM) and ADPbetaS (10 microM) was also antagonised by pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (10 microM) and reactive blue 2 (100 microM). The enhancement produced by alpha,beta-MeATP (10 microM) was not altered by NG-nitro-L-arginine methyl ester (100 microM), desipramine (1 microM) or idazoxan (0.1 microM) excluding, respectively, the possible involvement of nitric oxide, neuronal amine uptake or alpha2-autoinhibition of noradrenergic transmission. Contractions elicited by low (0.1 and 0.3 microM) but not by higher (1 and 3 microM) concentrations of exogenously applied noradrenaline were enhanced by alpha,beta-MeATP (10 microM). Neither the resting nor the stimulation-induced effluxes of radioactivity from [3H]-noradrenaline-labelled anococcygeus muscles were affected by alpha,beta-MeATP (10-100 microM). The findings suggest that P2-receptors subserve the enhancing actions of NADPH, alpha,beta-MeATP and ADPbetaS on sympathetic neuroeffector transmission; however, the subtype of P2-receptor involved and its location remain unclear.

Role of potassium channels in the nitrergic nerve stimulation-induced vasodilatation in the guinea-pig isolated basilar artery.

1. We studied the effects of various K+ channel blockers on the vasodilator responses of guinea-pig isolated basilar arteries to nitrergic nerve stimulation, the nitric oxide (NO) donor sodium nitroprusside (SNP), and the membrane permeable guanosine-3',5'-cyclic monophosphate (cyclic GMP) analogue 8-bromo-cyclic GMP (8-Br-cyclic GMP). 2. In endothelium-denuded preparations which were contracted with prostaglandin F2alpha (1 microM), electrical field stimulation (EFS, 10 Hz for 30 s) produced a vasodilatation which was totally blocked by the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester L-NAME; 100 microM) (n=3) and by the selective NO-sensitive guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ; 1 microM) (n=4). The vasodilator response to SNP (100 nM) was not reduced by L-NAME but was abolished by ODQ (1 microM) (n=4). 3. EFS-elicited vasodilatation was partly but significantly reduced by the non-selective K+ channel blockers tetraethylammonium (TEA, 1 and 3 mM) and 4-aminopyridine (4-AP, 3 mM), and by the large-conductance calcium-activated K+ channel (K(Ca) channel) blockers charybdotoxin (ChTX, 150 nM) and iberiotoxin (IbTX, 30 and 100 nM). In contrast, the ATP-sensitive K+ channel (K(ATP) channel) blocker glibenclamide (1-10 microM) and the small-conductance K(Ca) channel blocker apamin (100-500 nM) did not affect EFS-induced vasodilatation. 4. The vasodilator response elicited by SNP (10-100 nM) was significantly reduced by TEA (3 mM) and ChTX (150 nM) but not by apamin (500 nM) or glibenclamide (1 microM). The vasodilatation elicited by 8-Br-cyclic GMP (100 microM) was also reduced by TEA (3 mM) and ChTX (150 nM). 5. The results indicate that the vasodilatations induced by nitrergic nerve stimulation and the NO donor SNP in endothelium-denuded guinea-pig basilar artery depend on the formation of intracellular cyclic GMP. The increased cyclic GMP level activates large-conductance K(Ca) channels which partly mediate the vasodilator response. Neither K(ATP) channels nor apamin-sensitive small-conductance K(Ca) channels are involved in nitrergic transmitter-mediated vasodilatation.

Mechanisms of electrical field stimulation-induced vasodilatation in the guinea-pig basilar artery: the role of endothelium.

1. The role of endothelium in neuronal vasodilatation elicited by electrical field stimulation (EFS) was investigated in preparations of the isolated guinea-pig basilar artery in which the tone was raised with prostaglandin F2 alpha. 2. In preparations with intact endothelium, EFS produced frequency-dependent dilatations which were not affected by guanethidine but were slightly yet significantly reduced by atropine (1 microM), and were blocked by tetrodotoxin (1 microM) and the nitric oxide synthase inhibitor L-NAME (10 microM). 3. Dilatations were elicited by acetylcholine (3 microM); these were blocked by L-NAME and atropine (1 microM). 4. Dilatations were elicited by nicotine (100 microM); these were blocked by L-NAME and hexamethonium (100 microM). 5. Dilation elicited by sodium nitroprusside (SNP, 3 microM) was not affected by L-NAME. 6. The inhibitory effects of L-NAME were partially prevented by L-arginine (1 microM). 7. Removal of the endothelium resulted in a significant reduction of dilatations elicited by EFS, elimination of the dilator action of acetylcholine, but enhancement of that to SNP. 8. The results suggest that EFS-induced vasodilatation is mediated in part by the nitrergic transmitter and in part endothelium derived relaxing factor (EDRF) activated by acetylcholine released from cholinergic nerves.

Effects of hydroxocobalamin on nitrergic transmission in rat anococcygeus and bovine retractor penis muscles: sensitivity to light.

Hydroxocobalamin inhibited nitrergic nerve-induced relaxations in rat anococcygeus and bovine retractor penis muscles in a concentration-dependent manner. In the rat anococcygeus muscle, the inhibition was greater in light than in dark conditions, whereas in the bovine retractor penis the inhibition was similar under both conditions.