Role of nitric oxide on cholinergic component of bronchial tone in pig

In vitro studies demonstrated that stimulation of intrinsic nerves of airway smooth muscle results in a predominantly contractile response, followed by a relaxant response which involves cholinergic, adrenergic and non-adrenergic non-cholinergic (NANC) nerve activation. Thus, in this paper it is determined whether endogenous nitric oxide (NO) modulates cholinergic neurotransmission in isolated pig airway smooth muscle. Bronchial rings were suspended in organ baths for isometric measurement of tension and the contractions were induced using electrical field stimulation (EFS) techniques. Then, the effects of L-NG-nitroarginine (L-NOARG, 10 microM), an inhibitor of NO synthase, and L-arginine (L-ARG, 1 mM), a precursor of NO synthesis, were evaluated. The cholinergic contractions induced by electrical field stimulation (EFS: 60 V, 2 ms, 60 Hz) of pig lobar bronchial preparations increased (29%) in the presence of L-NOARG (10 microM). This effect may be released by nerves in pig large airways during EFS.     

Role of laparoscopy in the management of chronic pelvic pain

OBJECTIVE: The pharmacokinetics of N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthesis, was investigated in patients with septic shock. METHODS: Blood was sampled at intervals before, during and after 12-h infusion of L-NAME 1 mg x kg(-1) x h(-1) in nine septic shock patients for determination of plasma concentrations by high-performance liquid chromatography (HPLC). In three patients the renal clearance of the drug was determined. RESULTS: Incubation of L-NAME with plasma and blood in vitro revealed hydrolysis to N(G)-nitro-L-arginine (L-NOARG), the active inhibitor of NO synthesis. L-NOARG did not undergo further degradation. Continuous intravenous infusion of 1 mg x kg(-1) x h(-1) of L-NAME for 12 h in patients with septic shock increased blood pressure and resulted in increasing plasma concentrations of L-NOARG (Cmax 6.2 microg x ml(-1) at 12 h) whereas L-NAME concentrations reached a plateau within 1.5 h (Cmax 1.0 microg x ml(-1)). After the infusion was stopped L-NAME disappeared from the plasma rapidly (half-life 19.2 min) whereas L-NOARG concentration declined slowly (half-life 22.9 h). The calculated volume of distribution for L-NAME was 0.451 x kg(-1) body weight and 1.961 x kg(-1) for L-NOARG. The renal clearance for L-NOARG was 3.5% of total body clearance for L-NOARG, whereas L-NAME could not be detected in urine. CONCLUSION: We conclude that vasoconstriction with L-NAME in septic patients may result from hydrolysis to L-NOARG, the active inhibitor of NO synthesis. The long plasma half-life and large volume of distribution for L-NOARG suggests extensive distribution to extravascular tissues. Since renal excretion is minimal, elimination of the metabolite L-NOARG follows other pathways.     

alpha-bungarotoxin- and methyllycaconitine-sensitive nicotinic receptors mediate fast synaptic transmission in interneurons of rat hippocampal slices

The role of nitric oxide (NO) in the long-term serotoninergic neurotoxicity induced by (+/-)3,4-methylenedioxymethamphetamine (MDMA) in rats was investigiated. Pretreatment with Nomega-nitro-L-arginine (L-NOARG) (10 mg kg-1), a nitric oxide synthase (NOS) inhibitor, partially protected against long-term serotonin (5-HT) depletion induced by MDMA (40 mg kg-1) in frontal cortex and parietal cortex, but not in other brain regions examined. Brain NOS activities in these two regions were significantly elevated at 6 h after MDMA administration. Moreover, L-NOARG pretreatment caused significant inhibition of brain NOS activity but did not affect the acute 5-HT and dopamine (DA) changes or the hyperthermia induced by MDMA. These results suggest that it is the NOS inhibitory properties of L-NOARG, rather than its effects on the acute monoamine changes or the hyperthermia induced by MDMA, that are responsible for the prevention of neurotoxicity. The regional differences on the protection of L-NOARG and on the activation of NOS by MDMA indicate the unequal role that NO may play in MDMA-induced neurotoxicity in different brain regions.     

Nitric oxide-mediated changes in vascular reactivity in pregnancy in spontaneously hypertensive rats

1. To examine the mechanisms which may account for pregnancy-induced vasodilatation in spontaneously hypertensive rats (SHR), we have investigated the changes in vascular reactivity and the effects of endothelial nitric oxide (NO) inhibition in the in situ blood-perfused, mesenteric resistance vessels of 18-20 day pregnant SHR. The effects of NG-nitro-L-arginine (L-NOARG) were compared in pregnant and nonpregnant SHR and gestation matched normotensive Wistar-Kyoto (WKY) rats. 2. Intra-arterial mean blood pressures (MBP) were similar in pregnant and nonpregnant SHR. Basal perfusion pressures (BPP) were decreased in pregnant compared with nonpregnant SHR. Pregnant WKY had lower MBP and BPP than either pregnant or nonpregnant SHR. 3. Vasoconstrictor responses to electrical stimulation (ES) and intra-arterial noradrenaline (NA) were decreased in pregnant compared with nonpregnant SHR. These responses were still greater in pregnant SHR when compared with pregnant WKY. Vascular reactivity to angiotensin II (AII) in pregnant SHR was reduced to a similar level to that in pregnant WKY. 4. L-NOARG (5 mg kg-1, i.v.), an inhibitor of nitric oxide synthase, increased MBP and BPP in all groups. After L-NOARG, BPP were equalized between pregnant and nonpregnant SHR. Pregnant WKY still showed lower MBP and BPP than SHR groups. 5. L-NOARG potentiated vascular responses to ES, NA and AII in all groups. The blunted vascular responses to NA and ES were normalized and the reactivity to AII was only partially reversed in pregnant SHR compared with nonpregnant SHR. Pregnant WKY still had much lower vascular responses to ES and NA than either pregnant or nonpregnant SHR. L-NOARG enhanced vascular responses to All to a greater extent in pregnant SHR than in pregnant WKY.6. These results demonstrate that blunted responses to NA and ES were NO-dependent, while diminished reactivity to AII was only partially dependent on NO in the in situ blood perfused mesenteric resistance vessels of pregnant SHR.7. The present results in pregnant SHR differ from our previous finding with pregnant normotensive WKY, in which blunted responses to NA, but not to ES, were equalized by L-NOARG. Pregnancy induced vasodilatation in hypertensive rats appears to be more dependent on endothelial NO than in normotensive WKY. A defect of the endothelial NO generating pathway which promotes vasodilatation in pregnancy may contribute to the predisposition of women with essential hypertension to develop pre-eclampsia.     

Cocaine exposure prebreeding to weaning: maternal and offspring effects

1. The distribution of NADPH-diaphorase positive and catecholamine-containing nerve structures, and functional noradrenergic-nitrergic interactions, were studied in the rat anococcygeus muscle. 2. The morphological findings demonstrated NADPH-diaphorase positive neurons mostly as aggregates in intramural ganglia, nerve tracts and few single nerve fibres forming plexus-like structures. 3. The nitric oxide synthase inhibitor NG-nitro-L-arginine (L-NOARG) inhibited concentration-dependently the nitrergic relaxation, an effect reversed by L-arginine. The drug had dual effects on noradrenergic contractile responses: at lower concentrations (0.1-10 microM) it decreased the amplitude of contractions and this was not affected by L-arginine; higher concentrations (50-500 microM) potentiated the contractions, an effect that was prevented by L-arginine. 4. The electron acceptor, nitro blue tetrazolium (NBT) produced a rapid inhibition of the noradrenergic contractile responses (EC50 0.178 +/- 0.041 microM). The drug decreased the tone of the preparations. However, it potentiated concentration-dependently the nitrergic relaxations. 5. NBT (1 microM) had no significant effect on the relaxations induced by exogenously applied nitric oxide (NO)-donor sodium nitroprusside (SNP, 0.01-50 microM). However, the effect of NBT (0.1-10 microM) on the electrically induced relaxation was significantly decreased by L-NOARG (10 and 50 microM). The inhibition was of a non-competitive type. 6. Neither L-NOARG (100 microM) nor NBT (1 microM) had any effect on the spontaneous or electrically-induced release of 3H-radioactivity from the tissues preincubated in [3H]-noradrenaline. 7. It is concluded that L-arginine-NO pathway can modulate noradrenergic transmission in the rat anococcygeus muscle at postjunctional, but not prejunctional site(s).     

Excitatory transmission to the circular muscle of the guinea-pig ileum: evidence for the involvement of cannabinoid CB1 receptors

1. The effect of cannabinoid drugs has been investigated on cholinergic and non-adrenergic non-cholinergic (NANC) contractile responses to the circular smooth muscle of guinea-pig ileum elicited by electrical field stimulation (EFS). 2. The cannabinoid receptor agonist WIN 55,212-2 (1-1000 nM) and the putative endogenous ligand anandamide (0.1-100 microM) both produced a concentration-dependent inhibition of the cholinergic (9-57% and 1-51% inhibition) and NANC (9 55% and 2-57% inhibition) contractile responses. WIN 55,212-2 and anandamide did not modify the contractions produced by exogenous acetylcholine or substance P. 3. Apamin (30 nM), a blocker of Ca2+-activated K+ channels, reduced the inhibitory effect of WIN 55,212-2 on cholinergic, but not NANC, contractile response. NG-nitro-L-arginine methyl ester (100 microM), an inhibitor of nitric oxide synthase, or naloxone (1 microM), an opioid receptors antagonist, did not modify the inhibitory effect of WIN 55,212-2 on both cholinergic and NANC contractions. 4. The inhibitory effects of WIN 55,212-2 and anandamide on both cholinergic and NANC contractile response was competitively antagonized by the cannabinoid CB1 receptor antagonist SR 141716A (10-1000 nM). 5. In absence of other drugs, SR 141716A (1-1000 nM) enhanced cholinergic (1-45% increase) and NANC (2-38% increase) contractile responses elicited by electrical stimulation, but did not modify the contractions produced by acetylcholine or substance P. 6. It is concluded that activation of prejunctional cannabinoid CB1 receptors produces inhibition of cholinergic and NANC excitatory responses in the guinea-pig circular muscle. The inhibition of cholinergic (but not NANC) transmission involves activation of apamin-sensitive K+ channels. In addition, an endogenous cannabinoid ligand could inhibit cholinergic and NANC transmission in the guinea-pig ileal circular muscle.     

Regulation of the expression of follistatin in rat hepatocytes

The effect of lipopolysaccharide (LPS) treatment on noradrenergic responses elicited by electrical field stimulation (EFS) was investigated in the rabbit anococcygeus muscle. In the absence of LPS, EFS-induced contractions were enhanced and nitrergic relaxations were inhibited by NG-nitro-L-arginine (L-NOARG) but not by NG-monomethyl-L-arginine (L-NMMA). Administration of L-NMMA prior to L-NOARG inhibited the enhancement of EFS-induced contractions by L-NOARG and reversed the inhibitory effect of L-NOARG on nitrergic relaxations. Treatment with LPS induced a time-dependent loss of phenylephrine-induced tone which was inhibited by cycloheximide, dexamethasone, L-NMMA, or L-NOARG. Treatment of the anococcygeus muscle with LPS also resulted in a time-dependent loss in the magnitude of EFS-induced contractions and an increase in the delay of onset of contractions. These effects were reversible by pretreatment with cycloheximide or by treatment with L-NMMA. These results suggest that LPS induces a loss of tone and of noradrenergic responses through expression of the inducible NO synthase in the rabbit anococcygeus muscle. L-NMMA blocks these effects but does not affect nitrergic transmission, while L-NOARG is active against both.     

Different responsiveness to nitric oxide-cyclic guanosine monophosphate pathway in cholinergic and tachykinergic contractions of the rabbit iris sphincter muscle

PURPOSE: In the rabbit iris sphincter muscle, sodium nitroprusside (SNP), a nitric oxide (NO) donor, inhibits cholinergic contraction but does not affect tachykinergic contraction in vitro. The objectives of the current study were to clarify the mechanism for the different responsiveness to NO in cholinergic and tachykinergic muscular contractions, and to examine whether the mechanism for NO-induced inhibition of cholinergic muscular contraction is operative in vivo. METHODS: Iris sphincter muscle was dissected from the rabbit eye pretreated with or without endotoxin (lipopolysaccharide, LPS) in vivo. Cyclic guanosine monophosphate (cGMP) content in the iris sphincter muscle was determined by radioimmunoassay. The motor activity of the ring-shaped iris sphincter muscle was measured isometrically. Sodium nitroprusside, carboxy-2-phenyl-4,4,5,5,-tetramethyl-imidazoline-1-oxyl-3-oxide (C-PTIO, a scavenger of NO radicals), and 8-bromo cGMP (a permeable cGMP analogue) were administered between the first and second administrations of carbachol and neurokinin A, both of which had caused sustained contraction in the iris sphincter muscle. RESULTS: Sodium nitroprusside inhibited the contraction of the iris sphincter muscle caused by carbachol but had no effect on the contraction caused by neurokinin A. Application of C-PTIO significantly reduced SNP-induced cGMP accumulation in the muscle, as well as the SNP-induced inhibition of muscular contraction caused by carbachol. Neither carbachol nor neurokinin A influenced SNP-induced cGMP accumulation in the muscle. Induction of 8-bromo-cGMP significantly diminished the muscular contraction caused by carbachol but not that caused by neurokinin A. In vivo pretreatment of the eye with LPS increased, in a time-dependent manner, the cGMP accumulation in the iris sphincter muscle, which was significantly inhibited by pretreatment of NG-nitro-L-arginine methyl ester (an inhibitor of NO synthesis) in vivo. CONCLUSIONS: These results demonstrate that in rabbits the increase in cGMP accumulation induced by NO in the iris sphincter muscle is involved in the cholinergic contraction but not in the tachykinergic contraction, suggesting that different sensitivities to cGMP are essential for the different responsiveness to NO. Furthermore, the results of this study showed that the NO-cGMP pathway is operative in vivo and regulates iris sphincter muscle tone, at least when the eyes are infected with bacteria.     

Play, creativity, and adaptive functioning: implications for play interventions

This study focuses on the potential role for nitric oxide on the actions of the parasympathetic innervation to the heart. Earlier, we showed that the nitric oxide synthase (NOS) inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) reduced the bradycardia induced by stimulation of vagal efferent motor fibres and that these effects are reversible through administration of the NOS substrate L-arginine. In the present study, we show that D-arginine does not reverse the effects of the inhibitors and confirm that they are reversed by L-arginine. Another NOS inhibitor, NG-nitro-L-arginine (L-NOARG), produced similar effects which were not reversed by L-arginine. In an examination of the effect of increasing NO availability with the NO donor sodium nitroprusside the vagally induced bradycardia was enhanced at all frequencies tested. In a separate series, the effects of NOS inhibitors and NO donors on the dromotropic actions of the vagus were examined. The NOS inhibitor L-NAME, reduced the increase in atrio-ventricular conduction delay normally induced by efferent vagal stimulation at all frequencies tested both in the 'paced' and 'unpaced' heart. Further, sodium nitroprusside enhanced this delay. Overall the study indicates that NO has an important facilitatory role on both the chronotropic and dromotropic actions of the vagus nerve on the heart and that NO may be a rate-limiting factor in the cardiac responses to vagal stimulation.     

Stimulation by carbachol of mucus gel thickness in rat stomach involves nitric oxide

Instillation of carbachol (150 micrograms/kg) into the gastric lumen in vivo increased the thickness of the mucus gel layer. Intravenous administration of the inhibitor of nitric oxide (NO) synthase, NG-nitro-L- arginine methyl ester (L-NAME, 0.4-5 mg/kg) dose-dependently reduced the stimulation by carbachol, the half-maximal inhibitory dose being 0.57 mg/kg. This effect of L-NAME was abolished by administration of L-arginine but not by D-arginine (100 mg/kg i.v.). By contrast L-NAME (5 mg/kg) did not reduce the stimulatory effect of intraluminal 16,16-dimethyl prostaglandin E2 (50 micrograms/kg) on mucus gel thickness. These results implicate NO in the cholinergic activation of gastric mucus secretion.     

Endothelial calcium-calmodulin dependent nitric oxide synthase in the in vitro vascular hyporeactivity of portal hypertensive rats

BACKGROUND/AIMS: Increased nitric oxide production has been implicated in impaired vascular responsiveness to vasoconstrictors in portal hypertension. However, there is no firm evidence concerning the involved nitric oxide synthase isoform. The present study investigated the possible contribution of one nitric oxide synthase isoform, the endothelial constitutive Ca2+-calmodulin dependent, in the overproduction of nitric oxide in portal hypertension. METHODS: Vascular responses to norepinephrine and acetylcholine were evaluated in isolated thoracic aortic rings from normal and portal vein stenosed rats. RESULTS: An impaired concentration-dependent contraction to norepinephrine was observed in intact rings from portal hypertensive rats compared to controls. The hyporeactivity to norepinephrine was reversed after endothelium denudation, the inhibition of nitric oxide synthase with L-NOARG or the inhibition of calmodulin with W-7, but not after pre-incubation with indomethacin. Stimulation of intact rings with norepinephrine after the inhibition of calmodulin with calmidazolium was followed by a decreased vascular response in vessels from normal rats but not in those from portal hypertensive rats. Stimulation of intact rings with norepinephrine in a Ca2+-free medium was followed by a decreased vascular response in vessels from both portal hypertensive and normal rats. No difference in vasoconstrictive responses was observed between the two groups after calmidazolium or in a Ca2+-free medium. Relaxation induced by acetylcholine in norepinephrine-precontracted rings was more marked in rings from portal hypertensive rats than in controls. No differences in the vasodilator responses were observed after relaxations had been inhibited by the removal of the endothelium, pre-incubation with L-NOARG, indomethacin, W-7 or calmidazolium and in a Ca2+-free medium. CONCLUSIONS: This study demonstrates the involvement of the endothelial constitutive Ca2+-calmodulin dependent nitric oxide synthase isoform in the overproduction of nitric oxide in portal hypertension.     

RNA splicing ligase activity in the archaeon Haloferax volcanii

1. The double perfused mesentery was used to compare arterial and venous KCl- and acetylcholine (ACh)-induced responses in tissues taken from normotensive (WKY) and spontaneously hypertensive rats (SHR) in the presence or absence of inhibitors of nitric oxide (NO) synthase (NG-nitro-L-arginine (L-NOARG) and NG-nitro-L-arginine methyl ester (L-NAME)) and cyclo-oxygenase (indomethacin, mefenamic acid). 2. KCl (20 to 120 mM K+) caused concentration-dependent increases in arterial and venous perfusion pressures. The maximal arterial effects were significantly higher in the SHRs than in the WKY, with no differences in the venous pressor responses. 3. L-NAME and L-NOARG (100 microM) had no effect on the basal perfusion pressures in tissues from either WKY or SHRs, and mefenamic acid only induced a significant reduction of the basal perfusion pressures in the venous mesenteric vessels isolated from WKY. 4. L-NAME and L-NOARG (100 microM) potentiated the pressor responses to KCl to the same extent in the venous and arterial beds derived from WKY and SHR, while indomethacin and mefenamic acid (5 microM) only significantly decreased these responses in WKY. 5. Acetylcholine (ACh)-induced relaxations (1 nM to 10 microM) were significantly higher in arterial beds of WKY than in SHR, without differences in the venous relaxant responses. 6. L-NAME (100 microM) inhibited ACh-induced relaxations in arterial and venous beds from both groups of rats. Mefenamic acid was without effect on ACh-induced relaxations in either the arterial or the venous beds from WKY and SHR. 7. In conclusion, the liberation of NO in the perfused mesenteric vasculatures requires an active tone and no dysfunction of NO synthase activity is functionally apparent in the mesenteries isolated from SHRs. The cyclo-oxygenase pathway is only implicated in the KCl-induced responses of tissues derived from WKY, but not in the vasodilatations induced by ACh in either the arterial or the venous vasculatures from WKY and SHR.     

Multiple nitric oxide sources in neurogenic plasma extravasation in rat hindpaw skin

The contribution of nitric oxide (NO) to capsaicin-evoked plasma extravasation was studied in rat hindpaw skin. Two inhibitors of NO synthase were used: 7-nitroindazole, with a selectivity for nerve-derived NO, and the L-arginine derivative, N(omega)-nitro-L-arginine (L-NOARG), which is a non-selective inhibitor. Plasma extravasation was induced by intraplantar injection of 5 microg/50 microl capsaicin and measured by the Evans blue leakage technique. Both acute and chronic administration of 7-nitroindazole significantly reduced capsaicin-evoked plasma extravasation in rat hind-paw skin, whereas L-NOARG enhanced it. This enhancement was abolished non-stereospecifically by either L- or D-arginine. Our results suggest that NO production from different sources yields a complex action in maintaining the endothelial integrity in neurogenic plasma extravasation.     

Evidence that mechanisms dependent and independent of nitric oxide mediate endothelium-dependent relaxation to bradykinin in human small resistance-like coronary arteries

1. The effects of the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine (L-NOARG), the NO scavenger, oxyhaemoglobin (HbO) and high extracellular K+ upon endothelium-dependent relaxation to bradykinin were investigated in human isolated small coronary arteries. 2. Endothelium-dependent relaxations to bradykinin were compared in vessels contracted to approximately 50% of their maximum contraction to 124 mM KCl Krebs solution, regardless of treatments, with the thromboxane A2 mimetic, U46619 and acetylcholine. All relaxations were expressed as percentage reversal of the initial level of active force. 3. L-NOARG (100 microM) caused a small but significant, 12% (P < 0.01), decrease in the maximum relaxation (Rmax: 91.5 +/- 5.4%) to bradykinin but did not significantly affect the sensitivity (pEC50: 8.08 +/- 0.17). Increasing the concentration of L-NOARG to 300 microM had no further effect on the pEC50 or Rmax to bradykinin. HbO (20 microM) and a combination of HbO (20 microM) and L-NOARG (100 microM) reduced Rmax to bradykinin by 58% (P < 0.05) and 54% (P < 0.05), respectively. HbO (20 microM) and L-NOARG (100 microM, combined but not HbO (20 microM) alone, caused a significant 11 fold (P < 0.05) decrease in sensitivity to bradykinin. HbO (20 microM) decreased the sensitivity to the endothelium-independent NO donor, S-nitroso-N-acetylpenicillamine (SNAP), approximately 17 fold (P < 0.05). 4. Raising the extracellular concentration of K+ isotonically to 30 mM, reduced the Rmax to bradykinin from 96.6 +/- 3.1% to 43.9 +/- 10.1% (P < 0.01) with no significant change in sensitivity. A combination of HbO, L-NOARG and high K+ (30 mM) abolished the response to bradykinin. High K+ did not change either the sensitivity or maximum relaxation to SNAP. 5. In conclusion, L-NOARG does not completely inhibit endothelial cell NO synthesis in human isolated small coronary arteries. By comparison, HbO appeared to block all the effects of NO in this tissue and revealed that most of the relaxation to bradykinin was due to NO. The non-NO -dependent relaxation to bradykinin in the human isolated small coronary arteries appeared to be mediated by a K(+)-sensitive vasodilator mechanism, possibly endothelium-derived hyperpolarizing factor (EDHF).     

Nitrergic modulation of cholinergic responses in the opossum lower oesophageal sphincter

1. Electrical field stimulation (EFS) of the superfused lower oesophageal sphincter from opossum (Monodelphis domestica) elicited biphasic responses. The first phase (relaxation) was strictly dependent on the duration of the EFS. The second phase (contraction) started following termination of the EFS (< or = 15 Hz). EFS at frequencies above 15 Hz led only to contraction, which started immediately upon initiation of the stimulation. 2. In the presence of NG-nitro-L-arginine (L-NOARG; 0.1-300 microM), the relaxation phase was abolished and the contractile response started with the initiation of EFS (at all frequencies) and was greater in magnitude. The contractile response to EFS was completely blocked with scopolamine (10 microM). 3. Exogenous acetylcholine (1-100 microM) elicited concentration-dependent contractions of the sphincter in the presence of botulinum toxin. These contractions were abolished when EFS was applied during administration of acetylcholine. This inhibitory effect of EFS was completely reversed when the tissue was treated with L-NOARG (100 microM). 4. These results suggest that the cholinergic response in the opossum lower oesophageal sphincter is under nitrergic control.     

Characterization of the potassium channels involved in EDHF-mediated relaxation in cerebral arteries

1. In the presence of NG-nitro-L-arginine (L-NOARG, 0.3 mM) and indomethacin (10 microM), the relaxations induced by acetylcholine and the calcium (Ca) ionophore A23187 are considered to be mediated by endothelium-derived hyperpolarizing factor (EDHF) in the guinea-pig basilar artery. 2. Inhibitors of adenosine 5'-triphosphate (ATP)-sensitive potassium (K)-channels (KATP; glibenclamide, 10 microM), voltage-sensitive K-channels (Kv; dendrotoxin-1, 0.1 microM or 4-aminopyridine, 1 mM), small (SKCa; apamin, 0.1 microM) and large (BKCa; iberiotoxin, 0.1 microM) conductance Ca-sensitive K-channels did not affect the L-NOARG/indomethacin-resistant relaxation induced by acetylcholine. 3. Synthetic charybdotoxin (0.1 microM), an inhibitor of BKCa and Kv, caused a rightward shift of the concentration-response curve for acetylcholine and reduced the maximal relaxation in the presence of L-NOARG and indomethacin, whereas the relaxation induced by A23187 was not significantly inhibited. 4. A combination of charybdotoxin (0.1 microM) and apamin (0.1 microM) abolished the L-NOARG/ indomethacin-resistant relaxations induced by acetylcholine and A23187. However, the acetylcholine-induced relaxation was not affected by a combination of iberiotoxin (0.1 microM) and apamin (0.1 microM). 5. Ciclazindol (10 microM), an inhibitor of Kv in rat portal vein smooth muscle, inhibited the L-NOARG/ indomethacin-resistant relaxations induced by acetylcholine and A23187, and the relaxations were abolished when ciclazindol (10 microM) was combined with apamin (0.1 microM). 6. Human pial arteries from two out of four patients displayed an L-NOARG/indomethacin-resistant relaxation in response to substance P. This relaxation was abolished in both cases by pretreatment with the combination of charybdotoxin (0.1 microM) and apamin (0.1 microM), whereas each toxin had little effect alone. 7. The results suggest that Kv, but not KATP and BKCa, is involved in the EDHF-mediated relaxation in the guinea-pig basilar artery. The synergistic action of apamin and charybdotoxin (or ciclazindol) could indicate that both Kv and SKCa are activated by EDHF. However, a single type of K-channel, which may be structurally related to Kv and allosterically regulated by apamin, could also be the target for EDHF.     

No evidence for a significant non-nitrergic, hyperpolarising factor contribution to field stimulation-induced relaxation of the mouse anococcygeus

1. The aim of the study was to determine whether a nerve-derived hyperpolarizing factor (NDHF) might contribute to non-adrenergic, non-cholinergic (NANC) relaxations of the mouse anococcygeus when low concentrations of contractile agent are used to raise tone and low frequencies of field stimulation applied; such a non-nitrergic NDHF has been proposed to contribute to NANC relaxations of the rat anococcygeus and guinea-pig taenia coli. 2. Phenylephrine (0.1-100 microM) produced concentration-related contractions of the mouse isolated anococcygeus muscle; 0.2 microM phenylephrine (EC26) was used to raise tone in subsequent experiments. 3. Field stimulation (0.5, 1.0 and 5.0 Hz) produced frequency-dependent relaxations of phenylephrine-induced tone. In the presence of the nitric oxide synthase inhibitor L-NG-nitro-arginine (L-NOARG; 100 microM), the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one (ODQ; 5 microM), or a combination of these two drugs, relaxations to field stimulation were abolished at all frequencies studied. Relaxations to sodium nitroprusside (0.01-5 microM) were unaffected by L-NOARG but strongly inhibited by ODQ; neither enzyme inhibitor affected relaxations to 8-Br-cyclic GMP (10 microM). 4. Nifedipine (1 microM) reduced the contractile response to 0.2 microM phenylephrine by 38%; however, it had no effect on NANC relaxations. 5. It is concluded that NANC relaxations of the mouse anococcygeus are purely nitrergic and that there is no significant contribution from a putative NDHF.     

Differential responses of pulmonary arteries and veins to histamine and 5-HT in lung explants of guinea-pigs

1. The mechanisms by which histamine and 5-HT differentially contract pulmonary arteries and veins are unclear. In lung explants from 26 guinea-pigs, we compared responses of pulmonary arteries and vein to histamine, 5-HT and KCI, and examined potential determinants for the differential responses. Lungs were filled with agarose, sectioned into approximately 1 mm thick slices, and vascular luminal areas measured by image analysis. 2. Histamine and 5-HT produced a concentration-dependent constriction in arteries and veins, greater in the latter. KCl constricted arteries and veins equally. 3. The histamine H1 antagonist chlorpheniramine (10(-4) M) abolished contractions to histamine; the H2 antagonist cimetidine enhanced maximal responses and sensitivity of arteries and veins to histamine, and diminished the differences between their maximal responses; the NO synthase inhibitor Nomega-nitro-L-arginine (L-NOARG) increased the maximal responses of arteries and veins, and the differences between their responses; indomethacin had no effect. 4. Contractions to 5-HT were abolished in arteries and markedly reduced in veins by the 5-HT2 antagonist ketanserin (10(-4) M); L-NOARG potentiated the maximal responses of arteries but not of veins; indomethacin increased the maximal responses of arteries but reduced them in veins. 5. By morphometry, arteries had a greater medial thickness and luminal diameter than veins. 6. The data suggest that in guinea-pigs, H2 receptors are responsible for the differential contractile responses of pulmonary arteries and veins to histamine, whereas endothelium-derived vasoactive substances are responsible for their differential contractile responses to 5-HT.     

A broadening view of recombinational DNA repair in bacteria

The role of the cholinergic and peptidergic pathways in the impairment of gastric motility associated with diabetic gastroparesis was assessed at the postsynaptic level using isolated fundus smooth muscle strips. Maximal contractile responses to carbachol and galanin were significantly decreased in fundus strips isolated from rats rendered diabetic by a single intraperitoneal injection of streptozotocin (STZ, 70 mg/kg) 1, 4 and 8 weeks before experiments. We also observed notable decrements in the slopes and Hill's coefficients without conspicuous changes in the EC50 of the respective galanin concentration-response curves measured in strips obtained from STZ animals after 4 and 8 weeks. L-NAME reversed the above-mentioned alterations in an L-arginine-sensitive manner in STZ rats after 4 weeks but not in STZ rats after 8 weeks. The blood plasma nitrite/nitrate levels in STZ animals after 4 and 8 weeks were increased by 44.6 and 61.9%, respectively. Ca2+-independent nitric oxide synthase activity in gastric fundus strips and stomach corpus mucosa from STZ rats after 4 weeks was markedly enhanced by 37.4 and 31.9%, respectively, suggesting an enhanced nitric oxide production. In vivo insulin treatment prevented diabetes-induced alterations in smooth muscle contractility. We conclude that the smooth muscle dysfunction evoked by experimental diabetes causing diminished contractions of fundus strips to carbachol and galanin is at least partly due to the increased nitric oxide synthesis.     

Endothelium-dependent modulation of resistance vessel contraction: studies with NG-nitro-L-arginine methyl ester and NG-nitro-L-arginine

1. The effect of NG-nitro-L-arginine methyl ester (L-NAME) and NG-nitro-L-arginine (L-NOARG) on noradrenaline (NA)-induced contractility and acetylcholine (ACh)-induced endothelium-dependent relaxation was studied in rat mesenteric resistance arteries. 2. Third order branches of mesenteric arteries were dissected and mounted on two forty micron wires in a Mulvany myograph. 3. Incubation with L-NAME and L-NOARG (10 microM) caused a time-dependent shift in the 50% response to NA (ED50) (0.01 microM-10 microM) but was not associated with an increase in the maximum contractile response. 4. L-NAME and L-NOARG (10 microM) caused a time-dependent inhibition of ACh (1 microM)-induced relaxation with a maximum effect after 120 min. 5. Following endothelium removal, incubation with either L-NAME or L-NOARG caused no significant shift in the ED50, although the residual relaxation response to ACh (1 microM) was further attenuated. 6. Incubation with the cyclo-oxygenase inhibitor, indomethacin, enhanced the relaxation to ACh and reduced the inhibitory effects of L-NAME and L-NOARG. 7. In conclusion, L-NAME and L-NOARG are potent inhibitors of acetylcholine-induced endothelium-dependent relaxation in mesenteric resistance arteries. The shift in ED50 associated with these inhibitors suggests a probable role for the endothelium in modulating the contractility of the resistance vasculature.