Effects of escins Ia, Ib, IIa, and IIb from horse chestnuts on gastric emptying in mice

Inhibitory effects of the saponin fraction and its principal constituents, escins Ia, Ib, IIa, and IIb, from horse chestnuts on gastric emptying were investigated in mice loaded with a non-nutrient or nutrient meal. The saponin fraction and escins Ia-IIb inhibited gastric emptying of a 1.5% carboxymethyl cellulose sodium salt (CMC-Na) meal by 11.1-54.2% (12.5-200 mg/kg). Escins Ia-IIb (50 mg/kg) also inhibited gastric emptying of a 40% glucose meal by 21.1-23.5% except for escin Ia, a milk meal by 18.4-33.1%, and a 30% ethanol meal by 13.5-15.9%. The effects of escins Ia-IIb on gastric emptying of the CMC-Na meal were attenuated by pretreatment with streptozotocin (100 mg/kg, i.v.), capsaicin (75 mg/kg in total, s.c.), or insulin (1 U/kg, s.c.). The effect of insulin was reduced by glucose (2 g/kg, i.v.) which can directly nourish the brain, but not by fructose (2 g/kg, i.v.) which cannot be utilized by the brain. The effects of escins Ia-IIb (50 mg/kg) were overridden in 60% ethanol-loaded mice, in which the central nervous system was suppressed by ethanol. These results suggest that capsaicin-sensitive sensory nerves and central nervous system partly participate in the effects of escins Ia-IIb.     

Gastroprotections of escins Ia, Ib, IIa, and IIb on ethanol-induced gastric mucosal lesions in rats.

Effects of escins Ia, Ib, IIa, and IIb isolated from horse chestnuts on ethanol-induced gastric mucosal lesions and the roles of capsaicin-sensitive afferent neurons, endogenous nitric oxide (NO), sulfhydryls, prostaglandins, as well as gastric secretion and the sympathetic nervous system, were investigated in rats. Test samples were given orally to fasted rats 1 h before ethanol (1.5 ml/rat, p.o.) treatment or ligation of the pylorus. Escins Ia-IIb (10-50 mg/kg) potently inhibited ethanol-induced gastric mucosal lesions, whereas desacylescins I and II (50 mg/kg) showed no such effect. These active saponins (10 and 20 mg/kg) did not decrease the gastric secretion. The gastroprotections of escins Ia-IIb were attenuated by the pretreatment with capsaicin, N(G)-nitro-L-arginine methyl ester, and indomethacin, but not by N-ethylmaleimide. The effects of escins Ia-IIb were also attenuated in streptozotocin-induced diabetic rats, in which the activity of the sympathetic nervous system was abnormal. These results suggest that the gastroprotections of escins Ia-IIb on ethanol-induced gastric mucosal lesions are acid-independent, whereas endogenous prostaglandins, NO, capsaicin-sensitive afferent neurons, and the sympathetic nervous system participate.     

Methylmercury inhibits nitric oxide production mediated by Ca(2+) overload and protein kinase A activation

The importance of cytosolic free calcium level intracellular Ca(2+), [Ca(2+)]i, in neutrophil activation prompted us to investigate changes in [Ca(2+)]i of neutrophils caused by methylmercury (MeHg), which has been shown to have immunomodulatory properties. We have shown in this paper that MeHg increased [Ca(2+)]i in the mouse peritoneal neutrophil. The L-type calcium channel blocker verapamil can decrease the elevated [Ca(2+)]i caused by 10 microM MeHg, suggesting that Ca(2+)-influx through L-type Ca(2+) channel mediates the effect of MeHg. Moreover, MeHg potently decreased nitric oxide (NO) production but also the protein and mRNA level of NO synthase induced by lipopolysaccharide. Both verapamil (1 microM) and H-89 (10 microM) can antagonize the inhibitory effect of MeHg (10 microM) on NO production. These findings lead us to conclude that MeHg inhibits NO production mediated at least in part by Ca(2+)-activated adenylate cyclase-cAMP-protein kinase A pathway.     

Hg(2+) and Cu(2+) interfere with agonist-mediated Ca(2+) signaling in isolated Mytilus digestive gland cells

The effects of mercury and copper on agonist-mediated Ca-signaling were investigated in isolated cells from the marine mussel, Mytilus galloprovincialis Lam., by single cell fluorescence microscopy. In isolated digestive gland cells, short-term exposure (10 min) to both Hg(2+), a highly toxic metal and Cu(2+), an essential metal, in the nano-low μM range caused a sustained increase in cytosolic [Ca(2+)]. The effect of mercury on resting [Ca(2+)] was stronger than that of copper. The Hg-induced elevation in [Ca(2+)] seemed to be mainly due to an increased influx through Verapamil-sensitive Ca-channels, whereas the effect of Cu(2+) was related to a release from thapsigargin-sensitive intracellular stores. Agonists, such as epidermal growth factor (EGF), bradykinin (BK) and ATP, evoked Ca(2+) transients in isolated digestive gland cells through different mechanisms similar to those observed in mammalian cells, demonstrating the presence of common pathways of Ca-mediated cell signaling in both invertebrates and vertebrates. The agonist-mediated Ca(2+) response was affected by exposure to Hg(2+) and Cu(2+) in a concentration dependent manner: both metals significantly reduced the amplitude of the Ca(2+) spikes elicited by BK and ATP and decreased the percentage of EGF-responsive cells. The effects of Hg(2+) and Cu(2+) were apparently independent of their different type of interaction with the mechanisms involved in Ca(2+) homeostasis. The results clearly demonstrate that, in marine invertebrate cells, short-term exposure to heavy metal concentrations comparable to environmental exposure levels results in alterations of intracellular Ca(2+) homeostasis which compromise the cell response to extracellular stimuli involving Ca-mediated signaling. The mechanisms of heavy metal interference with Ca-homeostasis and signaling are discussed.     

Agmatine exerts anticonvulsant effect in mice: modulation by alpha 2-adrenoceptors and nitric oxide

The effect of agmatine, an endogenous polyamine metabolite, on seizure susceptibility was investigated in mice. Acute intraperitoneal administration of agmatine (5, 10, 20, 40 mg/kg) had a significant and dose-dependent inhibitory effect on pentylenetetrazole (PTZ)-induced seizures. The peak of this anticonvulsant effect was 45 min after agmatine administration. We further investigated the possible involvement of the alpha(2)-adrenoceptors and L-arginine/NO pathway in this effect of agmatine. The alpha(2)-adrenoceptor antagonist, yohimbine (0.5-2 mg/kg), induced a dose-dependent blockade of the anticonvulsant effect of agmatine. The nitric oxide synthase (NOS) substrate, L-arginine (60 mg/kg), inhibited the anticonvulsant property of agmatine and this effect was significantly reversed by NOS inhibitor N(G)-nitro-L-arginine (L-NAME, 30 mg/kg), implying an NO-dependent mechanism for L-arginine effect. We further examined a possible additive effect between agmatine (1 or 5 mg/kg) and L-NAME (10 mg/kg). The combination of L-NAME (10 mg/kg) with agmatine (5 but not 1 mg/kg) induced a significantly higher level of seizure protection as compared with each drug alone. Moreover, a combination of lower doses of yohimbine (0.5 mg/kg) and L-arginine (30 mg/kg) also significantly decreased the anticonvulsant effect of agmatine. In conclusion, the present data suggest that agmatine may be of potential use in seizure treatment.     

Down-regulation of phenobarbital-induced cytochrome P4502B mRNAs and proteins by endotoxin in mice: independence from nitric oxide production by inducible nitric oxide synthase

Multiple hepatic cytochrome P450 enzymes are down-regulated at the mRNA and protein levels during inflammation and infection. A body of evidence suggests that nitric oxide (NO) produced from inducible NO synthase (NOS2) is responsible for some of these effects. The current study was designed to examine the NO dependencies of the down-regulation of phenobarbital-induced CYP2B mRNAs and proteins by bacterial endotoxin (lipopolysaccharide, LPS) treatment in vivo, using an NOS2-null mouse model. Treatment of C57/BL6 mice with 0.3 mg/kg of LPS maximally suppressed phenobarbital-induced CYP2B9 and 2B10 mRNAs measured 12 hr after injection, whereas 1-10 mg/kg of LPS was required to elevate NO production. Down-regulation of CYP2B mRNAs by 1 mg/kg of LPS was equivalent in wild-type and NOS2-null mice. No effect of LPS in the dose range of 0.3 to 10 mg/kg was observed on microsomal CYP2B protein levels measured 12 hr after treatment, whereas 1 mg/kg of LPS suppressed CYP2B proteins 24 hr after treatment in both wild-type and NOS2-null mice. We conclude that the main mechanism for the down-regulation of CYP2B proteins in mouse liver following moderate- or high-dose LPS treatment is via NO-independent suppression of CYP2B9 and 2B10 mRNAs. Unlike rat hepatocytes, the contribution of a rapid, NO-dependent mechanism of CYP2B protein suppression in mouse liver appears to be minor or non-existent.     

Bile acids increase intracellular Ca(2+) concentration and nitric oxide production in vascular endothelial cells

The effects of bile acids on intracellular Ca(2+) concentration [Ca(2+)](i) and nitric oxide production were investigated in vascular endothelial cells. Whole-cell patch clamp techniques and fluorescence measurements of [Ca(2+)](i) were applied in vascular endothelial cells obtained from human umbilical and calf aortic endothelial cells. Nitric oxide released was determined by measuring the concentration of NO(2)(-). Deoxycholic acid, chenodeoxycholic acid and the taurine conjugates increased [Ca(2+)](i) concentration-dependently, while cholic acid showed no significant effect. These effects resulted from the first mobilization of Ca(2+) from an inositol 1,4,5-triphosphate (IP(3))-sensitive store, which was released by ATP, then followed by Ca(2+) influx. Both bile acids and ATP induced the activation of Ca(2+)-dependent K(+) current. Oscillations of [Ca(2+)](i) were occasionally monitored with the Ca(2+)-dependent K(+) current in voltage-clamped cells and Ca(2+) measurements of single cells. The intracellular perfusion of heparin completely abolished the ATP effect, but failed to inhibit the bile acid effect. Deoxycholic acid and chenodeoxycholic acid enhanced NO(2)(-) production concentration-dependently, while cholic acid did not enhance it. The bile acids-induced nitric oxide production was suppressed by N(G)-nitro-L-arginine methyl ester, exclusion of extracellular Ca(2+) or N-(6-aminohexyl)-5-chloro-l-naphthalenesulphonamide hydrochloride (W-7) and calmidazolium, calmodulin inhibitors. These results provide novel evidence showing that bile acids increase [Ca(2+)](i) and subsequently nitric oxide production in vascular endothelial cells. The nitric oxide production induced by bile acids may be involved in the pathogenesis of circulatory abnormalities in liver diseases including cirrhosis.     

Responsiveness to noradrenaline in aorta from wild-type,nitric oxide synthase-2, nitric oxide synthase-3 and alpha2A/D-adrenoceptor knockout mice

We have investigated the responsiveness of mouse aorta to noradrenaline (10 microM). In wild-type mice, noradrenaline produced an initial peak contraction (3.35+/-0.28 mN) and a significantly smaller plateau response (2.15+/-0.41 mN). The contractions were similar in aorta from nitric oxide synthase-2 (NOS-2) knockout mice. In vessels from NOS-3 knockout mice, noradrenaline contractions consisted of an early steeply rising phase with a later shallow rising phase to a maximum (10.21+/-0.84 mN), which was significantly greater than in wild-type and NOS-2 knockout mice, and resembled the contraction to phenylephrine (10 microM) in wild-type. In alpha(2A/D)-adrenoceptor knockout mice, the noradrenaline maximum was significantly smaller than in NOS-3 knockout but significantly larger than in wild-type. Following N(G)-nitro-L-arginine methyl ester (L-NAME, 10 microM), responses in wild-type and alpha(2A/D)-adrenoceptor knockout were as in NOS-3 knockout mice. The alpha(2D)-adrenoceptor antagonist BRL 44408 (2-((4,5-dihydro-1H-imidazole-2-yl)methyl)-2,3-di-hydro-1-methyl-1H-isoindole maleate; 1 microM) increased noradrenaline-induced contractions and the alpha(2)-adrenoceptor agonist xylazine reduced Prostaglandin F(2alpha)-induced contractions, in wild-type but not NOS-3 knockout. Contractions to noradrenaline in mouse aorta are modulated by NOS-3 and part of the effect involves activation of alpha(2A/D)-adrenoceptors.     

Evidence for the role of nitric oxide in nicotine-induced locomotor sensitization in mice

Rationale Nitric oxide (NO) is implicated in both acute effects of addictive drugs and development of dependence to them. We investigated the role of NO in nicotine-induced locomotor sensitization.Objectives The effects of N-nitro-l-arginine methyl ester (l-NAME), a NO synthase inhibitor, and a combination of a NO precursor l-arginine and l-NAME on nicotine-induced locomotor sensitization were investigated in Swiss Webster mice.Methods Sensitization to psychomotor stimulating effect of nicotine was rendered by seven injections of nicotine (1 mg/kg) on every other day. To investigate their effect on the development of sensitization to nicotine, l-NAME (15–60 mg/kg) and l-arginine (1 g/kg) were given before nicotine administration during the first seven sessions. To investigate the effect of these compounds on the expression of nicotine sensitization, after a 4-day drug-free period another group of mice received a challenge injection of nicotine on day 18.Results Nicotine (1 mg/kg) produced a robust locomotor sensitization in mice. The doses of 30 mg/kg and 60 mg/kg of l-NAME blocked the development of sensitization to nicotine; and, l-arginine (1 g/kg) pretreatment reversed this effect of l-NAME. Likewise, the doses of 30 mg/kg and 60 mg/kg of l-NAME inhibited the expression of sensitization to nicotine on day 18; and, l-arginine (1 g/kg) pretreatment reversed this inhibitory effect of l-NAME.Conclusions Our results suggest that NO is implicated in the development and expression of nicotine-induced locomotor sensitization in mice.     

Impaired vascular function in normoglycemic mice prone to autoimmune diabetes: role of nitric oxide

Type 1 diabetes is an immuno-inflammatory condition which increases the risk of cardiovascular disease, particularly in young adults. This study investigated whether vascular function is altered in mice prone to autoimmune diabetes and whether the nitric oxide (NO)-cyclic GMP axis is involved. Aortic rings suspended in organ chambers and precontracted with phenylephrine were exposed to cumulative concentrations of acetylcholine. To investigate the role of NO, some experiments were performed in the presence of either 1400W (N-(3-aminomethyl)benzyl-acetamidine hydrochloride), a selective inhibitor of the iNOS-isoform, L-NAME (N(G)-nitro-L-arginine methyl ester hydrochloride), an inhibitor of all three NOS-isoforms, or ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), a selective inhibitor of guanylate cyclase. Moreover, contractility to phenylephrine, big endothelin-1, and endothelin-1 was assessed and histological analysis and iNOS immunohistochemistry were performed. Endothelium-dependent relaxation was reduced in prediabetic NOD mice (78+/-4 vs. 88+/-2%, respectively, P<0.05 vs. control) despite normal plasma glucose levels (n.s. vs. control). Preincubation with 1400W further attenuated responses in prediabetic (P<0.05 vs. untreated) but not in diabetic or in control mice. In contrast, basal NO bioactivity remained unaffected until the onset of diabetes in NOD mice. Contractile responses to big endothelin-1 and endothelin-1 were reduced in prediabetic animals (P<0.05 vs. control), whereas in diabetic mice only responses to big endothelin-1 were decreased (P<0.05 vs. control). These data demonstrate that endothelium-dependent and -independent vascular function in NOD mice is abnormal already in prediabetes in the absence of structural injury. Early proinflammatory activation due to iNOS in diabetes-prone NOD mice appears to be one of the mechanisms contributing to impaired vasoreactivity.     

Enhancement by nitric oxide of neurogenic contraction in the mouse urinary bladder

In isolated detrusor strips from the mouse urinary bladder, contractile responses to electrical field stimulation were mostly mediated by neurally released acetylcholine and ATP. The aim of this study is to investigate the possible role of nitric oxide (NO) involved in the neurogenic detrusor contraction. Repetitive electrical field stimulation evoked muscle contractions of the isolated mouse detrusor strips, which could be abolished by tetrodotoxin (TTX). NO donors including sodium nitroprusside (SNP) and S-nitroso-N-acetyl-penicillamine (SNAP) as well as exogenous NO increased, while hemoglobin and NO synthase inhibitor N^G-nitro-L-arginine decreased the neurogenic detrusor contractions. The addition of L-arginine reversed the inhibitory effect of N^G-nitro-L-arginine. SNP failed to affect the contractions induced by carbachol and α,β-methylene ATP. These findings suggest that NO potentiated the excitatory neuromuscular transmission in electrically stimulated detrusor strips from the mouse. Received: 15 July 1997 / Accepted: 4 September 1997     

Evidence for the role of nitric oxide in caffeine-induced locomotor activity in mice

Rationale Nitric oxide (NO) is implicated in the acute locomotor activating effects of some addictive drugs such as amphetamine and cocaine, but has not been investigated in the case of caffeine.Objectives We investigated the effects of a nitric oxide synthase (NOS) inhibitor N-Nitro-l-arginine methyl ester (l-NAME) and a combination of l-arginine, a NO precursor, and l-NAME on caffeine induced locomotor activity in Swiss Webster mice.Methods Locomotor activity was recorded for 30 min immediately following caffeine (0.25–128 mg/kg) or saline administration. In a further study, l-NAME (15 and 30 mg/kg) was administered to another group of mice 30 min before caffeine (1 and 16 mg/kg) injections. Finally, l-arginine (1000 mg/kg), a nitric oxide precursor, was administered 20 min before the l-NAME (15 and 30 mg/kg) treatments and locomotor activities were again recorded immediately after caffeine (1 and 16 mg/kg) injections.Results Caffeine (0.5–16 mg/kg) significantly increased locomotor activity, while l-NAME (30 mg/kg) blocked caffeine (1 and 16 mg/kg)-induced locomotor activity. The low dose of l-NAME blocked only caffeine (1 mg/kg)-induced locomotor activity. l-Arginine reversed the inhibitory effects of l-NAME on caffeine-induced locomotor activity. l-NAME and l-arginine had no effect on the locomotor activity of the mice when given by themselves.Conclusions The results suggest that caffeine-induced locomotor activity might be modulated by NO in mice.     

Possible role of nitric oxide in anxiety following transient cerebral ischemia in mice

The possible role of nitric oxide (NO) in anxiety following transient cerebral ischemia by a 10-min bilateral carotid occlusion was examined in mice. Two days after the ischemia, mice showed a significant decrease in time spent on the open arms in the elevated plus-maze test; and likewise, they showed shortened social interaction time in the social interaction test, suggesting the induction of anxiety. Such anxiety behavior, however, was diminished 7 days after the treatment in both tests. A nonselective nitric oxide synthase (NOS) inhibitor, N( omega)-nitro-L-arginine methyl ester (L-NAME), and a selective inducible NOS (iNOS) inhibitor, S-ethylisothiourea (EIT), given twice after reperfusion, produced an anxiolytic effect in the elevated plus-maze test 2 days after the ischemia, while only the former produced antianxiety in the social interaction test. A relatively selective neuronal NOS (nNOS) inhibitor, 7-nitroindazole (7-NI), failed to decrease the level of anxiety in both tests. These results suggest that the production of NO participates in the anxiogenic behavior by the ischemia. Furthermore, NO generated by endothelial NOS (eNOS) or eNOS with iNOS, with no involvement of nNOS, plays an important role in the anxiety induced by the ischemia. Thus, we conclude that 10-min bilateral carotid occlusion provides a useful exploratory animal model for anxiety following transient cerebral ischemia.     

Induction and potential biological relevance of a Ca(2+)-independent nitric oxide synthase in the myocardium.

1. We have investigated whether the myocardium and isolated cardiac myocytes can express a Ca(2+)-independent NO synthase after treatment with endotoxin or cytokines. Nitric oxide synthesis was measured in cytosols from the left ventricular wall from rats treated with endotoxin, or from freshly isolated myocytes from adult rats treated in vitro with cytokines. 2. Cytosols from the ventricle of saline-treated control animals showed only Ca(2+)-dependent NO synthesis. After treatment with endotoxin, the expression of an inducible, Ca(2+)-independent NO synthase was observed. The activity of this enzyme was maximal at 6 h and returned towards control levels by 18 h; no alterations occurred in the Ca(2+)-dependent NO synthase activity. Parallel to this enzyme induction there was an increase in myocardial guanosine 3':5'-cyclic monophosphate (cyclic GMP) and plasma nitrite and nitrate (NOx-). All these changes were prevented by pretreatment of the rats with dexamethasone. 3. Myocytes possessed Ca(2+)-dependent NO synthase activity and expressed, after treatment with tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta), a Ca(2+)-independent NO synthase, the induction of which was prevented by dexamethasone and cycloheximide. 4. Since increases in cyclic GMP levels in the heart are associated with reduced myocardial contractility, it is possible that the enhanced production of NO by a Ca(2+)-independent enzyme accounts, at least in part, for the depression of myocardial contractility seen in septic shock, cardiomyopathies, allograft rejection, burn trauma, as well as during anti-tumour therapy with cytokines.     

Age-dependent acceleration of ischemic injury in endothelial nitric oxide synthase-deficient mice: potential role of impaired VEGF receptor 2 expression

Morbidity and mortality of peripheral arterial occlusive disease significantly increases with age, often exhibiting more severe disease pathology and decreased treatment effectiveness. Therapeutic angiogenesis with angiogenic growth factors may represent a valuable treatment option for the severely ill, older adult patient population. Aging is considered an independent cardiovascular risk factor, but pathomechanistically it is not well understood. Diminished endothelial nitric oxide (EDNO) production has been considered as a major contributor to the aging process. To investigate the effect of age on postischemic revascularization independent of changes in EDNO, we used endothelial nitric oxide synthase-deficient (ecNOS-KO) mice. We found an age-dependent acceleration in ischemic injury following unilateral femoral artery ligation in these animals compared to C57BL/J6 mice. Postischemic revascularization, quantified by measuring von Willebrand factor expression, was significantly impaired, suggesting that factors other than progressive EDNO deterioration are also involved in the age-dependent severe disease phenotype. Ischemia led to an increase in the expression of vascular endothelial growth factor receptor-2, KDR, in younger ecNOS-KO; however, this increase in KDR expression was absent in the older animals. Lack of increased KDR expression may provide a mechanistic explanation for the severe ischemic injury and perhaps can be used as a clinical marker to identify severe, vascular endothelial growth factor refractory patient population.     

Depression and cardiovascular disease: role of nitric oxide

Both depression and cardiovascular disease are major public health problems. Growing evidence shows that depression is a risk factor for the development of coronary artery disease (CAD). However, the exact mechanisms underlying the interplay between depression and CAD remain to be elucidated. Depression adversely affects autonomic and hormonal homeostasis, resulting in metabolic abnormalities, inflammation, increased platelet aggregation and endothelial dysfunction. All of these pathological features lead to atherothrombosis and cardiovascular events. However, there is no clear evidence that anti-depressant drugs or psychotherapy will reduce the risk or improve the outcome of CAD. Recent studies suggest that the L-arginine-nitric oxide (NO) pathway is involved in the genesis of depression. NO has many physiological functions, including vasodilatation, neurotransmission and platelet aggregation inhibition. It is synthesised from the cationic amino acid L-arginine by a family of enzymes: NO synthases (NOS). There are three NOS isoforms: inducible NOS (iNOS), endothelial NOS and neuronal NOS (nNOS). The availability and transport of L-arginine modulate rates of NO biosynthesis in circulating blood cells and vasculature, which provides a protective effect against cardiovascular disease. In depressive patients, the L-arginine-nitric oxide pathway seems to be impaired. The present review seeks a better understanding of the mechanisms that could identify depression as a cardiovascular risk factor and introduce new possible therapeutic interventions.     

Possible role of nitric oxide in the antinociceptive action of intraventricular bradykinin in mice

The i.c.v. administration of bradykinin (4, 8 and 16 μg) induced antinociception in mice which was resistant to naloxone; furthermore, the induction of tolerance to morphine by a single s.c. injection (100 mg/kg, 24 h before test doses of the peptide) did not affect antinociception. Since bradykinin is known to increase nitric oxide (NO) in peripheral tissues, we studied the possibility that its antinociceptive action may be related to NO effects in the central nervous system. Bradykinin effects were antagonized by previous treatment with N^G-nitro- -arginine or concomitant i.c.v. administration of bradykinin and methylene blue. The immediate precursor of NO, -arginine, which by itself produces analgesia, also reduced bradykinin effects; moreover, tolerance to -arginine significantly decreased the response to the peptide. These results suggest that NO is involved in antinociception induced by i.c.v. administration of bradykinin.     

Critical role of nitric oxide in the modulation of prepulse inhibition in Swiss mice

Rationale

Nitric oxide (NO) modulates the dopamine uptake and release processes and appears to be implicated in dopamine-related pathologies, such as schizophrenia. However, it is unclear whether there is excess or deficient NO synthesis in schizophrenia pathophysiology. Analyses of the intracellular pathways downstream of NO system activation have identified the cyclic nucleotide cyclic guanosine monophosphate (cGMP) as a possible target for drug development. Defects in the sensorimotor gating of the neural mechanism underlying the integration and processing of sensory information have been detected across species through prepulse inhibition (PPI).

Objectives

The aim of this study was to investigate the effects of NO/cGMP increase on sensorimotor gating modulation during dopamine hyperfunction.

Methods

Mice were treated with NO donors and subjected to the PPI test. Treatment with the NO donor sodium nitroprusside was preceded by pretreatment with a soluble guanylate cyclase (sGC) inhibitor. Additionally, the mice were treated with NO donors and phosphodiesterases inhibitors prior to amphetamine treatment.

Results

Pretreatment with the NO donors enhanced the PPI response and attenuated the amphetamine-disruptive effects on the PPI. The sGC inhibitor did not modify the sodium nitroprusside effects. Additionally, the cGMP increase induced by a specific phosphodiesterase inhibitor did not modify the amphetamine-disruptive effect.

Conclusions

This study provides the first demonstration that an increase in NO can improve the PPI response and block the amphetamine-disruptive effects on the PPI response. Our data are consistent with recent clinical results. However, these effects do not appear to be related to an increase in cGMP levels, and further investigation is thus required.     

Modulation of neuroeffector transmission by endogenous nitric oxide: a role for acetylcholine receptor-activated nitric oxide formation, as indicated by measurements of nitric oxide/ nitrite release

Nitric oxide (NO) synthase inhibitors enhanced nerve-mediated contractile responses in guinea pig ileum longitudinal muscle, likely via a prejunctional effect on substance P-like neuroeffector transmission. Supporting a modulatory role for NO, application of NO through administration of acid sodium nitrite evoked marked inhibitory effects on responses to transmural nerve stimulation. Substance P-like responses to nerve stimulation were abolished by substance P receptor antagonists and were enhanced by atropine, indicating a cholinergic influence on substance P-like neuroeffector transmission. Since acetylcholine can evoke release of NO from endothelium, the possible role of acetylcholine in NO release in ileum was examined. The release of NO/nitrite, determined by chemiluminescence, was inhibited by N^G-monomethyl-L-arginine (L-NMMA), by calcium removal, by tetrodotoxin or by atropine, indicating a nerve-mediated control of NO production. A basis for the NO release is likely to be spontaneous neuronal activity, where release of acetylcholine, with subsequent muscarinic receptor activation, contributes to stimulation of NO formation.     

Enhancement of nitric oxide production by methylecgonidine in cultured neonatal rat cardiomyocytes.

1. In the present experiments, we investigated the effects of methylecgonidine (MEG) on nitric oxide (NO) production in cultured neonatal rat cardiomyocytes. Incubation of cultured cardiomyocytes with carbachol or MEG for 48 h significantly enhanced NO production. No release was increased from 1.48+/-0.13 microM (mg protein)(-1) for control to 5.73+/-0.19 microM (mg protein)(-1) for 1 microM carbachol treated cells (P<0.001). In addition, incubation with 1 microM MEG enhanced NO production to 5.55+/-0.28 microM (mg protein)(-1). The effects of MEG on NO production were concentration-dependent. The muscarinic antagonist atropine prevented the enhancement of NO production induced by carbachol or MEG. Compared to MEG-induced NO production, cocaine was much less potent. 2. The enhancement of NO production by carbachol or MEG was even greater in cultured cardiomyocytes transfected with the M(2) cDNA. After 48-h incubation with 1 microM carbachol or 1 microM MEG, NO production was increased by 6.5 and 6.7 fold, respectively, in cardiomyocytes overexpressing M(2) receptors. Coincubation with atropine or N(G)-nitro-L-arginine methyl ester abolished the enhancement of NO production. In contrast, NO production enhanced by carbachol or MEG in M(1)- or M(3)-transfected cardiomyocytes was similar to the level in non-transfected cells. 3. Western blot analysis showed that the protein levels of M(1), M(2), and M(3) were significantly increased in cardiomyocytes transfected with the receptor cDNAs, but MEG had no effect on the expressions. It is interesting that both carbachol and MEG caused a significant increase in constitutive endothelial NO synthase (eNOS) only in M(2)-transfected cardiomyocytes, not in non-transfected, M(1)- or M(3)-transfected cells. Again, atropine blocked the MEG-produced induction of eNOS. 4. Our data demonstrate that MEG significantly enhanced NO production in cultured cardiomyocytes and that the enhancement of NO production may result from MEG stimulation of muscarinic M(2) receptors.