The nitric oxide-cyclic GMP signalling pathway in rat brain

Nitric oxide is a novel signalling molecule in the brain and a potent activator of the cyclic GMP-synthesising enzyme, soluble guanylate cyclase. To determine if stimulation of cyclic GMP formation is a widespread mechanism of nitric oxide signal transduction, we have compared the distribution of the nitric oxide-generating enzyme (nitric oxide synthase) with that of nitric oxide-stimulated cyclic GMP accumulation, throughout the rat brain. The former was done using NADPH diaphorase histochemistry and the latter by cyclic GMP immunohistochemistry following perfusion of the nitric oxide donor, nitroprusside, in vivo. At a gross level, there was generally a good match when the two were compared in adjacent sections. Although the relative staining intensity varied from area to area, in no grey matter region did we observe cyclic GMP accumulation in the absence of nitric oxide synthase staining. In detail, the locations were complementary rather than identical. In some areas, nitric oxide synthase was found in postsynaptic structures and cyclic GMP accumulation in presynaptic elements and fibres; in others, the locations were reversed. Glial cells and their processes also accumulated cyclic GMP in the cerebellum. The results suggest that soluble guanylate cyclase is a major nitric oxide "receptor" throughout the brain. They also support the hypothesis that nitric oxide generated therein primarily functions as a mediator of cell-cell signaling rather than as a conventional second messenger acting within the cells in which it is produced. The types of communication subserved by nitric oxide appear to be extraordinarily diverse.     

Region-specific developmental patterns of atrial natriuretic factor- and nitric oxide-activated guanylyl cyclases in the postnatal frontal rat brain

In the rat central nervous system, cyclic GMP can be produced by two isoforms of guanylyl cyclase: a cytosolic isoform, which is activated by nitric oxide, and a membrane-bound isoform, activated by atrial natriuretic factor. We studied the development of guanylyl cyclase activity upon maturation of the rat forebrain from postnatal days 4 to 24, using a combined immunocytochemical and biochemical approach. Atrial natriuretic factor-activated particulate guanylyl cyclase activity was found to decrease in the frontal cortex, in the lateral septum and in the piriform cortex upon maturation. A transient expression of atrial natriuretic factor-sensitive guanylyl cyclase activity was observed at postnatal day 8 in the caudate putamen complex, whereas an increase was observed in the lateral olfactory tract from postnatal days 8 to 24. Biochemical and immunocytochemical studies using the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester, or the inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinaloxin-1-one, indicated high levels of endogenous nitric oxide release at postnatal days 4 and 8. This activity decreased strongly in all brain areas examined. From postnatal day 8 onwards, atrial natriuretic factor-responsive cyclic GMP-immunoreactive cells could be characterized as astrocytes, with the exception of those in the the lateral olfactory tract, where the myelinated fibers became cyclic GMP producing. Furthermore, our results on activation of both guanylyl cyclases at postnatal day 8 leads to the suggestion that both isoforms might be found in the same cells. This study shows that there are pronounced differences between various frontal brain areas in the development of the responsiveness of both the particulate and soluble isoforms of guanylyl cyclase, and lends further support to the hypothesis that natriuretic peptides have a role in neuronal growth and plasticity of the rat brain.     

Behavioural, neurochemical and neuroanatomical effects of chronic postnatal N-nitro-L-arginine methyl ester treatment in neonatal and adult rats

In the present study we evaluated the consequences of interference with nitric oxide synthesis during development on brain function and behaviour in later life. Rat pups received a daily injection of the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME, 25 mg/kg, s.c.) from postnatal day 0 to 24. At postnatal day 8 L-NAME-treated rats had enlarged and heavier stomachs, while body weights appeared to be reduced. The stomachs were not affected in size and weight anymore at postnatal day 24, whereas the body weights were still reduced by the L-NAME treatment, although they soon recovered after termination of the treatment. At four months-of-age, rats were tested in non-cognitive (open field) and cognitive (Morris water escape, two-way active avoidance) tasks. Open field behaviour of adult rats postnatally treated with L-NAME was not affected. In the water escape task there were no differences between the saline and L-NAME-treated rats in spatial discrimination learning and spatial reversal learning. Furthermore, postnatal L-NAME treatment did not have an effect on the acquisition of the two-way active avoidance task. Subsequently, we tested rat pups during the L-NAME treatment at postnatal day 19 through 24 in the open field and the two-way active avoidance task. L-NAME treatment appeared to increase the behavioural activity in the open field. There was no difference in behaviour in the active avoidance task between saline and L-NAME-treated rats. Biochemical and immunocytochemical studies showed that at postnatal day 8 the basal cyclic GMP level was reduced, while the cyclic GMP formation due to incubation with the nitric oxide donor sodium nitroprusside appeared to be increased in the hippocampus, striatum and frontal cortex of L-NAME-treated rats. Hence, nitric oxide synthase was inhibited whereas the soluble guanylyl cyclase activity may be increased in sensitivity. At postnatal day 24 basal cyclic GMP levels and nitric oxide-mediated cyclic GMP formation in the brain structures of L-NAME-treated rats had normal values again. Taken together, the findings of this study suggest that postnatal inhibition of nitric oxide synthase has profound neurochemical effects during development and may have short-lasting effects on non-cognitive behaviour, but it does not affect behaviour and brain function in later life.     

Hypothermia during or after severe perinatal asphyxia prevents increase in cyclic GMP-related nitric oxide levels in the newborn rat striatum

The striatum is rich in nitric oxide synthase (NOS). It is present in a dense fiber network and in a few medium-sized non-spiny interneurons. Previous work showed chronic overexpression of NOS in the rat striatum after a severe perinatal asphyctic (SPA) insult. This was prevented by hypothermia. We investigated whether the overexpression of NOS was accompanied by increased NOS activity. As nitric oxide (NO) is a potent activator of the soluble isoform of guanylyl cyclase, we measured striatal 3',5'-cyclic monophosphate (cyclic GMP) synthesis in 10-day-old (P10) rat pups that were subjected to SPA during normothermia or hypothermia during or after the insult. Cyclic GMP levels in striatal tissue from control pups were approximately 25.8 pmol/mg protein and in the SPA group approximately 38.1 pmol/mg protein (p<0.01). Hypothermia, during as well as after insult, prevented this increase of cyclic GMP. Nomega-nitro-L-arginine (L-NAME) (0.1 mM) decreased cyclic GMP levels in control, SPA and hypothermia treated pups to similar low levels (approximately 8% of level without L-NAME). Sodium nitroprusside (SNP) stimulated cyclic GMP showed no differences between the four groups. This indicates that high cyclic GMP levels in the striatum of rats subjected to SPA are caused by increased NOS activity. Hypothermia after an asphyctic insult could be a promising treatment.     

Neuronal nitric oxide synthase activation by vasoactive intestinal peptide in bovine cerebral arteries

The participation of nitric oxide and vasoactive intestinal peptide (VIP) in the neurogenic regulation of bovine cerebral arteries was investigated. Nitrergic nerve fibers and ganglion-like groups of neurons were revealed by NADPH-diaphorase staining in the adventitial layer of bovine cerebral arteries. NADPH diaphorase also was present in endothelial cells but not in the smooth muscle layer. Double immunolabeling for neuronal nitric oxide synthase and VIP indicated that both molecules co-localized in the same nerve fibers in these vessels. Transmural nerve stimulation (200 mA, 0.2 milliseconds, 1 to 8 Hz) of endothelium-denuded bovine cerebral artery rings precontracted with prostaglandin F2 alpha, produced tetrodotoxin-sensitive relaxations that were completely suppressed by NG-nitro-L-arginine methyl ester (L-NAME) and by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline (ODQ), but were not affected by the adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536), nor by VIP tachyphylaxis induced by pretreatment with 1 mumol/L VIP. Transmural nerve stimulation also elicited increases in intracellular cyclic GMP concentration, which were prevented by L-NAME, and small decreases in intracellular cyclic AMP concentration. Addition of VIP to bovine cerebral artery rings without endothelium produced a concentration-dependent relaxation that was partially inhibited by L-NAME, ODQ, and SQ 22,536. The effects of L-NAME and SQ 22,536 were additive. VIP induced a transient increase in intracellular cyclic GMP concentration, which was maximal 1 minute after VIP addition, when the highest relaxation rate was observed, and which was blocked by L-NAME. It is concluded that nitric oxide produced by perivascular neurons and nerve fibers fully accounts for the experimental neurogenic relaxation of bovine cerebral arteries and that VIP, which also is present in the same perivascular fibers, acts as a neuromodulator by activating neuronal nitric oxide synthase.     

In vivo microdialysis study of a specific inhibitor of soluble guanylyl cyclase on the glutamate receptor/nitric oxide/cyclic GMP pathway

1. Nitric oxide (NO) is known to stimulate soluble guanylyl cyclase, thereby eliciting an elevation of guanosine 3':5'-cyclic monophosphate (cyclic GMP) in target cells. Recently, a selective inhibitor of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), has been identified and characterized in vitro. We have investigated the in vivo effects of ODQ on the glutamate receptor/NO/ cyclic GMP pathway by monitoring extracellular cyclic GMP during microdialysis of the cerebellum or the hippocampus of freely-moving adult rats. 2. Intracerebellar administration of ODQ (1-100 microM) via the microdialysis probe inhibited, in a concentration-dependent manner, the basal extracellular level of cyclic GMP. The maximal inhibition, measured after a 20 min perfusion with 100 microM ODQ, amounted to 80% and persisted unchanged as long as ODQ was perfused. When ODQ was removed from the perfusion stream after 20 min, the levels of cyclic GMP started to recover, suggesting reversibility of guanylyl cyclase inhibition by ODQ. 3. The cyclic GMP response evoked in the cerebellum by NMDA (200 microM) or by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA; 100 microM) was largely attenuated by 100 microM ODQ. The pattern of the inhibition curves suggests competition for guanylyl cyclase between ODQ and the NO generated by NMDA or AMPA receptor activation. 4. ODQ (100 microM) prevented the elevation of extracellular cyclic GMP levels provoked by intracerebellar infusion of the NO generator S-nitroso-N-acetylpenicillamine (SNAP; 1 mM). The inhibition of the SNAP effect was rapidly relieved when ODQ was removed from the perfusion fluid. However, ODQ (100 microM) was unable to affect the cyclic GMP response elicited by 5 mM SNAP, in keeping with the proposed idea that ODQ binds to the "NO receptor' in a reversible and competitive manner. 5. Infusion of ODQ (10, 100 or 300 microM) into the hippocampus of freely-moving rats diminished the basal extracellular level of cyclic GMP. The maximal inhibition amounted to 50% and was produced by 100 microM ODQ. 6. The cyclic GMP response observed when 1 mM SNAP was perfused in the hippocampus, similar in percentage terms to that seen in cerebellum, was dramatically reduced during co-infusion of 100 microM ODQ. 7. ODQ appears to act in vivo as a selective, reversible and possibly competitive inhibitor of the soluble guanylyl cyclase targeted by NO. This enzyme may generate most (about 80%) of the cyclic GMP found under basal conditions in the extracellular space of the cerebellum. In the hippocampus, about 50% of the basal cyclic GMP does not seem to originate from the ODQ-sensitive soluble guanylyl cyclase.     

Inhibition of endogenous nitric oxide synthesis activates particulate guanylyl cyclase in the rat renal glomeruli

Nitric oxide (NO) plays a crucial role in the regulation of kidney function and metabolism. Our previous study showed that dexamethasone, one of several known selective inhibitors of inducible nitric oxide synthase (NOS), had a stimulatory effect on soluble guanylyl cyclase in the glomeruli of rat kidney. However, in the presence of dexamethasone, the atrial natriuretic factor (ANF)-dependent system remained suppressed. The aim of the present study was to investigate whether inhibition of synthesis of endogenous NO modulates the activity of the guanylyl cyclase system(s) in glomeruli. In these studies, rats were injected with a non-selective NOS inhibitor, N-omega-nitro-L-arginine methyl ester (NAME; NAME-group), or saline solution (controls; C-group). Creatinine clearance (C(Cr)), and plasma and urinary nitrate/nitrite (NOx-) levels decreased in the NAME-group, but plasma and urinary guanosine 3',5'-cyclic monophosphate (cGMP) contents were unchanged. In the presence of 0.1 microM ANF, synthesis of cGMP in the NAME-group exceeded threefold the cGMP production in the C-group. In addition, the pre-contracted glomeruli of the NAME-group were fully relaxed at 0.1 microM ANF, but glomeruli obtained from the C-group were relaxed in the presence of a 10 times higher dose of ANF. The increased sensitivity of glomeruli to ANF was possibly due to the more than doubled activity of particulate guanylyl cyclase (pGC) in the NAME-group in comparison with the C-group. In the presence of 100 microM sodium nitroprusside (SNP), soluble guanylyl cyclase (sGC) generated significantly lower cGMP production in the NAME-group than in the C-group (1.61 +/- 0.33 vs. 2.91 +/- 0.69 nmol/mg protein/10 min, respectively). These results demonstrate that inhibition of the synthesis of endogenous NO may also have an inhibitory effect on the activity of sGC. In addition, increased activity of the pGC and ANF-dependent system appears to be compensatory to the altered activity of soluble guanylyl cyclase.     

Formation of olfactory memories mediated by nitric oxide

Sheep learn to recognize the odours of their lambs within two hours of giving birth, and this learning involves synaptic changes within the olfactory bulb. Specifically, mitral cells become increasingly responsive to the learned odour, which stimulates release of both glutamate and GABA (gamma-aminobutyric acid) neurotransmitters from the reciprocal synapses between the excitatory mitral cells and inhibitory granule cells. Nitric oxide (NO) has been implicated in synaptic plasticity in other regions of the brain as a result of its modulation of cyclic GMP levels. Here we investigate the possible role of NO in olfactory learning. We find that the neuronal enzyme nitric oxide synthase (nNOS) is expressed in both mitral and granule cells, whereas the guanylyl cyclase subunits that are required for NO stimulation of cGMP formation are expressed only in mitral cells. Immediately after birth, glutamate levels rise, inducing formation of NO and cGMP, which potentiate glutamate release at the mitral-to-granule cell synapses. Inhibition of nNOS or guanylyl cyclase activity prevents both the potentiation of glutamate release and formation of the olfactory memory. The effects of nNOS inhibition can be reversed by infusion of NO into the olfactory bulb. Once memory has formed, however, inhibition of nNOS or guanylyl cyclase activity cannot impair either its recall or the neurochemical release evoked by the learned lamb odour. Nitric oxide therefore seems to act as a retrograde and/or intracellular messenger, being released from both mitral and granule cells to potentiate glutamate release from mitral cells by modulating cGMP concentrations. We propose that the resulting changes in the functional circuitry of the olfactory bulb underlie the formation of olfactory memories.     

Mechanism of selective motor neuronal death after exposure of spinal cord to glutamate: involvement of glutamate-induced nitric oxide in motor neuron toxicity and nonmotor neuron protection

In this study, we analyzed the mechanism of selective motor neuronal death, a characteristic of amyotrophic lateral sclerosis, using embryonic rat spinal cord culture. When dissociated cultures were exposed to low-level glutamate (Glu) coadministered with the Glu transporter inhibitor L-trans-pyrrolidine-2,4-decarboxylate (PDC) for 24 hours, motor neurons were selectively injured through N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptors. Nitric oxide synthase (NOS) inhibitors attenuated this toxicity, and long-acting nitric oxide (NO) donors damaged motor neurons selectively. Nonmotor neurons survived after exposure to low-dose Glu/PDC, but Glu-induced toxicity was potentiated by coadministration of an NO-dependent guanylyl cyclase inhibitor. In addition, 8-bromo-cyclic GMP, a soluble cyclic GMP analogue, rescued nonmotor neurons, but not motor neurons, exposed to high-dose Glu/PDC. Twenty-four hours' incubation with PDC elevated the number of neuronal NOS-immunoreactive neurons by about twofold compared with controls, and a double-staining study, using the motor neuron marker SMI32, revealed that most of them were nonmotor neurons. These findings suggest that selective motor neuronal death caused by chronic low-level exposure to Glu is mediated by the formation of NO in nonmotor neurons, which inversely protects nonmotor neurons through the guanylyl cyclase-cyclic GMP cascade. Induction of neuronal NOS in nonmotor neurons might enhance both the toxicity of motor neurons and the protection of nonmotor neurons, which could explain the pathology of amyotrophic lateral sclerosis.     

Nocturnal decreases in nitric oxide and cyclic GMP contents in the chick brain and their prevention by light

The diurnal variations in the contents of nitric oxide (NO) and cyclic GMP were studied in the chick brain. NO and cyclic GMP contents in the chick brain were lower at night than during the day and were inversely correlated with high night-time tissue melatonin levels. Furthermore, when animals were kept in light at night, tissue melatonin levels remained at low diurnal values, whereas NO and cyclic GMP contents remained high. Since we have previously shown that physiological concentrations of melatonin inhibit nitric oxide synthase (NOS) activity in different brain areas, the nocturnal decrease in brain NO and cyclic GMP contents may be, in part, a consequence of the nocturnal inhibitory effect of melatonin on NOS activity.     

Nitric oxide-dependent production of cGMP supports the survival of rat embryonic motor neurons cultured with brain-derived neurotrophic factor

Trophic factor deprivation induces neuronal nitric oxide synthase (NOS) and apoptosis of rat embryonic motor neurons in culture. We report here that motor neurons constitutively express endothelial NOS that helps support the survival of motor neurons cultured with brain-derived neurotrophic factor (BDNF) by activating the nitric oxide-dependent soluble guanylate cyclase. Exposure of BDNF-treated motor neurons to nitro-L-arginine methyl ester (L-NAME) decreased cell survival 40-50% 24 hr after plating. Both low steady-state concentrations of exogenous nitric oxide (<0.1 microM) and cGMP analogs protected BDNF-treated motor neurons from death induced by L-NAME. Equivalent concentrations of cAMP analogs did not affect cell survival. Inhibition of nitric oxide-sensitive guanylate cyclase with 2 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reduced the survival of BDNF-treated motor neurons by 35%. cGMP analogs also protected from ODQ-induced motor neuron death, whereas exogenous nitric oxide did not. In all cases, cell death was prevented with caspase inhibitors. Our results suggest that nitric oxide-stimulated cGMP synthesis helps to prevent apoptosis in BDNF-treated motor neurons.     

Neurogenic nitric oxide mediates relaxation of canine corpus cavernosum

PURPOSE: The aim of the present study is to analyze mechanisms underlying neurogenic relaxation of the corpus cavernosum which are believed to participate in penile erection. MATERIALS AND METHODS: Mechanical responses to nerve stimulation by electrical pulses and nicotine were measured in strips of canine corpus cavernosum precontracted with phenylephrine. Cyclic guanosine monophosphate (GMP) contents in the strips were also measured by radioimmunoassay. Immunohistochemistry for nitric oxide synthase (NOS) and vasoactive intestinal polypeptide (VIP) was performed. RESULTS: Transmural electrical stimulation and nicotine produced relaxations in the isolated canine corpus. The neurogenic relaxation was abolished by N omega-nitro-L-arginine, a NOS inhibitor, and the inhibition was reversed by L-arginine. Relaxations induced by nerve stimulation and exogenous nitric oxide (NO) were depressed by oxyhemoglobin and methylene blue. Vasoactive intestinal polypeptide (VIP)-induced relaxations were not influenced by these inhibitors. In the controls strips and those made unresponsive to VIP by its repeated application, the responses to nerve stimulation did not differ. The content of cyclic GMP in the tissue increased in response to nicotine, the effect being abolished by the NO synthase inhibitor. Immunohistochemical study demonstrated neurons containing NOS and VIP. CONCLUSIONS: It appears that the relaxation induced by nerve stimulation is mediated solely by NO liberated from the nerve that activates soluble guanylate cyclase and increases the production of cyclic GMP in smooth muscle, whereas VIP does not play a role in the regulation of muscle tone under the experimental conditions used.     

Regulation of guanosine 3':5'-cyclic monophosphate in ovine tracheal epithelial cells

1. Guanosine 3':5'-cyclic monophosphate (cyclic GMP) is an important second messenger mediating the effects of nitric oxide (NO) and natriuretic peptides. Cyclic GMP pathways regulate several aspects of lung pathophysiology in a number of airway cells. The regulation of this system has not been extensively studied in pulmonary epithelial tissue. 2. We have studied the production of cyclic GMP by suspensions of ovine tracheal epithelial cells in response to activators of soluble guanylyl cyclase (sodium nitroprusside (SNP) and S-nitroso-N-acetyl-penicillamine (SNAP) and particulate guanylyl cyclase (atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP) and E. coli heat stable enterotoxin (STa)). 3. Both 10(-7)-10(-3) M and 10(-7)-10(-3) M SNAP generated a concentration-dependent marked elevation in cyclic GMP production when incubated with 10(-3) M 3-isobutyl-l -methylxanthine (IBMX) (both greater than 25 x baseline values with highest drug concentration). 4. The increase in production of cyclic GMP in response to 10(-6) M SNP and 10(-5) M SNAP was markedly inhibited by both 5 x 10(-5) M haemoglobin (102% and 92% inhibition) and 5 x 10(-5) M methylene blue (82% and 84% inhibition). 5. The increase in cyclic GMP in response to 10(-3) M SNP was measured following co-incubation with the phosphodiesterase inhibitors 10(-7)-10(-3) M IBMX, 10(-7)-10(-4) M milrinone and 10(-7)-10(-4) M SKF 96231. Only 10(-4)-10(-3) M IBMX significantly increased cyclic GMP levels. 6. Cyclic GMP production was also significantly elevated from baseline by 10(-5) M ANP, 10(-5) M BNP, 10(-5) M CNP and 200 iu ml-3 of E. coli STa toxin in the presence of 10(-3) M IBMX. Increases with these natriuretic peptides and STa toxin were smaller in magnitude (2-4 fold) than those seen with SNP and SNAP. CNP was the most potent of the natriuretic peptides studied suggesting type B membrane bound guanylate cyclase is the predominant form expressed. 7. These results suggest that ovine tracheal epithelial cells contain active guanylyl cyclases. The more marked response to SNP and SNAP than to natriuretic peptides suggests that soluble guanylyl cyclase predominates.     

Nicotine administration stimulates the in vivo N-methyl-D-aspartate receptor/nitric oxide/cyclic GMP pathway in rat hippocampus through glutamate release

1. The in vivo effects of nicotine on the nitric oxide (NO) synthase/cyclic GMP pathway of the adult rat hippocampus have been investigated by monitoring the levels of extracellular cyclic GMP during microdialysis in conscious unrestrained animals. 2. Intraperitoneal (i.p.) administration of nicotine caused elevation of cyclic GMP levels which was prevented by mecamylamine. The effect of nicotine was abolished by local infusion of the NO synthase inhibitor N(G)-nitro-L-arginine (L-NOARG) or by the soluble guanylyl cyclase blocker 1H-[1,2,4]oxadiazolo[4.3-a]quinoxaline-1-one (ODQ). 3. Local administration of the NMDA receptor antagonists cis-4-(phosphonomethyl)-2-piperidinecarboxylic acid (CGS19755) and dizocilpine (MK-801) inhibited by about 60% the nicotine-induced elevation of cyclic GMP. Nicotine was able to stimulate cyclic GMP outflow also when administered directly into the hippocampus; the effect was sensitive to mecamylamine, L-NOARG, ODQ or MK-801. 4. Nicotine, either administered i.p. or infused locally, produced augmentation of glutamate and aspartate extracellular levels, whereas the outflows of gamma-aminobutyric acid (GABA) and glycine remained unaffected. Following local administration of high concentrations of nicotine, animals displayed symptoms of mild excitation (sniffing, increased motor and exploratory activity) during the first 20-40 min of infusion, followed by wet dog shake episodes; these behavioural effects were prevented by mecamylamine or MK-801, but not by L-NOARG or by ODQ. 5. It is concluded that (a) nicotine stimulates the production of NO and cyclic GMP in the hippocampus; (b) this occurs, at least in part, through release of glutamate/aspartate and activation of NMDA receptors. Modulation of the NMDA receptor/NO synthase/cyclic GMP pathway may be involved in the cognitive activities of nicotine.     

Emergency medicine in the New Delhi area, India

OBJECTIVES: To examine the role of nitric oxide in the cardiovascular system in spontaneous hypertension. In particular, we wanted to know whether the production of nitric oxide in the cardiovascular system of the spontaneously hypertensive rat is different from that of the normotensive Wistar-Kyoto rat and whether nitric oxide is biologically effective in this system. DESIGN: We studied various aspects of the L-arginine-nitric oxide pathway in the cardiovascular system of spontaneously hypertensive rats and Wistar-Kyoto rats. METHODS: To address the first objective we analysed the expression of endothelial nitric oxide synthase in the heart by Western blotting and the activity of constitutive nitric oxide synthase in resistance microvessels obtained from the mesenterium, both from spontaneously hypertensive rats and Wistar-Kyoto rats aged 14-18 weeks. We also analysed the concentration of the oxidative product of nitric oxide, nitrate, in plasma from these rats. To address the second objective, that is, to assess the bioactivity of nitric oxide, we studied the accumulation in tissue of cyclic guanosine 3',5'-monophosphate (GMP), as well as the acute and chronic effects of withdrawing the nitric oxide vasodilatory tone with the inhibitor of nitric oxide synthesis NG-nitro-L-arginine methyl ester on Wistar-Kyoto rats and spontaneously hypertensive rats. RESULTS: We found that the expression of endothelial nitric oxide synthase in the heart, the activity of constitutive nitric oxide synthase in resistance microvessels and the concentration of nitrate in plasma were all significantly higher in the spontaneously hypertensive rats. In contrast, neither cyclic GMP levels nor the effects of NG-nitro-L-arginine methyl ester were greater in the spontaneously hypertensive rat than they were in the Wistar-Kyoto rat. CONCLUSIONS: The nitric oxide pathway is upregulated in the resistance circulation and the heart of the spontaneously hypertensive rat by a mechanism involving induction of the constitutive nitric oxide synthase and overproduction of nitric oxide. However, nitric oxide is not sufficiently bioactive to stimulate the formation of cyclic GMP and to maintain an adequate nitric oxide-dependent vasodilatory tone.     

Chronic exposure to aluminum impairs neuronal glutamate-nitric oxide-cyclic GMP pathway

Humans are exposed to aluminum from environmental sources and therapeutic treatments. However, aluminum is neurotoxic and is considered a possible etiologic factor in Alzheimer's disease and other neurological disorders. The molecular mechanism of aluminum neurotoxicity is not understood. We tested the effects of aluminum on the glutamate-nitric oxide-cyclic GMP pathway in cultured neurons. Neurons were exposed to 50 microM aluminum in culture medium for short-term (4 h) or long-term (8-14 days) periods, or rats were prenatally exposed, i.e., 3.7% aluminum sulfate in the drinking water, during gestation. Chronic (but not short-term) exposure of neurons to aluminum decreased glutamate-induced activation of nitric oxide synthase by 38% and the formation of cyclic GMP by 77%. The formation of cyclic GMP induced by the nitric oxide-generating agent S-nitroso-N-acetylpenicillamine was reduced by 33%. In neurons from rats prenatally exposed to aluminum but not exposed to it during culture, glutamate-induced formation of cyclic GMP was inhibited by 81%, and activation of nitric oxide synthase was decreased by 85%. The formation of cyclic GMP induced by S-nitroso-N-acetylpenicillamine was not affected. These results indicate that chronic exposure to aluminum impairs glutamate-induced activation of nitric oxide synthase and nitric oxide-induced activation of guanylate cyclase. Impairment of the glutamate-nitric oxide-cyclic GMP pathway in neurons may contribute to aluminum neurotoxicity.     

Identification and characterization of a novel beta subunit of soluble guanylyl cyclase that is active in the absence of a second subunit and is relatively insensitive to nitric oxide

Previously characterized soluble guanylyl cyclases form alpha-beta heterodimers that can be activated by the gaseous messenger, nitric oxide. In mammals, four subunits have been cloned, named alpha1, alpha2, beta1, and beta2. We have identified a novel soluble guanylyl cyclase isoform from the nervous system of the insect Manduca sexta that we have named M. sexta guanylyl cyclase beta3 (MsGC-beta3). It is most closely related to the mammalian beta subunits but has several features that distinguish it from previously identified soluble cyclases. Most importantly, MsGC-beta3 does not need to form heterodimers to form an active enzyme because guanylyl cyclase activity can be measured when it is expressed alone in COS-7 cells. Moreover, this activity is only weakly enhanced in the presence of the nitric oxide donor, sodium nitroprusside. Several of the amino acids in rat beta1 subunits, previously identified as being important in heme binding or necessary for nitric oxide activation, are substituted with nonsimilar amino acids in MsGC-beta3. There are also an additional 315 amino acids C-terminal to the catalytic domain of MsGC-beta3 that have no sequence similarity to any known protein. Northern blot analysis shows that MsGC-beta3 is primarily expressed in the nervous system of Manduca.     

Attenuation of contractions to acetylcholine in canine bronchi by an endogenous nitric oxide-like substance

1. The involvement was assessed of an endogenous nitric oxide-like substance in contractions of canine bronchi to acetylcholine. 2. Canine third order bronchial rings, in some of which the epithelium was removed mechanically, were suspended in organ chambers and isometric tension was recorded. In some experiments, the content of guanosine 3',5'-cyclic monophosphate (cyclic GMP) of the bronchi was also measured. 3. Acetylcholine induced concentration-dependent contractions. The contractions were potentiated by nitro-L-arginine (an inhibitor of the synthesis of nitric oxide), oxyhaemoglobin (a scavenger of nitric oxide), and methylene blue (an inhibitor of soluble guanylate cyclase). The magnitude of the potentiation to acetylcholine-induced contractions by these inhibitors were not significantly different between tissues with and without epithelium. 4. Acetylcholine induced a concentration-dependent increase in intracellular content of cyclic GMP, which was similar in bronchi with and without epithelium. These increases were abolished by nitro-L-arginine and methylene blue. 5. During contractions to acetylcholine, exogenous nitric oxide relaxed the canine bronchi. The relaxations were not affected by nitro-L-arginine, but were augmented by superoxide dismutase plus catalase, and were abolished by methylene blue. 6. These observations suggest that, during contraction evoked by acetylcholine, the production of an endogenous nitric oxide-like substance increases and in turn attenuates the response of the airways to the muscarinic agonist. However, the endogenous nitric oxide-like substance does not play a major role in the epithelium-dependent attenuation of the contraction to acetylcholine in canine bronchi.     

Involvement of K+ATP channels in nitric oxide-induced inhibition of spontaneous contractile activity of the nonpregnant human myometrium

Nitric oxide (NO), an important endogenous substance, is known to be a strong relaxant of smooth muscle, including myometrium. It has been postulated that the relaxing effect of NO on smooth muscle is achieved by the stimulation of soluble guanylyl cyclase, which leads to an increase in the cyclic guanosine 3',5'-monophosphate (cGMP) levels and hyperpolarization of the cellular membrane. The aim of our study was to investigate the involvement of K+ATP channels in the mechanism of cGMP-independent nitric oxide-induced inhibition of contractile activity of the nonpregnant human myometrium, obtained at hysterectomy. Nitric oxide's influence on contractile activity was recorded in the presence of methylene blue and glybenclamide, blockers of soluble guanylyl cyclase and K+ATP channels, respectively. Nitric oxide, generated by the NO donor DEA/NO, caused a dose-dependent inhibition of the spontaneous contractile activity of human nonpregnant myometrium. Preincubation with methylene blue (5 microM) did not prevent NO-induced relaxation of uterine strips, while 1.5 microM glybenclamide blocked this effect. Our results indicate that nitric oxide relaxes human non-pregnant uterus through K+ATP channels, independent of the cGMP pathway.