Endothelium-independent contractions to NG-monomethyl-L-arginine in canine basilar artery.

BACKGROUND AND PURPOSE: NG-substituted analogues of L-arginine are potent and selective inhibitors of nitric oxide synthase(s). The present study was designed to determine the effects of these analogues on the vascular smooth muscle of isolated canine basilar arteries. METHODS: Basilar artery rings without endothelium were suspended for isometric tension recording in Krebs-Ringer bicarbonate solution bubbled with 94% O2-6% CO2 (temperature = 37 degrees C, pH = 7.4). A radioimmunoassay technique was used to determine the levels of guanosine 3',5'-cyclic monophosphate (cyclic GMP). RESULTS: NG-Monomethyl-L-arginine (L-NMMA) caused concentration-dependent contractions, whereas the D-enantiomer and NG-nitro-L-arginine did not. Contractions to L-NMMA were reduced in the presence of L-arginine but not in the presence of D-arginine. Superoxide anions generated by xanthine plus xanthine oxidase in the presence of catalase abolished contractions to L-NMMA but did not affect contractions to the prostaglandin H2/thromboxane A2 agonist U46619. Zaprinast, a selective cyclic GMP phosphodiesterase inhibitor, caused concentration-dependent relaxations. L-NMMA selectively inhibited these relaxations. The inhibitory effect of L-NMMA was reversed in the presence of L-arginine. L-NMMA selectively reduced the basal production of cyclic GMP. CONCLUSIONS: These studies suggest that in cerebral arteries, contractions of smooth muscle cells to L-NMMA are mediated by inhibition of nitric oxide synthase with a resultant decrease in the basal production of nitric oxide.     

Effects of Nitric Oxide on the Survival and Neuritogenesis of Cerebellar Purkinje Neurons

Nitric oxide has been investigated widely both during neurodevelopment and in neurological diseases. However, whilst it has been established that nitric oxide-producing enzymes of nitric oxide synthase family are expressed in cerebellar Purkinje neurons, the effects of nitric oxide on the viability and morphology of these neurons remain unknown. Here, we have demonstrated that the activity of neuronal nitric oxide synthase, but not the inducible or endothelial forms of this enzyme, is required to support the survival of a proportion of cerebellar Purkinje neurons in vitro. We discovered that donation of high concentrations of exogenous nitric oxide reduces Purkinje neuron survival in culture and that peroxynitrite is also toxic to these cells. Finally, we demonstrated that exogenous nitric oxide and peroxynitrite reduce both the magnitude and the complexity of the neurite arbour extended by cerebellar Purkinje neurons. Taken together, these findings reveal that whilst a low level of endogenous nitric oxide, released by the activity of neuronal nitric oxide synthase, is beneficial to cerebellar Purkinje neurons in vitro, high levels of exogenous nitric oxide and peroxynitrite are detrimental to both the survival of these neurons and to their ability to extend processes and form functional neural networks.     

The effect of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and charybdotoxin (CTX) on relaxations of isolated cerebral arteries to nitric oxide

The mechanism underlying smooth muscle relaxations of cerebral arteries in response to nitric oxide is still not completely understood. The present study was designed to determine the role of soluble guanylate cyclase in the relaxations to a nitric oxide/nucleophile complex, diethylaminodiazen-1-ium-1,2-dioate (DEA-NONOate). Rings of canine middle cerebral arteries without endothelium were suspended in Krebs–Ringer bicarbonate solution for isometric tension recording. The levels of guanosine 3′,5′-cyclic monophosphate (cyclic GMP) were measured by radioimmunoassay technique. During contractions to uridine 5′-triphosphate (UTP), DEA-NONOate (10⁻¹⁰ to 10⁻⁵ M) caused concentration-dependent relaxations. Measurements of cyclic GMP levels in cerebral arterial wall demonstrated that DEA-NONOate is a potent stimulator of guanylate cyclase and subsequent formation of cyclic GMP. Increasing concentrations of a selective soluble guanylate cyclase inhibitor, 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), caused concentration-dependent reduction of both cyclic GMP production and relaxations to DEA-NONOate. Interestingly, in the presence of the highest concentration (3×10⁻⁶ M) of ODQ, production of cyclic GMP in response to 10⁻⁶ M of DEA-NONOate was abolished, whereas the same concentration of DEA-NONOate caused almost complete relaxation, suggesting that mechanisms independent of cyclic GMP production may mediate relaxing effect of high concentration of a nitric oxide donor. A selective Ca²⁺-activated potassium channel blocker charybdotoxin (CTX) significantly reduced relaxations to DEA-NONOate resistant to ODQ, supporting the idea that in cerebral arteries nitric oxide may activate potassium channels independently of cyclic GMP. The results of our study suggest that under physiological conditions, guanylate cyclase is a key mediator of cerebral arterial relaxations to nitric oxide. However, under pathological conditions associated with induction of nitric oxide synthase and increased biosynthesis of nitric oxide (e.g., cerebral ischemia, inflammation, sepsis), mechanisms other than formation of cyclic GMP may be activated.     

Cytochemical Evidence for Nitric Oxide/Cyclic GMP Signal Transmission in the Visual System of the Locust

Nitric oxide is a membrane-permeant messenger molecule which activates soluble guanylyl cyclase. Using NADPH diaphorase staining as a marker for the enzyme nitric oxide synthase and an antiserum against cyclic GMP (cGMP), we investigated the possible sites of nitric oxide and cGMP synthesis in the retina and lamina of Schisfocerca gregaria. The photoreceptor cells did not express NADPH diaphorase staining but monopolar cells of the lamina were strongly stained. After inhibition of phosphodiesterase activity and incubation of tissue in a nitric oxide donor, the photoreceptor cells showed cGMP immunoreactivity. In contrast to the photoreceptors, the monopolar cells of the lamina were not stained. Since the presynaptic photoreceptors were cGMP-immunoreactive and the postsynaptic targets of the monopolar cells did not express immunoreactivity, it is conceivable that nitric oxide released by monopolar cells may play a role as a retrograde messenger in visual information processing.     

Purification and characterization of a cyclic GMP-stimulated cyclic nucleotide phosphodiesterase from the cytosol of human platelets

A cyclic GMP-stimulated cyclic nucleotide phosphodiesterase was purified to near homogeneity from the 150,000 g supernatant fraction of human platelets by a combination of DEAE-cellulose chromatography and cyclic GMP affinity chromatography. Overall purification was about 7400-fold with a 10% to 15% recovery of activity. On NaDodSO4-containing polyacrylamide gels, the purified enzyme migrates as a single band Mr = 105,000. Phosphodiesterase activity co-migrates with the protein band on native polyacrylamide gels. Both Mg2+ and Mn2+ support the activity of this phosphodiesterase. The enzyme hydrolyzes both cyclic AMP and cyclic GMP with similar maximal rates. The hydrolysis of both nucleotides exhibits positive homotropic cooperativity with S0.5 values of 50 +/- 12 microM for cyclic AMP and 35 +/- 15 microM for cyclic GMP and Hill coefficients of 1.2 to 1.5 for both nucleotides. Low levels of cyclic GMP stimulate the rate of cyclic AMP hydrolysis from 3- to 10-fold. The activity of this phosphodiesterase is not stimulated by the calcium binding protein, calmodulin. The cyclic GMP stimulation of cyclic AMP hydrolysis by this phosphodiesterase may provide a possible regulatory link between the metabolism of these two nucleotides in platelets.     

Hypobaric hypoxia modifies constitutive nitric oxide synthase activity and protein nitration in the rat cerebellum

Ischemic hypoxia provokes alterations in the production system of nitric oxide in the cerebellum. We hypothesize that the nitric oxide system may undergo modifications due to hypobaric hypoxia and that may play a role in high altitude pathophysiology. Therefore, changes in the nitric oxide system of the cerebellum of rats submitted to acute hypobaric hypoxia were investigated. Adult rats were exposed for 7 h to a simulated altitude of 8235 m (27000 ft.) and then killed after 0 h or 1, 3, 5 and 10 days of reoxygenation. Nitric oxide synthase calcium-dependent and -independent activity, immunoblotting and immunohistochemistry of neuronal, endothelial, and inducible nitric oxide synthase, and nitrotyrosine were evaluated. Immunoreactivity for neuronal nitric oxide synthase slightly increased in the baskets of the Purkinje cell layer and in the granule cells, after 0 h of reoxygenation, although no changes in neuronal nitric oxide synthase immunoblotting densitometry were detected. Calcium-dependent activity significantly rose after 0 h of reoxygenation, reaching control levels in the following points, and being coincident with a peak of eNOS expression. Nitrotyrosine formation showed significant increments after 0 h and 1 day of reoxygenation. Nitrotyrosine immunoreactivity showed an intracellular location change in the neurons of the cerebellar nuclei and in addition, an appearance of nitration in the soma of the Purkinje cells was detected. No changes in inducible nitric oxide synthase activity, immunoblotting or immunohistochemistry were detected. We conclude that at least part of the nitric oxide system is involved in cerebellum responses to hypobaric hypoxia.     

Compartmentation of NADPH-diaphorase activity in the mouse cerebellar cortex

The mammalian cerebellum is built around an array of parasagittal bands of Purkinje cells that can be demonstrated by immunocytochemical staining for the differentiation antigen zebrin II. Climbing and Mossy fiber afferents also terminate in bands, and the afferent terminal fields and the Purkinje cell bands are aligned. The convergence of mossy and climbing fiber pathways onto the Purkinje cells, which are the sole output of the cerebellar cortex, is a characteristic feature of cerebellar circuitry. Previous studies showed that when both afferent pathways are activated synchronously there develops a long-term depression of synaptic efficacy at the parallel fiber-Purkinje cell synapse. Two second messenger pathways mediate long-term depression: one involves diacylglyceroland protein kinase C, and the other involves nitric oxide that is generated by a nitric oxide synthase. We have studied the distribution of nitric oxide synthase in the adult Mouse cerebellum by using nicatinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry. NADPH-diaphorase activity is found mainly in the granule and basket cells. Within the granular layer NADPH-diaphorase activity is expressed nonuniformly patches of granule cells and synaptic glomeruli. The patches are yseen in all lobules, are reproducible from individual to individual, and are topographically ordered with respect to the Purkinje cell compartments as revealed by using anti-zebrin II immunocytochemistry. These data imply that nitric oxide-dependent, long-term depression may only involve a subset of mossy fiber/granule cell projections, and that one role for nitric oxide may be to refine cerebellar receptive fields. © 1994 Wiley-Liss, Inc.     

NADPH-Diaphorase histochemistry in the postnatal mouse cerebellum suggests specific developmental functions for nitric oxide

NADPH-diaphorase histochemistry has been applied for the localization of nitric oxide synthase during the postnatal development of the mouse cerebellum. Staining for NADPH-diaphorase during the first week after birth was confined to some but not all laminae of the immature cerebellum: NADPH-diaphorase activity was located in the molecular/Purkinje cell layer and in inner parts of the internal granular layer. The external granular layer and the developing white matter were essentially unstained. Expression was earliest and very strong in parallel fibers and in the internal granule layer of the ventral part of the pyramis and the dorsal part of the uvula. Staining in the Purkinje cell layer was observed throughout the cerebellum. The presence of formazan deposits within Purkinje cells was verified by colocalization with calbindin D-28k immunoreactivity. The distribution of NADPH-diaphorase activity changed into the adult pattern between 8 and 12 days of age: Within the molecular layer, basket cells and their processes became strongly stained. Reaction product within Purkinje cells gradually disappeared. Likewise, strongly stained parallel fibers were no longer detectable. These results suggest that nitric oxide is involved in different processes in cerebellar development. © 1993 Wiley-Liss, Inc.     

Evidence that nitric oxide mediates the cyclic GMP response to synaptic activity in the rat superior cervical ganglion.

Preganglionic nerve stimulation in the rat superior cervical ganglion (SCG) caused an increase in guanosine 3':5'-cyclic monophosphate (cyclic GMP) in a Ca(2+)-dependent manner. This increase was inhibited by oxyhaemoglobin, and blocked stereoselectively by an inhibitor of nitric oxide synthase, NG-nitro-L-arginine. Thus, nitric oxide or a similar substance appears to mediate the neuronal cyclic GMP response to synaptic activity in the rat SCG.     

Electrophysiology of Purkinje neurons in the weaver mouse: iontophoresis of neurotransmitters and cyclic nucleotides, and stimulation of the nucleus locus coeruleus.

We compared the Purkinje cells of adult normal and weaver mutant (wv/wv) mice by iontophoretic and electrophysiological tests. Although weaver Purkinje cells fire spontaneously at a rate (38 Hz) similar to normal mouse neurons (40 Hz), several abnormalities of firing were seen: high frequency bursts of single (simple) spikes occurred in 5-10-sec episodes in 38% of weaver cells, compared to 8% in normal mice; spontaneous complex spikes (climbing fiber-like burst responses) occurred in several different forms in a given Purkinje cell. As in normal mice and rats, the spontaneous single spike activity is readily depressed by electrical stimulation of the locus coeruleus, the presumed source of a dense noradrenergic plexus in the weaver cerebellar cortex. In a preliminary experiment the adrenergic blocking agent, fluphenazine, antagonized the responses to locus coeruleus stimulation. Iontophoresis of norepinephrine (NE), GABA and serotonin (5-HT) also uniformly depressed Purkinje cell single spike activity in all normal and weaver mice; cyclic AMP depressed 55% of normal and 70% of weaver Purkinje cells. Glutamate was always excitatory. The only qualitative difference was seen with acetylcholine, which was mostly inhibitory in normal mouse, but increased the firing rate in 42% of weaver Purkinje neurons. Cyclic GMP was predominantly excitatory in both types. Thus, despite the absence of parallel fibers, weaver Purkinje neurons grossly resemble normal Purkinje cells electrophysiologically as well as morphologically. Since several sites of indirect presynaptic actions are eliminated in weaver, our results further substantiate the direct post-synaptic inhibitory nature of GABA, 5-HT and NE, and the noradrenergic pathway from locus coeruleus to Purkinje cells. Similarly, consistent inhibitory responses to cyclic AMP in the weaver support the previously hypothesized role of cyclic AMP in the post-synaptic inhibitory response to NE.     

Cytokines Regulate L-Arginine-Dependent Cyclic GMP Production in Rat Glial Cells

We have previously demonstrated that primary astrocyte cultures from neonatal rat cortex and C6 glioma cells express a calcium-independent nitric oxide synthase (NOS) on induction with bacterial endotoxin (lipopolysaccharide, LPS). One hypothesis regarding the mechanism of the LPS induction is that it causes release of cytokines from these cells which then induce the enzyme directly. Such cytokine induction of NOS has been demonstrated in many extraneural cell types. l -Arginine-dependent increases in cyclic GMP correlate with smaller increases in accumulation of nitrite, the major oxidation product of nitric oxide, and hence can serve as a more sensitive measure of nitric oxide production. Here we provide evidence that interferon-γ (IFN-γ), interleukin (IL)-1β and tumour necrosis factor-α induce l -arginine-dependent cyclic GMP synthesis in C6 cells and that a combination of IFN-γ and IL-1β induce l -arginine-dependent cyclic GMP synthesis in astrocyte cultures, indicating that these cytokines induce NOS. In both cell types the induction by cytokines was less sensitive to inhibition by dexamethasone, IL-10 and IL-4 than was induction by LPS. These data suggest that cytokines can also induce a NOS in glial cells and that the mechanism of this induction may be more direct than that of LPS, since it is less sensitive to modulation by immunosuppressors. Due to the close associations of astrocytes with neurons and microvasculature, cytokine-induced NOS could have potentially important pathophysiological effects in the central nervous system.     

Endothelin stimulates nitric oxide-dependent cyclic GMP formation in rat cerebellar astroglia

Endothelins (ETs) elicit a diversity of cellular responses in cultured astrocytes that suggest an important role of these peptides in glial cell function. Stimulation of astroglial ET receptors induces phosphoinositide (PI) hydrolysis and intracellular calcium mobilization, but little is known about the signalling events that occur downstream of this system. Here we show that in rat cerebellar astroglia in culture ETs produce a receptor-mediated stimulation of cyclic GMP (cGMP) formation that is rapid and totally dependent on nitric oxide synthase (NOS) activity. The effect is blocked by an inhibitor of PI phospholipase C, compound U73122, and by depletion of intracellular calcium stores with thapsygargin. These results indicate that calcium released by inositol trisphosphate is responsible for NOS activation and subsequent cGMP formation.     

Dipyridamole potentiates platelet inhibition by nitric oxide.

In a placebo-controlled double blind cross-over experiment the adenosine uptake inhibitor dipyridamole (400 mg/day) did not affect ex vivo platelet aggregation induced by collagen or adenosine-diphosphate (ADP) in an electronic whole blood aggregometer (WBA). Dipyridamole was also inactive in vitro, unless red blood cell injury was deliberately enhanced, thereby increasing the level of free adenine nucleotides. Since dipyridamole also inhibits cyclic guanosine monophosphate (GMP) phosphodiesterase (PDE), we used platelet rich plasma (PRP) to study its interaction with authentic and endothelium-derived nitric oxide (NO). The latter inhibits platelets by increasing cyclic GMP. Dipyridamole (1 to 30 microM), either alone or in combination with a subthreshold concentration of prostacyclin (PGI2), was inactive. However, when combined with a subthreshold concentration of NO, dipyridamole caused a concentration-dependent platelet suppression, which became more pronounced when PGI2 was present as well. It is concluded that dipyridamole could reduce the threshold for platelet suppression by NO through inhibition of cyclic GMP PDE.     

Long-Term Depression in Rat Cerebellum Requires both NO Synthase and NO-sensitive Guanylyl Cyclase

Long-term depression (LTD) of synaptic transmission between parallel fibres and Purkinje cells is a well-known example of synaptic plasticity taking place in the cerebellum. Nitric oxide (NO) has been implicated in synaptic plasticity in other brain areas, but its function in cerebellar LTD is controversial. Even when an involvement is suggested, the NO signal transduction pathway is unclear. One candidate is the cyclic GMP-synthesizing enzyme, soluble guanylyl cyclase, whose activity in the brain and elsewhere is powerfully stimulated by NO. By recording intracellularly from Purkinje cells in cerebellar slices, we demonstrate that blockade of NO synthase completely inhibits LTD induced by pairing parallel fibre stimulation with postsynaptic Ca²⁺ spike firing. LTD was also blocked by intracellular application of 1H-[1, 2, 4]oxadiazolo[4, 3-a]quinoxalin-1-one, a recently identified potent and selective inhibitor of soluble guanylyl cyclase. These findings indicate that soluble guanylyl cyclase is required for cerebellar LTD and suggest that this enzyme, located within Purkinje cells, transduces the NO signal in this form of synaptic plasticity.     

Nitric oxide regulates the proliferation of chick embryo retina cells by a cyclic GMP-independent mechanism

Nitric oxide (NO) is an intercellular messenger involved in many physiological and pathological processes of vertebrate and invertebrate animal tissues. In the embryonic chick retina, nitric oxide synthase (NOS) activity and a system for l-arginine transport between neurons and glial cells were described, supporting the idea that nitric oxide is a critical molecule during retinal development. In the present work we show that nitric oxide is a modulator of cell proliferation in chick embryo retina. Mixed cultures of retinal neurons and glial cells were submitted to [(3)H]-thymidine incorporation after drug treatment. Incubation for 24h with the NO donors S-nitroso-N-acetyl-penicillamine (SNAP) or Spermine nitric oxide (SpNO) complex promoted a decrease of approximately 70% of [(3)H]-thymidine incorporation in a dose-dependent manner. SNAP did not increase Lactate dehydrogenase release and its effect was not mimicked by 8-bromo cyclic GMP, or blocked by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), indicating that the effect was not due to cell death or mediated by increases of cyclic GMP levels. The inhibition was completely prevented by dithiotreitol (DTT), strongly indicating the participation of an S-nitrosylation mechanism. SNAP blocked the increase of [(3)H]-thymidine incorporation induced by ATP. Using purified cultures of glial cells we showed that the NO donor SNAP produced an inhibition of 50% in cell proliferation and did stimulate ERK1/2 phosphorylation, indicating that the inhibition of this pathway was not involved in its cytostatic effect. [(3)H]-Thymidine autoradiography of mixed cultures showed labeling of oval nuclei of glial flat cells. The injection of eggs with SNAP also did promote an intense inhibition of [(3)H]-thymidine incorporation in retinas from 9-day-old embryos. These data suggest that nitric oxide affects the proliferation of chick embryo retina glial cells in culture or "in vivo" through cyclic GMP and ERK-independent pathways.     

Pharmacological shift of the ambiguous nitric oxide action from neurotoxicity to cyclic GMP-mediated protection

Abstract

Objectives: The effect of intracellular cyclic guanosine monophosphate (GMP) increase on neuronal damage was tested using a newly developed nitric oxide-related injury model of cultured spinal cord neurons.

Methods: Neuronal damage after 24-hour-exposure to sodium nitroprusside (SNP), a nitric oxide (NO) donor, was evaluated by measuring the activity of released lactate dehydrogenase from injured neurons.

Results: Oxygen radical scavengers had a protective effect, indicating that the neuronal damage, elicited by 10 μM SNP, was largely due to peroxynitrite formation. Alternatively, a strong inhibition of the NO-induced damage could also be achieved by an intracellular cyclic GMP increase resulting from the addition of 100 μM 8-bromo-cyclic GMP. Propentofylline (PPF, 1–100 μM), a xanthine derivative and rather selective phosphodiesterase (PDE) inhibitor, enhanced intracellular cyclic GMP elevation induced by SNP exposure. The neuronal damage induced by 10 μM SNP exposure for 24 hours was almost completely blocked in the presence of 1 μM PPF.

Discussion: These results suggest that NO has an ambiguous action, i.e. toxic by favoring the formation of, but protective by intracellular cyclic GMP elevation which can be reinforced by PDE inhibition. Therefore, PDE inhibitors, such as PPF, may be useful therapeutic drugs to limit oxidative neuronal damage in the central nervous system.     

mRNA expression patterns of the cGMP-hydrolyzing phosphodiesterases types 2, 5, and 9 during development of the rat brain

Recent evidence indicates that cGMP plays an important role in neural development and neurotransmission. Since cGMP levels depend critically on the activities of phosphodiesterase (PDE) enzymes, mRNA expression patterns were examined for several key cGMP-hydrolyzing PDEs (type 2 [PDE2], 5 [PDE5], and 9 [PDE9]) in rat brain at defined developmental stages. Riboprobes were used for nonradioactive in situ hybridization on sections derived from embryonic animals at 15 days gestation (E15) and several postnatal stages (P0, P5, P10, P21) until adulthood (3 months). At all stages PDE9 mRNA was present throughout the whole central nervous system, with highest levels observed in cerebellar Purkinje cells, whereas PDE2 and PDE5 mRNA expression was more restricted. Like PDE9, PDE5 mRNA was abundant in cerebellar Purkinje cells, although it was observed only on and after postnatal day 10 in these cells. In other brain regions, PDE5 mRNA expression was minimal, detected in olfactory bulb, cortical layers, and in hippocampus. PDE2 mRNA was distributed more widely, with highest levels in medial habenula, and abundant expression in olfactory bulb, olfactory tubercle, cortex, amygdala, striatum, and hippocampus. Double immunostaining of PDE2, PDE5, or PDE9 mRNAs with the neuronal marker NeuN and the glial cell marker glial fibrillary acidic protein revealed that these mRNAs were predominantly expressed in neuronal cell bodies. Our data indicate that three cGMP-hydrolyzing PDE families have distinct expression patterns, although specific cell types coexpress mRNAs for all three enzymes. Thus, it appears that differential expression of PDE isoforms may provide a mechanism to match cGMP hydrolysis to the functional demands of individual brain regions.     

Lesion-induced neuronal nitric oxide synthase in Purkinje cells of the rat cerebellar cortex: Histochemical and in situ hybridization study

Lesion-induced induction of neuronal nitric oxide synthase (nNOS) was examined in the rat cerebellum. The stab-lesioned cerebellar cortex was examined with NADPH-diaphorase (NADPH-d) histochemistry and in situ hybridization using nNOS cRNA probe at 1, 3, 7, 14, 35 days post-lesion. NADPH-d- and nNOS mRNA-positive Purkinje cells appeared adjacent to the lesion by 3 days after the lesion. The area of distribution expanded and the number of positive cells increased at 7 days after the lesion, and at 14 days post-lesion, shrunken NADPH-d-positive Purkinje cells with irregular surface appeared. NADPH-d activity and nNOS mRNA signal could not be detected in Purkinje cells after 35 days post-lesion. Combined NADPH-d histochemistory and in situ hybridization using glutamic acid decarboxylase (GAD) cRNA probe revealed that nNOS-expressing Purkinje cells showed fewer GAD mRNA signals than those in normal Purkinje cells. The atrophic contour and the lower expression of GAD mRNA signals in NADPH-d positive Purkinje cells suggest that nNOS is expressed under a degenerating process.     

Nitrite,nitrate and cGMP in the cerebrospinal fluid in degenerative neurologic diseases

Summary To investigate whether nitric oxide (NO) plays a role in degenerative neurologic disease (DND), we measured nitrite, nitrate and cyclic GMP in cerebrospinal fluid (CSF) samples from patients with Parkinson's disease (PD), spinocerebellar ataxia (SCA) and amyotrophic lateral sclerosis (ALS). We found no significant change in CSF nitrite, nitrate or cyclic GMP in patients with any DND compared with control values. These results suggest that NO production is preserved in PD, SCA and ALS.     

Long-term Depression Requires Nitric Oxide and Guanosine 3':5'Cyclic Monophosphate Production in Rat Cerebellar Purkinje Cells

In patch-clamped Purkinje cells, bath application of the nitric oxide synthase inhibitor N _G-methyl- l -arginine consistently prevents the induction of long-term depression (LTD) of parallel fibre-mediated excitatory postsynaptic potentials (EPSPs) induced by their pairing with calcium spikes. On the other hand, bath application of nitric oxide donors and of 8-bromoguanosine 3':5'cyclic monophosphate is able to reproduce an LTD-like phenomenon. LTD of parallel fibre-mediated EPSPs also occurs when nitric oxide donors or guanosine 3':5'cyclic monophosphate are directly dialysed into Purkinje cells, and this effect partially occludes LTD induced by pairing protocols. These results show that nitric oxide does play a role in LTD induction, and demonstrate for the first time that its site of action is probably the soluble guanylate cyclase of Purkinje cells.