Nitric oxide-evoked glutamate release and cGMP production in cerebellar slices: control by presynaptic 5-HT1D receptors

We previously reported that pre- and postsynaptic 5-hydroxytryptamine (5-HT) receptors effectively control glutamatergic transmission in adult rat cerebellum. To investigate where 5-HT acts in the glutamate ionotropic receptors/nitric oxide/guanosine 3',5'-cyclic monophosphate (cGMP) pathway, in the present study 5-HT modulation of the cGMP response to the nitric oxide donor S-nitroso-penicillamine (SNAP) was studied in adult rat cerebellar slices. While cGMP elevation produced by high-micromolar SNAP was insensitive to 5-HT, 1 microM SNAP, expected to release nitric oxide in the low-nanomolar concentration range, elicited cGMP production and endogenous glutamate release both of which could be prevented by activating presynaptic 5-HT1D receptors. Released nitric oxide appeared responsible for cGMP production and glutamate release evoked by 1 microM SNAP, as both the effects were mimicked by the structurally unrelated nitric oxide donor 2-(N,N-diethylamino)-diazenolate-2-oxide (0.1 microM). Dependency of the 1 microM SNAP-evoked release of glutamate on external Ca2+, sensitivity to presynaptic release-regulating receptors and dependency on ionotropic glutamate receptor functioning, suggest that nitric oxide stimulates exocytotic-like, activity-dependent glutamate release. Activation of ionotropic glutamate receptors/nitric oxide synthase/guanylyl cyclase pathway by endogenously released glutamate was involved in the cGMP response to 1 microM SNAP, as blockade of NMDA/non-NMDA receptors, nitric oxide synthase or guanylyl cyclase, abolished the cGMP response. To conclude, in adult rat cerebellar slices low-nanomolar exogenous nitric oxide could facilitate glutamate exocytotic-like release possibly from parallel fibers that subsequently activated the glutamate ionotropic receptors/nitric oxide/cGMP pathway. Presynaptic 5-HT1D receptors could regulate the nitric oxide-evoked release of glutamate and subsequent cGMP production.     

Metabotropic glutamate receptor 5 modulates the nitric oxide-cGMP pathway in cerebellum in vivo through activation of AMPA receptors

Metabotropic glutamate receptors (mGluRs) modulate important processes in cerebellum including long-term depression, which also requires formation of nitric oxide (NO) and cGMP. Some reports suggest that mGluRs could modulate the NO-cGMP pathway in cerebellum. However this modulation has not been studied in detail. The aim of this work was to assess by microdialysis in freely moving rats whether activation of mGluR5 modulates the NO-cGMP pathway in cerebellum in vivo and to analyze the underlying mechanisms. We show that mGluR5 activation increases extracellular glutamate, citrulline and cGMP in cerebellum. Blocking NMDA receptors with MK-801 does not prevent any of these effects, indicating that NMDA receptors activation is not required. However in the presence of MK-801 the effects are more transient, returning faster to basal levels. Blocking AMPA receptors prevents the increase in citrulline and cGMP induced by mGluR5 activation, but not the increase in glutamate. The release of glutamate is prevented by tetrodotoxin but not by fluoroacetate, indicating that glutamate is released from neurons and not from astrocytes. Activation of AMPA receptors increases citrulline and cGMP. These data indicate that activation of mGluR5 induces an increase of extracellular glutamate which activates AMPA receptors, leading to activation of nitric oxide synthase and increased NO, which activates guanylate cyclase, increasing cGMP. The response mediated by AMPA receptors desensitize rapidly. Activation of AMPA receptors also induces a mild depolarization, allowing activation of NMDA receptors which prolongs the duration of the effect initiated by activation of AMPA receptors. These data support that the three types of glutamate receptors: mGluR5, AMPA and NMDA cooperate in the modulation of the grade and duration of activation of the NO-cGMP pathway in cerebellum in vivo. This pathway would modulate cerebellar processes such as long-term depression.     

Arginine Availability Controls the N-Methyl-d-Aspartate-Induced Nitric Oxide Synthesis: Involvement of a Glial-Neuronal Arginine Transfer

Abstract: The neuronal nitric oxide (NO) synthase generates NO from arginine. NO mediates its physiological effects mainly by stimulating the synthesis of cyclic GMP. We have investigated the role of the arginine availability on the NMDA-induced cyclic GMP accumulation in immature rat brain slices. The effect of NMDA was blocked by the inhibitor of the NO synthase, N ^G-nitro- l -arginine, and by the antagonist of ionotropic non-NMDA receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). This inhibition was not due to a direct interaction of CNQX with the NMDA receptor, and it was overcome by the presence of exogenously applied arginine. CNQX also blocked the NMDA-evoked release of [³H]arginine from cerebellar slices. Moreover, the arginine uptake inhibitor l -lysine reduced the cyclic GMP response to NMDA significantly. Therefore, the extracellular arginine availability, which is dependent on the activation of ionotropic non-NMDA receptors, determines the rate of the NO biosynthesis by the neuronal NO synthase. Together with the reported release of arginine from glial cells upon activation of glial ionotropic non-NMDA receptors and the predominant glial localization of arginine, these data provide the first evidence of an essential role of the arginine transfer from glial cells to neurons for the biosynthesis of NO.     

Glutamate triggers neurosecretion and apoptosis in bovine chromaffin cells through a mechanism involving NO production by neuronal NO synthase activation

Previous work from our group stated that nitric oxide (NO), via cytokines, induces apoptosis in chromaffin cells by a mechanism involving iNOS, nNOS, and NF-κB. In this paper the involvement of glutamate as a possible intracellular trigger of neurosecretion and NO-mediated apoptosis has been evaluated. We show that chromaffin cells express different ionotropic and metabotropic glutamate receptors, this exerting different effects on the regulation of basal and glutamate-induced catecholamine secretion, via NO/cGMP. In addition, we studied the effects of endogenously generated NO, both basal and glutamate-stimulated, on apoptosis of chromaffin cells. Our results show that glutamate agonists are able to induce cell death and apoptosis in bovine chromaffin cells, parallel to an increase in NO production. Such effects were reversed by NOS inhibitors and glutamate receptor antagonists. Under basal conditions, iNOS inhibitors did not have any effect on apoptosis, whereas nNOS inhibitors induced apoptosis, indicating a neuroprotective effect of constitutive nNOS-generated NO. In contrast, glutamate-induced apoptosis was strongly reversed by nNOS inhibitors and weakly by iNOS inhibitors, thus indicating nNOS involvement in glutamate-mediated apoptosis. These results were confirmed by the fact that nNOS expression, but not iNOS, is specifically activated by glutamate. Finally, our results suggest the participation of PKG, PKA, PKC, and MAPK pathways in glutamate-mediated nNOS activation in chromaffin cells and point out the involvement of both PKA and PKC signaling pathways in the apoptotic effect of glutamate.     

A long-term depression of AMPA currents in cultured cerebellar Purkinje neurons.

Cerebellar long-term depression (LTD) is a model of synaptic plasticity in which conjunctive stimulation of parallel fiber and climbing fiber inputs to a Purkinje neuron induces a persistent depression of the parallel fiber-Purkinje neuron synapse. We report that an analogous phenomenon may be elicited in the cultured mouse Purkinje neuron when iontophoretic glutamate application and depolarization of the Purkinje neurons are substituted for parallel fiber and climbing fiber stimulation, respectively. The induction of LTD in these cerebellar cultures requires activation of both ionotropic (AMPA) and metabotropic quisqualate receptors, together with depolarization in the presence of external Ca2+. This postsynaptic alteration is manifest as a depression of glutamate or AMPA currents, but not aspartate or NMDA currents. These results strengthen the contention that the expression of cerebellar LTD is at least in part postsynaptic and provide evidence that activation of both ionotropic and metabotropic quisqualate receptors are necessary for LTD induction.     

Retinal Cell Death Induced by TRPV1 Activation Involves NMDA Signaling and Upregulation of Nitric Oxide Synthases

The activation of the transient receptor potential vanilloid type 1 channel (TRPV1) has been correlated with oxidative and nitrosative stress and cell death in the nervous system. Our previous results indicate that TRPV1 activation in the adult retina can lead to constitutive and inducible nitric oxide synthase-dependent protein nitration and apoptosis. In this report, we have investigated the potential effects of TRPV1 channel activation on nitric oxide synthase (NOS) expression and function, and the putative participation of ionotropic glutamate receptors in retinal TRPV1-induced protein nitration, lipid peroxidation, and DNA fragmentation. Intravitreal injections of the classical TRPV1 agonist capsaicin up-regulated the protein expression of the inducible and endothelial NOS isoforms. Using 4,5-diaminofluorescein diacetate for nitric oxide (NO) imaging, we found that capsaicin also increased the production of NO in retinal blood vessels. Processes and perikarya of TRPV1-expressing neurons in the inner nuclear layer of the retina were found in the vicinity of nNOS-positive neurons, but those two proteins did not colocalize. Retinal explants exposed to capsaicin presented high protein nitration, lipid peroxidation, and cell death, which were observed in the inner nuclear and plexiform layers and in ganglion cells. This effect was partially blocked by AP-5, a NMDA glutamate receptor antagonist, but not by CNQX, an AMPA/kainate receptor antagonist. These data support a potential role for TRPV1 channels in physiopathological retinal processes mediated by NO, which at least in part involve glutamate release.     

Two Pathways of Cyclic GMP Production Through Glutamate Receptor-Mediated Nitric Oxide Synthesis

The selective agonists for the metabotropic glutamate receptor and the ionotropic non-N-methyl-D-aspartate (NMDA) glutamate receptor, (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD) and (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), respectively, increased the cyclic GMP (cGMP) content in cerebellar slices prepared from adult rats. The ACPD-induced rise in cGMP level was blocked by compounds known to antagonize metabotropic glutamate receptors, such as DL-2-amino-3-phosphonopropionic acid and L-2-amino-4-phosphonobutyric acid, but not by ionotropic glutamate receptor antagonists, D-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), whereas the AMPA-induced rise in cGMP level was suppressed by CNQX. Both rises in cGMP level involved nitric oxide synthase (NOS), because NG-methyl-L-arginine (NMLA), an inhibitor of NOS, blocked both cGMP level rises, and excess L-arginine reversed the effect of NMLA. After lithium chloride treatment, which could exhaust phosphatidylinositol phosphates, ACPD no longer increased cGMP levels, whereas AMPA was still effective. In a calcium-free medium, ACPD still induced a rise in cGMP level, whereas AMPA did not. When the molecular layer was isolated to determine the cGMP content separately from that in the rest of the cerebellar cortex, it was found that ACPD raised the cGMP level mainly in the molecular layer, whereas AMPA raised it in both sections.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of modification of excitatory and inhibitory inputs in various neurons of olivary-cerebellar network

It is known from the experimental data that at different cerebellar neurons there are voltage-dependent Ca2+ channels, NMDA receptors, metabotropic glutamate and GABAB receptors. This receptor arrangement ensures that activation of excitatory and inhibitory input results in changes in activity of protein kinases and phosphatases and subsequent modification of synaptic efficacy. The mechanism of synaptic plasticity is advanced that in accordance with the known experimental data concerning the modification of excitatory and inhibitory inputs to Purkinje cells, granule cells, and deep cerebellar nuclei cells. The mechanism is based on a postulate that phosphorylation/dephosphorylation of AMPA (GABAA) receptors on cerebellar cells causes the LTP/LTD of excitatory (LTD/LTP of inhibitory) transmission. It is assumed that modification rules for Purkinje cells, granule cells, and deep cerebellar nuclei cells, wherein cGMP-dependent protein kinase G is involved in synaptic plasticity, are distinct from those of hippocampal/neocortical cells, wherein cAMP-dependent protein kinase A is involved in synaptic plasticity, since cGMP (cAMP) concentration decreases (increases) with Ca2+ rise.     

Climbing fiber innervation of NG2-expressing glia in the mammalian cerebellum

The molecular layer of the cerebellar cortex is populated by glial progenitors that express ionotropic glutamate receptors and extend numerous processes among Purkinje cell dendrites. Here, we show that release of glutamate from climbing fiber (CF) axons produces AMPA receptor currents with rapid kinetics in these NG2-immunoreactive glial cells (NG2+ cells) in cerebellar slices. NG2+ cells may receive up to 70 discrete inputs from one CF and, unlike mature Purkinje cells, are often innervated by multiple CFs. Paired Purkinje cell-NG2+ cell recordings show that one CF can innervate both cell types. CF boutons make direct synaptic junctions with NG2+ cell processes, indicating that this rapid neuron-glia signaling occurs at discrete sites rather than through ectopic release at CF-Purkinje cell synapses. This robust activation of Ca2+-permeable AMPA receptors in NG2+ cells expands the influence of the olivocerebellar projection to this abundant class of glial progenitors.     

The role of nitric oxide in hippocampal long-term potentiation.

Long-term potentiation is a long-lasting, use-dependent increase in the strength of synaptic connections. We investigated the role of nitric oxide (NO) in determining the duration of potentiation induced by high frequency stimulation of afferents in the CA1 region of the rat hippocampus. The calcium/calmodulin-dependent production of NO can be initiated by activation of excitatory amino acid receptors and results in increased levels of cGMP in target cells. Here we report that only a relatively short-term potentiation can be induced in the presence of nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor. The effects of L-NAME on the duration of potentiation are partially reversed by coadministration of L-arginine, a precursor of neuronal NO, and by dibutyryl cGMP. Hemoglobin, which binds extracellular NO, also shortens the duration of stimulus-induced potentiation. The results suggest a role for NO in the maintenance of activity-dependent synaptic enhancements, possibly via the generation of cGMP.     

Long-term depression of mGluR1 signaling

Glutamate produces both fast excitation through activation of ionotropic receptors and slower actions through metabotropic receptors (mGluRs). To date, ionotropic but not metabotropic neurotransmission has been shown to undergo long-term synaptic potentiation and depression. Burst stimulation of parallel fibers releases glutamate, which activates perisynaptic mGluR1 in the dendritic spines of cerebellar Purkinje cells. Here, we show that the mGluR1-dependent slow EPSC and its coincident Ca transient were selectively and persistently depressed by repeated climbing fiber-evoked depolarization of Purkinje cells in brain slices. LTD(mGluR1) was also observed when slow synaptic current was evoked by exogenous application of a group I mGluR agonist, implying a postsynaptic expression mechanism. Ca imaging further revealed that LTD(mGluR1) was expressed as coincident attenuation of both limbs of mGluR1 signaling: the slow EPSC and PLC/IP3-mediated dendritic Ca mobilization. Thus, different patterns of neural activity can evoke LTD of either fast ionotropic or slow mGluR1-mediated synaptic signaling.     

Excitatory amino acid signal transduction in the hippocampus: role of noradrenergic afferents and nitric oxide in cGMP increases in vivo.

Previous studies have demonstrated that excitatory amino acid (EAA)-dependent increases in cerebellar cGMP are dependent upon the prior activation of nitric oxide (NO) synthase. Additionally, the actions of NMDA, but not kainate or quisqualate, in elevating cerebellar cGMP have been shown to be dependent upon intact noradrenergic innervation of the cerebellum. In the current study we extended these observations to the hippocampus and again found that EAA-dependent increases in hippocampal cGMP also involve prior formation of NO. And as in the case of the cerebellum, NMDA-dependent increases in hippocampal cGMP involve prior release of norepinephrine which in turn apparently activates an alpha 1-adrenergic receptor to elicit cGMP increases. In toto, these data suggest that a key role of NMDA receptors in these brain regions is to presynaptically regulate the release of norepinephrine, thereby modulating the tone of this monoaminergic system. This may be a general principle which needs experimentation in other terminal fields of noradrenergic pathways.     

cGMP production is coupled to Ca-dependent nitric oxide generation in rabbit parotid acinar cells

We investigated the mechanism of guanosine 3′,5′-monophosphate (cGMP) production in rabbit parotid acinar cells. Methacholine, a muscarinic cholinergic agonist, stimulated cGMP production in a dose-dependent manner but not isoproterenol, a β-adrenergic receptor stimulant. Methacholine-stimulated cGMP production has been suggested to be coupled to Ca²⁺ mobilization, because intracellular Ca ²⁺ elevating reagents, such as thapsigargin and the Ca²⁺ ionophore A23187, mimicked the effect of methacholine. The cGMP production induced by Ca²⁺ mobilization has also been suggested to be coupled to nitric oxide (NO) generation because the effects of methacholine, thapsigargin and A23187 on cGMP production were blocked by N^G-nitro-L-arginine methyl ester (L-NAME), a specific inhibitor of nitric oxide synthase (NOS), and hemoglobin, a scavenger of nitric oxide (NO). Sodium nitroprusside (SNP), a NO donor, stimulated cGMP production. Furthermore, methacholine stimulated NO generation, and NOS activity in the cytosolic fraction in rabbit parotid acinar cells was exclusively dependent on Ca²⁺. These findings suggest that cGMP production induced by the activation of muscarinic cholinergic receptors is coupled to NO generation via Ca²⁺ mobilization.     

Elements of the nitric oxide/cGMP pathway expressed in cerebellar granule cells: biochemical and functional characterisation

It is known that the nitric oxide (NO)/cGMP pathway affects neuronal development and the expression of the different proteins is developmentally dependent in several brain areas. However, so far there are no data on the expression of the proteins involved in this signalling system during the development of the cerebellar granule cell, one of the most widely used models of neuronal development. This study was accordingly designed to analyse the developmental regulation of neuronal nitric oxide synthase (nNOS), soluble guanylyl cyclase subunits (alpha1, alpha2 and beta1) and cGMP-dependent protein kinases (cGK I and cGK II) in cerebellar granule cells through real time-polymerase chain reaction (RT-PCR) and Western blotting. We were able to detect guanylyl cyclase subunits and cGK I and cGK II in cerebellar granule cells at every stage of development examined (cells freshly isolated from 7-day-old rat pups, and cells cultured for 7 days or 14 days). Expression levels, nevertheless, varied significantly at each stage. nNOS, alpha2 and beta1 and cGK II levels increased during granule cell development, while alpha1 and cGK I showed an opposite behaviour pattern; the levels of these latter proteins diminished as the cells matured. The functionality of this pathway was assessed by stimulating cells kept in culture for 7 days with DEA/NO or with N-methyl-D-aspartate (NMDA). Cells responded by increasing intracellular cGMP and activating cGMP-dependent protein kinase activity, which effectively phosphorylated two well-known substrates of this activity, the vasodilator stimulated phosphoprotein (VASP) and the cAMP response element binding protein (CREB). In summary, through both functional and biochemical tests, this is the first demonstration of a complete NO/cGMP signalling transduction pathway in cerebellar granule cells. Our results also indicate the developmental regulation of the proteins in this system.     

Involvement of Nitric Oxide in Adenosine Release in the Developing and Adult Mouse Hippocampus

The novel type of neurotransmitter/neuromodulator nitric oxide (NO) is linked to activation of the N-methyl-D-aspartate (NMDA) class of glutamate receptors and has been shown to modify transmitter release in the brain. The inhibitory neuromodulator adenosine has been thought to act as an endogenous neuroprotectant against cerebral ischemia and neuronal damage. The effects of NO-generating compounds on the release of preloaded [3H]adenosine from hippocampal slices from developing (7-day-old) and adult (3-month-old) mice were investigated, using a superfusion system, under normal conditions and in vitro ischemia. The release of adenosine was markedly potentiated at both ages by the NO-producing compounds S-nitroso-N-acetylpenicillamine, sodium nitroprusside, and hydroxylamine. The evoked releases were reduced by the NO synthase inhibitors nitroarginine and 7-nitroindazole at both ages. They were also reduced by the inhibitor of soluble guanylyl cyclase 1H-(1,2,4-oxadiazolo(4,3a)quinoxalin-1-one (ODQ) in adults, indicating that the NO/cGMP pathway is involved in this release. Release of adenosine was also evoked when the cGMP levels were increased by superfusing slices with the phosphodiesterase inhibitor zaprinast. The markedly enhanced adenosine release under ischemic conditions was further potentiated by the ionotropic glutamate receptor agonists and NO-generating compounds, whereas zaprinast and ODQ had no effect, rendering unlikely the involvement of cGMP in the ischemic release. Moreover, NO was able to provoke substantial release of adenosine in the presence of NMDA under both normal and ischemic conditions, which could significantly add to the neuroprotective potential of this neuromodulator in both adult and developing hippocampus.     

Kinetics of nitric oxide-cyclic GMP signalling in CNS cells and its possible regulation by cyclic GMP

Physiologically, nitric oxide (NO) signal transduction occurs through soluble guanylyl cyclase (sGC), which catalyses cyclic GMP (cGMP) formation. Knowledge of the kinetics of NO-evoked cGMP signals is therefore critical for understanding how NO signals are decoded. Studies on cerebellar astrocytes showed that sGC undergoes a desensitizing profile of activity, which, in league with phosphodiesterases (PDEs), was hypothesized to diversify cGMP responses in different cells. The hypothesis was tested by examining the kinetics of cGMP in rat striatal cells, in which cGMP accumulated in neurones in response to NO. Based on the effects of selective PDE inhibitors, cGMP hydrolysis following exposure to NO was attributed to a cGMP-stimulated PDE (PDE 2). Analysis of NO-induced cGMP accumulation in the presence of a PDE inhibitor indicated that sGC underwent marked desensitization. However, the desensitization kinetics determined under these conditions described poorly the cGMP profile observed in the absence of the PDE inhibitor. An explanation shown plausible theoretically was that cGMP determines the level of sGC desensitization. In support, tests in cerebellar astrocytes indicated an inverse relationship between cGMP level and recovery of sGC from its desensitized state. We suggest that the degree of sGC desensitization is related to the cGMP concentration and that this effect is not mediated by (de)phosphorylation.     

Characteristics of GABA release modified by glutamate receptors in mouse hippocampal slices

The major part of hippocampal innervation is glutamatergic, regulated by inhibitory GABA-releasing interneurons. The modulation of [(3)H]GABA release by ionotropic and metabotropic glutamate receptors and by nitric oxide was here characterized in superfused mouse hippocampal slices. The ionotropic glutamate receptor agonists kainate, N-methyl-D-aspartate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate potentiated the basal GABA release. These effects were blocked by their respective antagonists 6-nitro-7-cyanoquinoxaline-2,3-dione (CNQX), dizocilpine and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo(f)quinoxaline-7-sulfonamide (NBQX), indicating receptor-mediated mechanisms. The NO-generating compounds S-nitroso-N-acetylpenicillamine (SNAP), sodiumnitroprusside and hydroxylamine enhanced the basal GABA release. Particularly the sodiumnitroprusside-evoked release was attenuated by the NO synthase inhibitor N(G)-nitro-L-arginine (L-NNA) and the inhibitor of soluble guanylyl cyclase 1H-(1,2,4)oxadiazolo(4,3a)quinoxalin-1-one (ODQ), indicating the involvement of the NO/cGMP pathway. This inference is corroborated by the enhancing effect of zaprinast, a phosphodiesterase inhibitor, which is known to increase cGMP levels. The K(+)-stimulated hippocampal GABA release was reduced by the groups I and III agonists of metabotropic glutamate receptors (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylate (t-ACPD) and L-(+)-2-amino-4-phosphonobutyrate (L-AP4), which effects were abolished by their respective antagonists (RS)-1-aminoindan-1,5-dicarboxylate (AIDA) and (RS)-2-cyclopropyl-4-phosphonophenylglycine (CPPG), again indicating modification by receptor-mediated mechanisms.     

A Kainate Receptor Linked to Nitric Oxide Synthesis from Arginine

In slices of young rat cerebellum, the glutamate analogue kainate induced a large accumulation of cyclic GMP, which was inhibited by non-N-methyl-D-aspartate antagonists. Quisqualate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate evoked only small cyclic GMP responses and inhibited the effect of kainate. When tested in cerebellar cell suspensions, glutamate was also a potent antagonist of the cyclic GMP response to kainate. Superoxide dismutase enhanced the response in the isolated cells, whereas haemoglobin and methylene blue were inhibitory. The response in slices was Ca2+ dependent, augmented by arginine, and inhibited by L-NG-monomethylarginine in a manner that could be reversed by additional arginine. It is concluded that stimulation of kainate receptors leads to activation of the enzyme that synthesizes nitric oxide from arginine and that activation of soluble guanylate cyclase by the released nitric oxide accounts for the cyclic GMP generation.