On the selectivity of neuronal NOS inhibitors.

BACKGROUND AND PURPOSE: Isoform-selective inhibitors of NOS enzymes are desirable as research tools and for potential therapeutic purposes. Vinyl-l-N-5-(1-imino-3-butenyl)-l-ornithine (l-VNIO) and N(ω) -propyl-l-arginine (NPA) purportedly have good selectivity for neuronal over endothelial NOS under cell-free conditions, as does N-[(3-aminomethyl)benzyl]acetamidine (1400W), which is primarily an inducible NOS inhibitor. Although used in numerous investigations in vitro and in vivo, there have been surprisingly few tests of the potency and selectivity of these compounds in cells. This study addresses this deficiency and evaluates the activity of new and potentially better pyrrolidine-based compounds. EXPERIMENTAL APPROACH: The inhibitors were evaluated by measuring their effect on NMDA-evoked cGMP accumulation in rodent hippocampal slices, a response dependent on neuronal NOS, and ACh-evoked cGMP synthesis in aortic rings of the same animals, an endothelial NOS-dependent phenomenon. KEY RESULTS: l-VNIO, NPA and 1400W inhibited responses in both tissues but all showed less than fivefold higher potency in the hippocampus than in the aorta, implying useless selectivity for neuronal over endothelial NOS at the tissue level. In addition, the inhibitors had a 25-fold lower potency in the hippocampus than reported previously, the IC(50) values being approximately 1 µM for l-VNIO and NPA, and 150 µM for 1400W. Pyrrolidine-based inhibitors were similarly weak and nonselective. CONCLUSION AND IMPLICATIONS: The results suggest that l-VNIO, NPA and 1400W, as well as the newer pyrrolidine-type inhibitors, cannot be used as neuronal NOS inhibitors in cells without stringent verification. The identification of inhibitors with useable selectivity in cells and tissues remains an important goal.

Inhibition of nitric oxide-activated guanylyl cyclase by calmodulin antagonists.

BACKGROUND AND PURPOSE: Nitric oxide (NO) controls numerous physiological processes by activation of its receptor, guanylyl cyclase (sGC), leading to the accumulation of 3'-5' cyclic guanosine monophosphate (cGMP). Ca(2+)-calmodulin (CaM) regulates both NO synthesis by NO synthase and cGMP hydrolysis by phosphodiesterase-1. We report that, unexpectedly, the CaM antagonists, calmidazolium, phenoxybenzamine and trifluoperazine, also inhibited cGMP accumulation in cerebellar cells evoked by an exogenous NO donor, with IC(50) values of 11, 80 and 180 microM respectively. Here we sought to elucidate the underlying mechanism(s). EXPERIMENTAL APPROACH: We used cerebellar cell suspensions to determine the influence of CaM antagonists on all steps of the NO-cGMP pathway. Homogenized tissue and purified enzyme were used to test effects of calmidazolium on sGC activity. KEY RESULTS: Inhibition of cGMP accumulation in the cells did not depend on changes in intracellular Ca(2+) concentration. Degradation of cGMP and inactivation of NO were both inhibited by the CaM antagonists, ruling out increased loss of cGMP or NO as explanations. Instead, calmidazolium directly inhibited purified sGC (IC(50)= 10 microM). The inhibition was not in competition with NO, nor did it arise from displacement of the haem moiety from sGC. Calmidazolium decreased enzyme V(max) and K(m), indicating that it acts in an uncompetitive manner. CONCLUSIONS AND IMPLICATIONS: The disruption of every stage of NO signal transduction by common CaM antagonists, unrelated to CaM antagonism, cautions against their utility as pharmacological tools. More positively, the compounds exemplify a novel class of sGC inhibitors that, with improved selectivity, may be therapeutically valuable.

Probing the presence of the ligand-binding haem in cellular nitric oxide receptors.

BACKGROUND AND PURPOSE: Nitric oxide (NO) acts on receptors coupled to guanylyl cyclase (GC), leading to cGMP accumulation. The NO binding site is a haem group, oxidation or loss of which diminishes NO-stimulated activity. Agonists reportedly engaging both these NO-insensitive forms have emerged. Here we characterize the effect of a prototype compound (BAY 58-2667) and use it to assess the haem status of cellular GC. EXPERIMENTAL APPROACH: GC activity measurements were made on the purified protein and on rat platelets. KEY RESULTS: Experiments on purified GC showed that the target for BAY 58-2667 is the haem-free GC, not the haem-oxidized form. The efficacy of BAY 58-2667 was about half that shown normally by NO. In platelets, BAY 58-2667 was a potent GC activator (EC50 approximately 15 nM) but the maximum effect was only about 1% of that achievable with NO. Nevertheless, it was enough to evoke cGMP-dependent protein phosphorylation. Profound (85 %) desensitization of NO-evoked GC activity did not alter the effectiveness of BAY 58-2667. Haem oxidation, however, increased the efficacy of BAY 58-2667 by 22-fold, implying that about half the cellular GC was then haem-free. Oxidation appeared to enhance the rate of haem dissociation from purified GC. CONCLUSIONS AND IMPLICATIONS: Compounds such as BAY 58-2667 are useful for probing the occupancy of the haem pocket of NO receptors in cells but not for distinguishing oxidized from reduced haem. In vivo, such compounds are likely to be particularly effective in conditions where there is deficient haem incorporation or enhanced haem loss.

Inactivation of nitric oxide by rat cerebellar slices.

Nitric oxide (NO) functions as an intercellular messenger throughout the brain. For this role to be performed efficiently, there must be a mechanism for neutralizing NO, but whether an active biological process exists, or whether NO is lost mainly through diffusion is unclear. To investigate this issue, rat cerebellar slices were exposed to constant levels of NO and the cGMP generated within the slice used as an indicator of NO concentrations therein. NO was about 1000-fold less potent in slices (EC50, 1 microM) than in separated cells from the same tissue (EC50, 1.6 nM), consistent with access of NO to the slice interior being greatly hindered by inactivation. Supporting this interpretation, immunohistochemical analysis indicated a marked concentration gradient of cGMP across the thickness of slices exposed to subsaturating NO concentrations, signifying a marked NO gradient. Several known NO-degrading processes, including reaction with lipid peroxyl radicals, erythrocytes and superoxide ions, were eliminated as contributing factors, indicating a novel mechanism. A diffusion-inactivation model was used to estimate the kinetics of NO consumption by the slices. The best fits to experimental data indicated a Michaelis-Menten-type reaction having a Vmax of 1-2 microM s-1 and a Km of around 10 nM. The rates predict that inactivation would impose a very short half-life (<10 ms) on NO in physiological concentrations (up to 10 nM) and that it would play an important role in shaping the NO concentration profiles when it is synthesized by multiple nearby sites.

Pathological consequences of inducible nitric oxide synthase expression in hippocampal slice cultures.

The generation of toxic concentrations of nitric oxide by the inducible nitric oxide synthase expressed in microglia and other brain cell types is frequently invoked as a causative factor in neurodegeneration. Experiments were carried out on slice cultures of rat hippocampus to test this hypothesis. Exposure of the slices to bacterial lipopolysaccharide plus interferon-gamma led to a time-dependent expression of functional inducible nitric oxide synthase that was found only in microglia. Microglial activation by other means, such as physical damage, was not associated with inducible nitric oxide synthase expression. Damage and cell death in slices expressing inducible nitric oxide synthase was evaluated over a period of 6 days, but none was found. Consistent with this result, cGMP measurements indicated that the average local nitric oxide concentration remained in the low nanomolar range. When the microglial population was expanded to a density three-fold above normal by applying granulocyte-macrophage colony stimulating factor, however, lipopolysaccharide plus interferon-gamma provoked neurodegeneration that could be blocked by an inducible nitric oxide synthase inhibitor. The associated nitric oxide concentration in the slices was saturating for guanylyl cyclase-coupled nitric oxide receptors, signifying at least 10 nM. It is concluded that inducible nitric oxide synthase is expressed in microglia only in response to specific stimuli involving the innate immune system, and that the resulting level of nitric oxide in intact brain tissue is normally too low to inflict damage directly. Quantities of nitric oxide sufficient to contribute directly or indirectly to pathology could be produced should the density of microglia become high enough, although caution must be exercised in extrapolating this finding to the human brain in vivo.

Pathological implications of iNOS expression in central white matter: an ex vivo study of optic nerves from rats with experimental allergic encephalomyelitis.

Excessive nitric oxide (NO) production from the inducible isoform of nitric oxide synthase (iNOS) has been invoked as a causative factor in many neurodegenerative disorders, including multiple sclerosis. This hypothesis has been supported by in vitro studies showing that glial iNOS expression results in toxic NO concentrations (near 1 microm). To investigate the relevance of such findings, experiments were carried out ex vivo on optic nerves from rats with exacerbated experimental allergic encephalomyelitis, a model of multiple sclerosis. The nerves displayed characteristic immunopathology and expression of iNOS in macrophages and/or microglia and there was overt axonal damage in localized regions of the optic chiasm. The resulting NO levels in the optic nerve were sufficient to cause activation of guanylyl cyclase-coupled NO receptors, resulting in marked cGMP accumulation in axons throughout the nerve. Nevertheless, calibration of cGMP levels against those evoked by exogenous NO indicated that the nerves were not compromised metabolically and that their ambient NO concentration was only approximately 1 nm. Consistent with this observation, electrophysiological tests indicated that there was no ongoing malfunctioning of the type that can be elicited by high exogenous NO concentrations. It is concluded that, with iNOS expressed in physiological locations and levels, the tissue levels of NO remain at concentrations far lower than those shown to have toxic effects, despite continuous NO synthesis. The fact that NO can rise to much higher levels in dispersed cultures in vitro may be attributable to a deficiency in NO inactivation in such preparations.

Nitric oxide toxicity in CNS white matter: an in vitro study using rat optic nerve.

Excessive nitric oxide formation may contribute to the pathology occurring in diseases affecting central white matter, such as multiple sclerosis. The rat isolated optic nerve preparation was used to investigate the potential toxicity of the molecule towards such tissue. The nerves were exposed to a range of concentrations of different classes of nitric oxide donor for up to 23 h, with or without a subsequent period of recovery, and the damage assessed by quantitative histological methods. Degeneration of axons and macroglia occurred in a time- and concentration-dependent manner, the order of susceptibility being: axons>oligodendrocytes>astrocytes. Use of NONOate donors differing in half-life indicated that nitric oxide delivered in an enduring manner at relatively low concentration was more toxic than the same amount supplied rapidly at high concentration. The mechanism by which nitric oxide affects axons was studied using a donor [3-(n-propylamino)propylamine/NO adduct, PAPA/NO] with an intermediate half-life that produced selective axonopathy after a 2-h exposure (plus 2 h recovery). Axon damage was abolished if, during the exposure, Na(+) or Ca(2+) was removed from the bathing medium or the sodium channel inhibitors tetrodotoxin or BW619C89 (sipatrigine) were added. In electrophysiological experiments, the donor elicited a biphasic depolarisation. The second, larger component (occurring after 7-10 min) was associated with a block of nerve conduction and could be inhibited by tetrodotoxin. Coincident with the secondary depolarisation was a reduction in ATP levels by about 50%, an effect that was also inhibited by tetrodotoxin. It is concluded that nitric oxide, in submicromolar concentrations, can kill axons and macroglia in white matter. The findings lend support to the hypothesis that nitric oxide may be of importance to white matter pathologies, particularly those in which inducible nitric oxide synthase is expressed. The axonopathy, at least when elicited over relatively short time intervals, is likely to be caused by metabolic inhibition. As in anoxia and anoxia/aglycaemia, nitric oxide-induced destruction of axons is likely to be caused by the Ca(2+) overload that follows a reduction in ATP levels in the face of continued influx of Na(+) through voltage-dependent channels.

Soluble guanylyl cyclase activator YC-1 protects white matter axons from nitric oxide toxicity and metabolic stress, probably through Na(+) channel inhibition.

In the rat isolated optic nerve, nitric oxide (NO) activates soluble guanylyl cyclase (sGC), resulting in a selective accumulation of cGMP in the axons. The axons are also selectively vulnerable to NO toxicity. The experiments initially aimed to determine any causative link between these two effects. It was shown, using a NONOate donor, that NO-induced axonal damage occurred independently of cGMP. Unexpectedly, however, the compound YC-1, which is an allosteric activator of sGC, potently inhibited NO-induced axonopathy (IC(50) = 3 microM). This effect was not attributable to increased cGMP accumulation. YC-1 (30 microM) also protected the axons against damage by simulated ischemia, which (like NO toxicity) is sensitive to Na(+) channel inhibition. Although chemically unrelated to any known Na(+) channel inhibitor, YC-1 was effective in two biochemical assays for activity on Na(+) channels in synaptosomes. Electrophysiological recording from hippocampal neurons showed that YC-1 inhibited Na(+) currents in a voltage-dependent manner. At a concentration giving maximal protection of optic nerve axons from NO toxicity (30 microM), YC-1 did not affect normal axon conduction. It is concluded that the powerful axonoprotective action of YC-1 is unrelated to its activity on sGC but is explained by a novel action on voltage-dependent Na(+) channels. The unusual ability of YC-1 to protect axons so effectively without interfering with their normal function suggests that the molecule could serve as a prototype for the development of more selective Na(+) channel inhibitors with potential utility in neurological and neurodegenerative disorders.

The shaping of nitric oxide signals by a cellular sink.

1. The functioning of nitric oxide (NO) as a biological messenger necessitates that there be an inactivation mechanism. Cell suspensions from a rat brain region rich in the NO signalling pathway (cerebellum) were used to investigate the existence of such a mechanism and to determine its properties. 2. The cells consumed NO in a manner that could not be explained by reaction with O(2), superoxide ions or contaminating red blood cells. Functionally, the mechanism was able to convert constant rates of NO formation into low steady-state NO concentrations. For example, with NO produced at 90 nM min(-1), the cells (20 x 10(6) ml(-1)) held NO at 20 nM. Various other cell types behaved similarly. 3. The influence of NO inactivation on the ability of NO to access its receptor, soluble guanylyl cyclase, was explored by measuring cGMP accumulation in response to the clamped NO concentrations. The extrapolated steady-state EC(50) for NO was 2 nM, a concentration readily achieved by low NO release rates, despite inactivation. 4. When confronted by higher NO release rates for several minutes, the clamping mechanism failed, resulting in a progressive rise in NO concentration. While the clamp was maintained, cellular respiration was unaffected but, as it failed, respiration became inhibited by NO. The IC(50) was measured to be 120 nM (at 100-140 microM O(2)). 5. It is concluded that cerebellar (and other) cells possess a powerful NO inactivation mechanism that, extrapolated to the whole tissue, would impose on NO a half-life of around 100 ms. This and other properties of the device appear ideal for shaping low-level NO signals for activating its receptor, soluble guanylyl cyclase, whilst avoiding adverse effects on mitochondrial function. The exhaustibility of the mechanism provides a scenario for NO to become toxic.

mGlu1 receptors mediate a post-tetanic depression at parallel fibre-Purkinje cell synapses in rat cerebellum.

Metabotropic glutamate (mGlu) receptors are located pre- and postsynaptically at central synapses. Activation of the receptors by exogenous agonists usually results in a reversible depression of fast glutamatergic neurotransmission. Evidence that synaptically released glutamate has such an action, however, is scarce. Sharp microelectrode recordings were used to investigate the modulatory role of mGlu receptors at a well-studied glutamatergic synapse, the one between parallel fibres and Purkinje cells in rat cerebellar slices. Brief, tetanic stimulation of the parallel fibres caused a depression of subsequent fast EPSPs. This post-tetanic depression (PTD) reached its maximum 4.5 s after the tetanus. Measured at this point, PTD was frequency-dependent; 10 stimuli at 20 Hz produced no significant depression, whereas, at 100 Hz the same number of stimuli was maximally effective (approximately 50% depression). The nonselective mGlu antagonist, (S)-alpha-methyl-4-carboxyphenylglycine 1 mm or the GABAB antagonist, CGP35348 (1 mm), both decreased the magnitude of the PTD. In the presence of CGP35348 the mGlu1 antagonist, 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (300 microm), inhibited PTD further. A group II/III mGlu antagonist had no effect. These observations indicate that synaptically activated mGlu1 receptors not only generate a slow EPSP and induce Ca2+ mobilization in Purkinje cells, as reported previously, but also produce a transient depression of fast synaptic transmission. This short-term plasticity may be important for shaping the output of cerebellar circuits and/or it could provide a substrate for long-term depression when additional mechanisms are superimposed.

Exogenous nitric oxide causes potentiation of hippocampal synaptic transmission during low-frequency stimulation via the endogenous nitric oxide-cGMP pathway.

Nitric oxide (NO) is a putative participant in synaptic plasticity and demonstrations that exogenous NO can elicit the same plastic changes have been taken to support such a role. The experiments, carried out on the CA1 region of rat hippocampal slices, were aimed at testing this interpretation. A major component of tetanus-induced long-term potentiation (LTP) was lost in response to L-nitroarginine, which inhibits NO synthase, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), which inhibits NO-sensitive soluble guanylyl cyclase (sGC). At 0.2 Hz afferent fibre stimulation, exogenous NO produced, concentration-dependently, a synaptic depression that reverted on washout to a persistent potentiation that occluded tetanus-induced LTP. The NO concentrations necessary (estimated in the 100-nM range), however, were mostly supramaximal for stimulating hippocampal slice sGC activity. Nevertheless the potentiation, but not the preceding depression, was blocked by ODQ. L-nitroarginine and an NMDA antagonist were similarly effective, indicating mediation by the endogenous NMDA receptor-NO synthase-sGC pathway. At a concentration normally too low to affect synaptic transmission but sufficient to stimulate sGC (estimated to be 50 nM), exogenous NO reversed the effect of L-nitroarginine and caused a potentiation which was blocked by ODQ. At a concentration inducing the depression/potentiation sequence, NO partially inhibited hippocampal slice oxygen consumption. It is concluded that, at physiological levels, exogenous NO can directly elicit a potentiation of synaptic transmission through sGC, provided that the synapses are suitably primed. At higher concentrations, NO inhibits mitochondrial respiration, which can result in an enduring synaptic potentiation due to secondary activation of the endogenous NO-cGMP pathway.

Metabotropic glutamate receptor subtypes modulating neurotransmission at parallel fibre-Purkinje cell synapses in rat cerebellum.

The actions of reportedly group-selective metabotropic glutamate (mGlu) receptor agonists and antagonists on neurotransmission at parallel fibre-Purkinje cell synapses in the rat cerebellum have been characterised using sharp microelectrode recording and an in vitro slice preparation. Application of the group I agonist (S)-3,5-dihydroxyphenylglycine (DHPG) or the group III selective agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4) depressed synaptic transmission in a reversible and concentration-dependent manner (EC(50)=18 and 5 microM, respectively). The depression produced by DHPG was unrelated to the depolarisation observed in some Purkinje cells. The group II agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV, 1 microM) had no effect. The effects of DHPG were inhibited by the group I-selective antagonist 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt), but not by the group II/III antagonist alpha-methyl-4-phosphonophenylglycine (MPPG). The effect of L-AP4 was inhibited by MPPG, but not by the group I/II antagonist (S)-alpha-methyl-4-carboxyphenylglycine (MCPG). By themselves, the antagonists did not affect the EPSPs, suggesting that neither receptor is activated during low frequency neurotransmission. It is concluded that, in addition to the excitatory role for group I receptors described previously, both group I and III (but not group II) mGlu receptors operate at this synapse to inhibit synaptic transmission. The specific receptor subtypes involved are likely to be mGlu1 and mGlu4.

Nitric oxide-induced potentiation of CA1 hippocampal synaptic transmission during baseline stimulation is strictly frequency-dependent.

Nitric oxide (NO) has been hypothesised to serve a signalling role in certain types of synaptic plasticity. If so, exogenously applied NO should be able to elicit those same plastic changes under appropriate conditions. In the case of hippocampal long-term potentiation (LTP), however, existing evidence is discrepant. Field recordings of synaptic transmission in the CA1 area of rat hippocampal slices were used to re-examine this issue. Under 0.2 Hz afferent fibre stimulation, NO (delivered using two different NONOates) produced, concentration-dependently, a depression of synaptic transmission. On washout of NO, the depression gave way to a persistent potentiation, the amplitude of which was also graded with NONOate concentration. Tetanus-induced LTP, induced an hour after washout, was occluded in proportion to the degree of prior NO-induced potentiation. At a lower stimulation frequency of 0.033 Hz, the depression was unaltered but no rebound potentiation took place and subsequent tetanus-induced LTP was normal. Tests indicated that there is a clear time-window during which 0.2 Hz stimulation needs to be applied relative to the delivery of NO to produce a potentiation. The findings explain previous divergent results and indicate that exogenous NO-triggered potentiation depends critically on the frequency of synaptic transmission.

Subunits of the nitric oxide receptor, soluble guanylyl cyclase, expressed in rat brain.

Despite the widespread use of nitric oxide as a signalling molecule in the central nervous system, the molecular makeup of its receptor, soluble guanylyl cyclase (sGC), therein is poorly understood. Accordingly, RT-PCR and in situ hybridization were used to identify sGC subunits expressed in rat brain. In addition to the expected mRNA for alpha 1 and beta1 subunits, message for the beta 2 subunit was detected in the cerebellum at all developmental stages investigated (1--150 days postnatum). The use of degenerate primers allowed the identification of mRNA coding for the rat alpha 2 subunit, which was also expressed at every age studied. All but beta 2 were detected by in situ hybridization in the brains of both 8-day-old and adult rats. The distribution patterns indicated that in some areas, e.g. caudate-putamen and nucleus accumbens, sGC probably exists mainly as the alpha 1 beta 1 heterodimer. In others, e.g. hippocampus and olfactory bulb, alpha 2 beta 1 is likely to be dominant. In the cerebellum, alpha 1 and beta 1 message was strong in the Purkinje cell layer but was not confined to Purkinje cells: smaller cells, presumed to be the Bergmann glia, were also labelled. In contrast, alpha 2 mRNA was concentrated in cerebellar granule cells. Western blotting indicated an excess of alpha 1 over beta 1 protein in the cerebellum, the reverse of what was found in the lung. It is concluded that, in molecular terms, sGC is likely to be more complex and exhibit more regional variation in the brain than previously thought. The functional consequences of this heterogeneity require investigation.

Sub-second kinetics of the nitric oxide receptor, soluble guanylyl cyclase, in intact cerebellar cells.

Soluble guanylyl cyclase (sGC) catalyzes cGMP synthesis and serves as a physiological receptor for nitric oxide (NO). Recent evidence indicates that key properties of sGC within cells differ from those of purified sGC. We have devised a technique for resolving NO-stimulated sGC activity in cells on a sub-second time scale, enabling the first quantitative description of the kinetics of the enzyme within its natural environment. Upon release of NO from a caged derivative, sGC became activated without any lag observable at a 20-ms sampling time. Deactivation of sGC on removal of NO occurred with a rate constant of 3.7 s(-)(1), which is 25-fold faster than the fastest estimate for purified sGC. Desensitization of sGC occurred with a time constant of 6.9 s at an estimated 70 nm NO and became faster at a higher concentration, indicating that NO accelerates desensitization. The concentration-response curve for NO consequently became increasingly bell-shaped with time, a phenomenon that causes the apparent potency of NO to increase with time. The results indicate that sGC within cells behaves in a highly dynamic fashion, allowing the NO-cGMP pathway to operate within a kinetic framework more resembling that of neurotransmission than the properties of purified sGC suggest.

"cAMP-specific" phosphodiesterase contributes to cGMP degradation in cerebellar cells exposed to nitric oxide.

Nitric oxide (NO) functions as a diffusible messenger in the central nervous system and elsewhere, exerting many of it physiological effects by activating soluble guanylyl cyclase, so increasing cellular cGMP levels. Hydrolysis of cyclic nucleotides is achieved by phosphodiesterases (PDEs) but the enzyme isoforms responsible for degrading cGMP in most cells have not been identified. We have devised a method for quantitatively monitoring the rate of breakdown of cGMP within intact cells and have applied it to rat cerebellar cell suspensions previously stimulated with NO. In contrast to previous findings in cultured cerebellar cells, there was no evidence from the use of selective inhibitors that PDE 1 participated importantly in cGMP hydrolysis. Moreover, procedures expected to increase PDE 1 activity by raising cytosolic Ca2+ concentrations (neurotransmitter agonists, Ca2+ ionophore) failed to influence cGMP breakdown. Instead, through the use of inhibitors selective for different PDE families, two isoforms were implicated: a "cGMP-specific" PDE (PDE 5), inhibited by sildenafil and zaprinast, and a "cAMP-specific" PDE (PDE 4), inhibited by low concentrations of rolipram and Ro-20-1724 and by milrinone. An explanation is offered for a participation of PDE 4 based on the high estimated intracellular cGMP concentration (approximately 800 microM) and the low affinity of the enzyme for cGMP. In accordance with predictions, recombinant PDE 4 was shown to hydrolyze high cGMP concentrations in a rolipram-sensitive manner. The widespread use of rolipram to test for a specific involvement of cAMP in cellular phenomena must therefore be questioned.

Rapid desensitization of the nitric oxide receptor, soluble guanylyl cyclase, underlies diversity of cellular cGMP responses.

A major receptor for nitric oxide (NO) is the cGMP-synthesizing enzyme, soluble guanylyl cyclase (sGC), but it is not known how this enzyme behaves in cells. In cerebellar cells, NO (from diethylamine NONOate) increased astrocytic cGMP with a potency (EC(50)

Nitric oxide stimulates cGMP formation in rat optic nerve axons, providing a specific marker of axon viability.

A major transduction pathway for nitric oxide (NO) is stimulation of soluble guanylyl cyclase and the generation of cyclic GMP (cGMP). In the central nervous system, the NO-cGMP pathway has previously been associated primarily with synapses, particularly glutamatergic synapses. We report here that NO caused a large increase in the levels of cGMP in a central white matter tract devoid of synapses, namely in the rat isolated optic nerve. Cyclic GMP immunohistochemistry indicated that this response was confined to the axons. Accordingly, nerves previously subjected to 1 h of oxygen/glucose deprivation, which leads to irreversible axonal damage, displayed an 80% reduction in their subsequent capacity to generate cGMP in response to NO and a corresponding reduction in the numbers of cGMP-immunostained axons. Protection of the axon cGMP response against this insult was achieved by omission of Ca2 + or Na + from the incubation medium, and by the pharmacological agents tetrodotoxin, lamotrigine, BW619C89 and BW1003C87, all of which protect axonal structure from oxygen/glucose deprivation-induced damage. The results suggest that the NO-cGMP pathway has a hitherto unsuspected function in the optic nerve. Additionally, the expression of NO-stimulated guanylyl cyclase in optic nerve axons provides a simple, sensitive and specific marker of their functional integrity that is likely to be valuable in investigating the mechanisms responsible for axon degeneration in ischaemia and other conditions.

Molecular characterization and in situ localization of a full-length cyclic nucleotide-gated channel in rat brain.

Ion channels gated directly by cyclic nucleotides are required for the transduction of sensory signals in photoreceptor cells and olfactory cells. Cyclic nucleotide-gated (CNG) channels may also be expressed in the central nervous system because partial transcripts that share homology with CNG channels have been found therein. We have now isolated and cloned a full-length CNG channel cDNA from adult rat brain. The sequence is identical to that of the alpha-subunit originally found in the olfactory epithelium (CNCalpha3). In situ hybridization, using probes specific for the CNCalpha3 mRNA, suggest that this channel is expressed widely in the rat brain, albeit mostly at relatively low levels. Certain neuronal populations, however, such as deep cerebellar nuclei, olfactory bulb mitral cells and cerebellar Purkinje neurons, appeared specially enriched. The study demonstrates for the first time that a full-length CNG channel mRNA is expressed in the brain, supporting the possibility that CNG channels are involved in central neural communication and plasticity. The sequence reported in this paper has been deposited in the GenBank data base (accession no. AF126808).

Regulation of synaptic transmission in the mossy fibre-granule cell pathway of rat cerebellum by metabotropic glutamate receptors.

The role of metabotropic glutamate receptors (mGluRs) in the mossy fibre-granule cell pathway in rat cerebellum was studied using slice preparations and electrophysiological techniques. Application of the group I selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) evoked, in a concentration-dependent manner (EC50 = 33 microM), a depolarising/hyperpolarising complex response from granule cells which was preferentially inhibited by the group I selective antagonist (S)-4-carboxyphenylglycine (4CPG). The group III selective agonist L-amino-4-phosphonobutyrate (AP4) evoked a hyperpolarising response (EC50 = 10 microM) which was inhibited by the group II/III selective antagonist (S)-alpha-methyl-4-phosphonophenylglycine (MPPG). The group II agonist (2S,2'R,3'R)-2-(2',3'-dicarboxylcyclopropyl)glycine (DCG-IV) elicited no measurable voltage change. The amplitude of the synaptically-mediated mossy fibre response in granule cells was unaffected during application of AP4, was reduced by DHPG and was enhanced by DCG-IV (EC50 = 80 nM). These effects were inhibited by the group selective antagonists 4CPG and (2S,1'S,2'S,3'R)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine (PCCG-4), respectively. Further investigation using patch-clamp recording revealed that DCG-IV potently inhibited spontaneous GABAergic currents. We conclude that group I and III (but not group II) mGluRs are functionally expressed by granule cells, whereas unexpectedly group II or III mGluRs do not appear to be present presynaptically on mossy fibre terminals. Group II mGluRs are located on Golgi cell terminals; when activated these receptors cause disinhibition, a function which may be important for gating information transfer from the mossy fibres to the granule cells.