Cerebellar cGMP Research Articles

Increasing extracellular cGMP in cerebellum in vivo reduces neuroinflammation, GABAergic tone and motor in-coordination in hyperammonemic rats

Hyperammonemia is a main contributor to cognitive impairment and motor in-coordination in patients with hepatic encephalopathy. Hyperammonemia-induced neuroinflammation mediates the neurological alterations in hepatic encephalopathy. Intracerebral administration of extracellular cGMP restores some but not all types of cognitive impairment. Motor in-coordination, is mainly due to increased GABAergic tone in cerebellum. We hypothesized that extracellular cGMP would restore motor coordination in hyperammonemic rats by normalizing GABAergic tone in cerebellum and that this would be mediated by reduction of neuroinflammation.The aims of this work were to assess whether chronic intracerebral administration of cGMP to hyperammonemic rats: 1) restores motor coordination; 2) reduces neuroinflammation in cerebellum; 3) reduces extracellular GABA levels and GABAergic tone in cerebellum; and also 4) to provide some advance in the understanding on the molecular mechanisms involved.The results reported show that rats with chronic hyperammonemia show neuroinflammation in cerebellum, including microglia and astrocytes activation and increased levels of IL-1b and TNFa and increased membrane expression of the TNFa receptor. This is associated with increased glutaminase expression and extracellular glutamate, increased amount of the GABA transporter GAT-3 in activated astrocytes, increased extracellular GABA in cerebellum and motor in-coordination. Chronic intracerebral administration of extracellular cGMP to rats with chronic hyperammonemia reduces neuroinflammation, including microglia and astrocytes activation and membrane expression of the TNFa receptor. This is associated with reduced nuclear NF-κB, glutaminase expression and extracellular glutamate, reduced amount of the GABA transporter GAT-3 in activated astrocytes and reduced extracellular GABA in cerebellum and restoration of motor coordination.The data support that extracellular cGMP restores motor coordination in hyperammonemic rats by reducing microglia activation and neuroinflammation, leading to normalization of extracellular glutamate and GABA levels in cerebellum and of motor coordination.

Preclinical findings predicting efficacy and side-effect profile of LY2940094, an antagonist of nociceptin receptors.

Nociceptin/Orphanin FQ (N/OFQ) is a 17 amino acid peptide whose receptor is designated ORL1 or nociceptin receptor (NOP). We utilized a potent, selective, and orally bioavailable antagonist with documented engagement with NOP receptors in vivo to assess antidepressant‐ and anxiolytic‐related pharmacological effects of NOP receptor blockade along with measures of cognitive and motor impingement. LY2940094 ([2‐[4‐[(2‐chloro‐4,4‐difluoro‐spiro[5H‐thieno[2,3‐c]pyran‐7,4′‐piperidine]‐1′‐yl)methyl]‐3‐methyl‐pyrazol‐1‐yl]‐3‐pyridyl]methanol) displayed antidepressant‐like behavioral effects in the forced‐swim test in mice, an effect absent in NOP −/− mice. LY2940094 also augmented the behavioral effect of fluoxetine without changing target occupancies (NOP and serotonin reuptake transporter [SERT]). LY2940094 did not have effects under a differential‐reinforcement of low rate schedule. Although anxiolytic‐like effects were not observed in some animal models (conditioned suppression, 4‐plate test, novelty‐suppressed feeding), LY2940094 had effects like that of anxiolytic drugs in three assays: fear‐conditioned freezing in mice, stress‐induced increases in cerebellar cGMP in mice, and stress‐induced hyperthermia in rats. These are the first reports of anxiolytic‐like activity with a systemically viable NOP receptor antagonist. LY2940094 did not disrupt performance in either a 5‐choice serial reaction time or delayed matching‐to‐position assay. LY2940094 was also not an activator or suppressor of locomotion in rodents nor did it induce failures of rotarod performance. These data suggest that LY2940094 has unique antidepressant‐ and anxiolytic‐related pharmacological effects in rodents. Clinical proof of concept data on this molecule in depressed patients have been reported elsewhere.

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.

N-(4-((2-(trifluoromethyl)-3-hydroxy-4-(isobutyryl)phenoxy)methyl)benzyl)-1-methyl-1H-imidazole-4-carboxamide (THIIC), a Novel Metabotropic Glutamate 2 Potentiator with Potential Anxiolytic/Antidepressant Properties: In Vivo Profiling Suggests a Link between Behavioral and Central Nervous System Neurochemical Changes

The normalization of excessive glutamatergic neurotransmission through the activation of metabotropic glutamate 2 (mGlu2) receptors may have therapeutic potential in a variety of psychiatric disorders, including anxiety/depression and schizophrenia. Here, we characterize the pharmacological properties of N-(4-((2-(trifluoromethyl)-3-hydroxy-4-(isobutyryl)phenoxy)methyl)benzyl)-1-methyl-1H-imidazole-4-carboxamide (THIIC), a structurally novel, potent, and selective allosteric potentiator of human and rat mGlu2 receptors (EC(50) = 23 and 13 nM, respectively). THIIC produced anxiolytic-like efficacy in the rat stress-induced hyperthermia assay and the mouse stress-induced elevation of cerebellar cGMP and marble-burying assays. THIIC also produced robust activity in three assays that detect antidepressant-like activity, including the mouse forced-swim test, the rat differential reinforcement of low rate 72-s assay, and the rat dominant-submissive test, with a maximal response similar to that of imipramine. Effects of THIIC in the forced-swim test and marble burying were deleted in mGlu2 receptor null mice. Analysis of sleep electroencephalogram (EEG) showed that THIIC had a sleep-promoting profile with increased non-rapid eye movement (REM) and decreased REM sleep. THIIC also decreased the dark phase increase in extracellular histamine in the medial prefrontal cortex and decreased levels of the histamine metabolite tele-methylhistamine (t-MeHA) in rat cerebrospinal fluid. Collectively, these results indicate that the novel mGlu2-positive allosteric modulator THIIC has robust activity in models used to predict anxiolytic/antidepressant efficacy, substantiating, at least with this molecule, differentiation in the biological impact of mGlu2 potentiation versus mGlu2/3 orthosteric agonism. In addition, we provide evidence that sleep EEG and CSF t-MeHA might function as viable biomarker approaches to facilitate the translational development of THIIC and other mGlu2 potentiators.

Pharmacological Characterization of cGMP Regulation by the Biarylpropylsulfonamide Class of Positive, Allosteric Modulators of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptors

The biarylpropylsulfonamide class of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) potentiators represented by N-2-(4-(4-cyanophenol)phenol)propyl-2-propanesulfonamide (LY404187) and (R)-4'-[1-fluoro-1-methyl-2-(propane-2-sulfonylamino)-ethyl]-biphenyl-4-carboxylic acid methylamide (LY503430) are positive, allosteric AMPA receptor activators, which enhance AMPA receptor-mediated neurotransmission by reducing desensitization of the ion channel. Although these compounds have efficacy in in vivo rodent models of cognition, depression, and Parkinson's disease, little is known about biochemical pathways activated by these agents. Given the well established regulation of the nitric oxide/cGMP pathway by excitatory neurotransmission, the current study characterized AMPA receptor potentiator-mediated cGMP response in mouse cerebellum. Acute treatment by both LY404187 and LY503430 [2.0, 5.0, or 10 mg/kg subcutaneously (s.c.)] elevated basal cerebellar cGMP levels in a dose-dependent manner. Pretreatment with the noncompetitive, allosteric AMPA receptor-selective antagonist 7H-1,3-dioxolo[4,5-h][2,3]benzodiazepine-7-carboxamide, 5-(4-aminophenyl)-8,9-dihydro-N,8-dimethyl-monohydrochloride-(9CI) (GYKI 53655) [3.0 mg/kg intraperitoneally (i.p.)], completely blocked the effect of LY404187, demonstrating that activation of AMPA receptors induces cGMP levels. Interestingly, pretreatment with the N-methyl-d-aspartate (NMDA) open channel blocker dizocilpine (0.3 and 1.0 mg/kg i.p.) also abolished the AMPA receptor potentiator-mediated cGMP accumulation, indicating that activation of AMPA receptors leads to NMDA receptor-mediated transmission involved in cGMP regulation. Pharmacological augmentation of the endogenous glutamate tone via the alkaloid harmaline (20-60 mg/kg i.p.) synergized with AMPA potentiator activity and provided further direct evidence of in vivo allosteric activation of AMPA receptors by LY404187. The synergism between harmaline and LY404187 was specific, since cGMP accumulation induced by foot-shock stress was not augmented by the AMPA receptor potentiator. Taken together, these data indicate that the cGMP system may play an important role in pharmacological efficacy of the biarylpropylsulfonamide class of AMPA receptor potentiators.

P.4.d.005 Pregabalin acts at calcium channel alpha2-delta type 1 proteins to prevent acute stress-induced increases in cerebellar cGMP

P.4.d.005 Pregabalin acts at calcium channel alpha2-delta type 1 proteins to prevent acute stress-induced increases in cerebellar cGMP

Dual effect of diazepam on cGMP levels in rat brain slices.

The effect of diazepam on NO-mediated cGMP synthesis was studied in rat brain slices. It was found that diazepam dose-dependently decreased cGMP synthesis in cerebellar slices, with an inhibition of 90% at 1 mM diazepam. cGMP levels in the presence of diazepam were not restored to control levels by the addition of 0.1 mM sodium nitroprusside, whereas the decrease in cerebellar cGMP levels induced by 0.1 mM L-NAME was restored by the simultaneous application of NO-donors. In addition to the decrease of cGMP levels in neuronal structures induced by 1 mM diazepam, we observed increased cGMP immunoreactivity in glial cells in the cerebellum, the hippocampus, and the cerebral cortex. The significance of this observation is discussed.

Sevoflurane MAC and Cerebellar Cyclic GMP

Department of Anesthesia and Critical Care; Massachusetts General Hospital; Boston, Massachusetts 02114;ichinose@etherdome.mgh.harvard.eduIn Reply:-I thank Drs. Haddad, Johns, and Pajewski for their interest and valuable comments regarding our recent article. [1] I have to admit that our speculation regarding the role of cyclic guanosine monophosphate (cGMP) in the mechanism of sevoflurane minimum alveolar concentration (MAC) reduction is based on rather weak evidence. I also agree with Haddad et al. regarding the possible development of compensatory mechanisms as we described in our discussion. However, we would like to point out that, although we found a correlation between the reduction of cGMP and sevoflurane MAC after acute 7-NI, there was a marked difference in the magnitude of reduction of the two parameters. Together with the dissociation of the two parameters after long-term 7-NI administration, these observations may suggest that the relation between cGMP and MAC is not linear. It is conceivable that only a small amount of nitric oxide or cGMP, or both, is necessary to maintain normal nociception. If this is the case, variation of nitric oxide synthase (NOS) activity or cGMP concentrations, or both, in the brain may not closely correlate with MAC of volatile anesthetics. I would also like to point out the limitation of the studies of our own [1] and others [2] that tested the effects of NOS inhibitors on anesthetic potencies and NOS activities. In two studies in which cGMP or NOS activity, or both, were measured after NOS inhibitor administration, cGMP concentration and NOS activity were not (and could not be) measured during tail pinch, but were measured in the cerebellum of unstimulated animals that had received the same dose of NOS inhibitors as those that had tail pinch. [1,2] Therefore, although cerebellar cGMP concentration and NOS activity should reflect those in the spinal cord (proposed nociceptive pathway in tail pinch, see discussion of Ichinose et al. [1]), [3] no data are available regarding NOS activity or cGMP concentrations during MAC measurements (tail pinch).Fumito Ichinose, M.D.Department of Anesthesia and Critical Care; Massachusetts General Hospital; Boston, Massachusetts 02114;ichinose@etherdome.mgh.harvard.edu(Accepted for publication December 4, 1998.)

Interaction of the α-2 adrenergic- and opioid receptor with the cGMP system in the mouse cerebellum

The α-2 adrenergic agonist dexmedetomidine (Dex), 3–300 μg/kg, i.p., decreased cerebellar cGMP in a dose-dependent manner. Fentanyl (F), an opioid agonist, increased cerebellar cGMP at 0.3 mg/kg, s.c., and decreased it at doses ≥1 mg/kg. The inhibitory effect was receptor specific, that of Dex being blocked by the α-2 adrenergic antagonist yohimbine, 5 mg/kg, i.p.; that of F by the opioid antagonist naloxone, 5 mg/kg, i.p. In contrast the stimulatory effect of F was blocked by both naloxone and yohimbine. Yohimbine also enhanced the inhibitory effect of F. In mice pretreated with pertussis toxin, 2 μg/mouse, given i.c.v. 72 h before the agonists, the decrease in cGMP induced by Dex or F was not affected, while the stimulatory effect of F was reversed to an inhibitory effect. When inhibiting doses of F and Dex were administered together, the cGMP response was smaller than the sum of the individual responses. Dex attenuated in a dose-dependent manner the decrease in cGMP induced by F, and unmasked or enhanced the stimulatory effect of F. These results show that the α-2 adrenergic- and opioid-receptors are coupled to the cGMP effector system and suggest that the two pathways converge at a common post-receptor site in the cascade of events transducing the receptor signal to cGMP regulation.

7-Nitroindazole, a selective inhibitor of neuronal nitric oxide synthase: effect on sevoflurane MAC and cerebellar cyclic GMP in mice.

Considerable evidence suggests that nitric oxide (NO) plays a role in synaptic transmission in the central and peripheral nervous systems. However, whether inhibition of NO synthesis decreases minimum alveolar concentration (MAC) of inhalational anesthetics is controversial. We examined the effects of 7-nitroindazole (7-NI), a selective inhibitor of neuronal NOS (nNOS), on the MAC of sevoflurane and cerebellar cyclic guanosine monophosphate (cGMP) levels in mice. Sevoflurane MAC and cerebellar cGMP levels were determined in mice after acute intraperitoneal or weeklong gavage feeding of 7-NI. Sevoflurane MAC and cerebellar cGMP levels after chronic treatment were measured on days 1, 4, and 7 and were repeated after an acute intraperitoneal dose of nitro g -L-arginine methylester (L-NAME). Acute and chronic treatment with 7-NI decreased the sevoflurane MAC by 20%-30%. Reduction of cerebellar cGMP levels was greater after intraperitoneal administration of NOS inhibitors than after gavage feeding of 7-NI. Acute or chronic selective inhibition of neuronal NOS decreases the sevoflurane MAC and cerebellar cGMP levels in mice. 7-NI permitted probing of the role of NO in perception of noxious stimuli.

Lack of correlation between the reduction of sevoflurane MAC and the cerebellar cyclic GMP concentrations in mice treated with 7-nitroindazole.

Although inhibition of nitric oxide synthase (NOS) has been reported to be antinociceptive and to reduce the threshold of general anesthesia, the mechanism of action is largely unknown. Specifically, the relation between the minimum alveolar concentration (MAC)-reducing effects of NOS inhibition and cyclic guanosine monophosphate (cGMP) concentrations in the brain has not been defined. To further characterize the effects of NOS inhibition, the authors studied the relation between the MAC of sevoflurane and the cGMP concentration of the brain after acute and chronic treatment with a neuronally selective NOS inhibitor, 7-nitroindazole (7-NI). Sevoflurane MAC and cerebellar cGMP concentrations were determined in mice after acute intraperitoneal administration or after 1, 2, 3, and 4 days of gavage feeding of 7-NI. After acute or chronic treatment with 7-NI, the mice were separated into two groups. Sevoflurane MAC was measured by a tail-clamp method in the first group, and cerebellar cGMP concentrations were measured by enzyme-linked immunosorbent assay in the second group of the mice. In mice, acute intraperitoneal administration of 7-NI dose dependently decreased sevoflurane MAC and cerebellar cGMP; and 4-day-long gavage feeding with 7-NI (500 mg/ kg, every 8 h) time dependently decreased cerebellar cGMP, but sevoflurane MAC was reduced only for the first 2 days and returned to its baseline after 3 days of 7-NI feeding. Although an acute selective inhibition of neuronal NOS decreases sevoflurane MAC and cerebellar cGMP concentrations in mice, there was a dissociation between the two parameters during long-term neuronal NOS inhibition. There may be cGMP-independent compensatory mechanisms that mediate nociception when NOS is chronically inhibited.

This Month in Anesthesiology

This Month in Anesthesiology

Suppression of cyclic guanosine monophosphate formation in rat cerebellar slices by propofol, ketamine and midazolam.

The nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) system is involved in glutamatergic neurotransmission. The current study determined the effects of propofol, ketamine and midazolam on rat cerebellar cGMP formation, attempting to clarify whether the effect was due to suppression of NO-cGMP system or to direct interaction with glutamatergic receptors. Cerebellar slices, obtained from six- to eight-day-old Wistar rats, were pretreated with propofol (10 microM-1 mM), ketamine (10-100 microM) or midazolam (1-100 microM) for 30 min. and then stimulated with L-glutamate (3 mM), N-methyl-D-aspartate (NMDA, 0.1 mM), kainate (0.1 mM) or sodium nitroprusside (SNP, 0.3 mM) (n = 5-11 for each group). The levels of cGMP were determined by radioimmunoassay. None of the anaesthetics studied altered cGMP levels when no stimulant was given. Propofol (10 microM-1 mM) suppressed L-glutamate-, NMDA-, kainate- and SNP stimulated cGMP formation in a concentration-dependent manner, the sensitivity to propofol was in the order of NMDA > kainate > L-glutamate. SNP. Ketamine (10-100 microM) suppressed L-glutamate- and NMDA-stimulated cGMP formation, but did not suppress kainate- or SNP-stimulated cGMP formation. Midazolam (10-100 microM) did not affect NMDA-, L-glutamate- or SNP-stimulated cGMP formation, but suppressed kainate-induced formation. The inhibitory effects of propofol, ketamine and midazolam on cGMP formation in rat cerebellar slices are due mainly to interaction with receptors for excitatory amines, and not due to the suppression of nitric oxide synthase or guanylate cyclase activities.

The Novel Anxiolytic U-101017: In Vitro and Ex Vivo Binding Profile and Effect on Cerebellar cGMP

The binding affinities ( K i) of U-101017 and diazepam for the GABA A receptor in rat cortical membranes were determined using [ 3H]flunitrazepam ([ 3H]FNZ) as the ligand. The inhibition constants of U-101017 and diazepam were 3.78 nM and 6.36 nM, respectively. Brain uptake of U-101017 was studied by the ex vivo [ 3H]FNZ binding assay. A significant ex vivo inhibition of [ 3H]FNZ binding was observed 10 min after oral administration of U-101017, and the effect lasted for at least 240 min (the last time point of investigation). The potential anxiolytic activity of U-101017 and diazepam was investigated in nonstressed and stressed (electric foot shock) mice by quantitative estimation of cerebellar cyclic 3′,5′-guanosine monophosphate (cGMP). Both U-101017 and diazepam dose-dependently decreased cGMP and attenuated stress-induced elevations in cGMP. These effects were antagonized by the GABA A receptor antagonist flumazenil. U-101017 was about two orders of magnitude more potent in stressed animals than in controls. The results of our investigation indicate that the anxiolytic-like activity of U-101017 is mediated via GABA A receptors.

Anxiolytic-like effects of PNU-101017, a partial agonist at the benzodiazepine receptor.

PNU-101017 is a chemically novel ligand at the benzodiazepine recognition site of cloned GABAA receptors. It was reported to potentiate GABA-mediated chloride current in cultured cells with a moderate intrinsic activity and a biphasic dose-response relationship. In this study, we confirmed that PNU-101017 has a partial agonist-like effect in the antagonism of metrazole-induced seizures in mice. It produced no sedation or ataxia, but did antagonize diazepam-induced motor deficit of mice in the rotarod test. PNU-101017 was weakly active in anti-conflict anxiolytic tests, but attenuated the plasma corticosteroid response to mild stress in rats. It also antagonized stress-induced elevation of cerebellar cGMP levels in mice. Like chlordiazepoxide, it increased drinking of saline solution in thirsty rats. PNU-101017 did not potentiate the CNS-depressant effects of ethanol, and produced no evidence of physical dependence when administered repeatedly. Agonists with low intrinsic activity at the benzodiazepine receptor, such as PNU-101017, should be further explored for therapeutic uses.

Regulation of cyclic GMP levels in the rat frontal cortex in vivo: effects of exogenous carbon monoxide and phosphodiesterase inhibition

A microdialysis method combined with a sensitive radioimmunoassay was used to monitor cGMP release in the frontal cortex of the anesthetized rats in vivo. We assessed the relative contribution of endogenous nitric oxide (NO), and effects of exogenous carbon monoxide (CO) and phosphodiesterase activity, as possible regulators of cortical CGMP levels. Perfusion with CO-saturated aCSF (≈1 mM CO) failed to significantly stimulate cortical cGMP levels. For comparison, cerebellar cGMP levels increased by 2-fold during CO stimulation, followed by a prolonged response that was fully reversible with the NO synthase inhibitor N G-nitro- l-arginine methyl ester ( l-NAME). Cortical perfusion with zinc protopophyrin-IX (100 μM), a widely used inhibitor of the CO-generating enzyme heme oxygenase, suppressed cGMP levels by 50%, a response that spontaneously recovered in spite of the continuous presence of the metalloporphyrin. Perfusion with isobutylmethyl xanthine IBMX (1 mM) resulted in 5-fold increase in cortical cGMP levels, as compared to basal levels without IBMX. In the presence of IBMX, l-NAME suppressed basal cortical cGMP levels by 70% indicating that NO synthase activity generates the bulk of cGMP in this brain region, as previously shown for basal cGMP production in the hippocampus and the cerebellum. These data also emphasize a crucial role for phosphodiesterase activity in the maintenance of cGMP levels in vivo in the frontal cortex. The relatively weak responses to exogenous CO lend little support for a role of this gas in regulating basal cortical cGMP levels in vivo.

Neuroprotective effects of the dopamine agonists pramipexole and bromocriptine in 3-acetylpyridine-treated rats

The neuroprotective effects of pramipexole, a dopamine agonist, were investigated in 3-acetylpyridine (3-AP)-treated Wistar rats. Bromocriptine was used as a reference compound to compare the results obtained with pramipexole. A significant reduction ( P<0.01) in cerebellar cGMP and ATP was observed 96 h after treatment with 3-AP (500 μmol/kg, i.p.). Both pramipexole and bromocriptine significantly attenuated 3-AP-induced reduction in cerebellar cGMP and ATP. Consistent with the neurochemical effect, both pramipexole and bromocriptine prevented 3-AP-induced loss of motor coordination. 3-Acetylpyridine produced a significant ( P<0.01) loss of neurons in the inferior olivary nucleus. Treatment with pramipexole and bromocriptine partially, but significantly ( P<0.01), prevented the loss of inferior olivary neurons. There was no reduction in the temperature of the animals, indicating that hypothermia was not responsible for neuroprotection. © 1997 Elsevier Science B.V. All rights reserved.

NMDA receptor-mediated increase in cyclic GMP in the rat cerebellum in vivo is blocked by alaproclate and GEA-857.

The effects of alaproclate and GEA-857 (2-(4-chlorophenyl)-1,1-dimethylethyl 2-amino-3-methylbutanoate) on the production of cyclic GMP in the rat cerebellum in vivo induced by stimulation of N-methyl-D-aspartate (NMDA) receptors were studied. Alaproclate per se at a dose of 20 mg/kg subcutaneously, did not influence the basal cGMP level. The increase in cGMP induced by harmaline (20 mg/kg subcutaneously) was dose-dependently antagonized by alaproclate (5-40 mg/kg subcutaneously). S-(-)-Alaproclate was 2-5 times more potent than the R-(+)-enantiomer. GEA-857 which in contrast to alaproclate is a very weak 5-HT uptake inhibitor shared the ability of alaproclate to inhibit the effect of harmaline on cGMP accumulation with similar potency to S-(-)-alaproclate. Alaproclate at 15 mg/kg subcutaneously blocked the increase in cGMP in cerebellum caused by NMDA itself at 200 mg/kg subcutaneously. In contrast to alaproclate, the K+ channel antagonist, 4-aminopyridine, 5 mg/kg subcutaneously, produced per se an increase in cGMP levels in the rat cerebellum by 300% which was antagonized by the NMDA receptor antagonists, dizocilpine, phencyclidine and (+/-)-CCP, the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester and by alaproclate. Alaproclate. Alaproclate and GEA-857 antagonized seizures induced by NMDA, 200 mg/kg subcutaneously at doses similar to those antagonizing the harmaline- and NMDA-induced elevation of cerebellar cGMP. Neither alaproclate nor GEA-857 caused any behavioural effects typical for uncompetitive NMDA receptor antagonists except a slight increase in motor activity and sniffing. The effect of alaproclate on the NMDA receptor-mediated increase in cGMP in rat cerebellum in vivo might be due to blockade of the cation channel of the NMDA receptor complex previously observed in in vitro experiments and these compounds seems to belong to the group of low-affinity uncompetitive NMDA receptor antagonists that might have clinical interest.

Involvement of glutamate and γ-aminobutyric acid (GABA)-ergic systems in thyrotropin-releasing hormone-induced rat cerebellar cGMP formation

The increase in cyclic guanosine 3′,5′-monophosphate (cGMP) caused by subcutaneous injection of thyrotropin-releasing hormone (TRH) tartrate was observed in a region-specific manner in the rat cerebellum. TRH tartrate (TRH-T) (2.8, 7.0 and 17 mg/kg as free TRH, s.c.) produced dose-dependent increases in cGMP levels markedly in the cerebellar superior and inferior vermis, and a smaller but still significant increase in the cerebellar hemispheres and brainstem but no significant increases in other brain regions. The TRH-induced increase in the cGMP level in the cerebellum was suppressed by pretreatment with muscimol, THIP (4,5,6,7-tetrahydroisoxazolo[5,4- c]pyridin-3(2 H)-one) or MK-801 (dizocilpine maleate) and partially suppressed by atropine but was not suppressed by chlordiazepoxide, oxazepam, phentolamine, propranolol, cyproheptadine, haloperidol, baclofen or DNQX (6,7-dinitroquinoxaline-2,3-dione), suggesting the possible involvement of GABA (γ-aminobutyric acid)(A)-ergic, N-methyl- d-aspartate (NMDA)-type glutamatergic and cholinergic systems. These results suggest that excitatory amino acids may be involved in the cGMP formation caused by TRH in the cerebellar areas, and that cGMP formation is inhibited by enhancement of GABA A receptor function.

Alpha-2 adrenoceptor agonists decrease cyclic guanosine 3',5'-monophosphate in the mouse brain.

In the central nervous system neurotransmitters, drugs or conditions that excite increase cyclic guanosine 3',5'-monophosphate (cGMP), an effect mediated by the neuromodulator nitric oxide, whereas those that sedate decrease cGMP. Volatile anesthetics were shown to decrease cerebellar cGMP, an effect that correlates with their anesthetic and anticonvulsant effect. Because alpha-2 adrenoceptor agonists have anesthetic properties, the role of the nitric oxide-cGMP pathway in the action of the alpha-2 adrenoceptor agonists clonidine and dexmedetomidine was investigated. Groups of mice were given, intraperitoneally, one dose of either 30-600 micrograms/kg clonidine, or 3-300 micrograms/kg D-medetomidine (dexmedetomidine) or L-medetomidine. The alpha-2 adrenoceptor antagonists, 0.3-5 mg/kg yohimbine or 1 mg/kg atipamezole, 1 mg/kg of the alpha-1 antagonist prazosin, and 10-300 mg/kg of the nitric oxide synthase inhibitors, N omega-nitro-1-arginine methylester and N omega-nitro-1-arginine, were given 10-20 min before the agonist. The mice were killed by microwave radiation focused to the head. Cyclic GMP was measured by radioimmunoassay in deproteinized extracts from different brain areas. Clonidine and dexmedetomidine, at sedative doses, dose-dependently decreased cerebellar cGMP (ED50: 100 and 50 micrograms/kg for clonidine and dexmedetomidine, respectively). This effect was inhibited by yohimbine and atipamezole, but not by prazosin, confirming the alpha-2 nature of the response to the agonists. L-medetomidine, which has no sedative/hypnotic effect, did not decrease cGMP. Pretreatment of the mice with a maximum dose of 100 mg/kg of a nitric oxide synthase antagonist abolished the cGMP response to the agonists. Similar results were obtained in the cerebral cortex, hippocampus and caudate nucleus. The results suggest that the nitric oxide-cGMP pathway is an effector system coupled to the alpha-2 adrenoceptor mediating sedation.