Cerebellar cGMP Research Articles

Neuroprotective Effects of the Pyrrolopyrimidine U-104067F in 3-Acetylpyridine-Treated Rats

The neuroprotective effects of U-104067F [(9-(2-morpholinyl)ethyl)2,4-di-l-pyrrolidinyl-9H-pyrimidino(4,5-b)indole monohydrochloride hydrate], a pyrrolopyrimidine antioxidant, were investigated in 3-acetylpyridine (3-AP)-treated Wistar rats. A significant (P<0.01) reduction in cerebellar cGMP and ATP was observed at 96 h after treatment with 3-AP (500 μmol/kg ip). Oral administration of U-104067F significantly attenuated 3-AP-induced reductions in cGMP and ATP, and this effect was dose related. Consistent with the neurochemical effect, U-104067F 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 U-104067F partially, but significantly (P<0.01), prevented the loss of inferior olivary neurons. The physiological and neurochemical parameters were maintained within normal limits by U-104067F in 3-AP-treated rats despite only partial preservation of inferior olivary neurons.

Comparative dopaminergic and muscarinic antagonist activity of clozapine and haloperidol

Clozapine is an atypical antipsychotic drug, and its dopamine and muscarinic antagonist activity has been compared with haloperidol in rodents. Elevation in rat striatal acetylcholine (ACh) and mice cerebellar cGMP has been used as an agonist response for oxotremorine and quinpirole. Pretreatment with clozapine significantly blocked oxotremorine-induced elevation in striatal ACh ( p < 0.01) and cerebellar cGMP ( p < 0.05). At the same doses, clozapine had no significant effect on quinpirole-induced increases in ACh and cGMP levels. Pretreatment with haloperidol significantly antagonized quinpirole-induced elevation in striatal ACh ( p < 0.01) and cerebellar cGMP ( p < 0.05), and haloperidol had no significant effect on oxotremorine-induced agonist responses. Thus, clozapine is antimuscarinic at a dose level that lacks dopamine antagonist properties, whereas haloperidol is a dopamine antagonist and lacks antimuscarinic activity. The atypical neuroleptic profile of clozapine may be due to its high antimuscarinic and low antidopaminergic activity.

Determination of brain nitric oxide synthase inhibition in vivo: Eex vivo assays of nitric oxide synthase can give incorrect results

The in vivo potencies of N ω-nitro- l-arginine ( l-NA), N ω-monomethyl- l-arginine ( l-NMMA) and N ω-iminoethyl- l-ornithine ( l-NIO) against brain nitric oxide synthase (NOS) were determined by assessing their ability to inhibit harmaline-induced increases in rat cerebellar cGMP. l-NA, l-NIO and l-NMMA were all able to completely prevent the harmaline-induced increase in cGMP with ID 50s of 0.5,30 and 55 mg/kg, respectively, and with the same order of potency as that seen for inhibition of cerebellar NOS in vitro. The inhibitory effects of low doses of l-NA on cerebellar cGMP were maintained for at least 8 hr. The ID 50 of l-NA for inhibition of cerebellar cGMP in vivo was similar to its ID 50 for inhibition of cerebellar NOS ex vivo but only when NOS activity was assayed as an initial rate. However, doses of l-NMMA and l-NIO that inhibited harmaline-induced increases in cerebellar cGMP in vivo by 50% failed to inhibit NOS ex vivo. The methyl ester of l-NA, l-NAME, produced substantial inhibition of cerebellar NOS ex vivo when given either orally, intraperitoneally or intravenously but with a slower onset of action than l-NA. These results demonstrate that measurement of NOS activity ex vivo can accurately reflect the degree of inhibition of NOS in vivo with inhibitors that dissociate slowly from the enzyme such as l-NA, but only when the initial rate of NOS activity is measured.

Differential regulation of cyclic GMP levels in the frontal cortex and the cerebellum of anesthetized rats by nitric oxide: an in vivo microdialysis study

A microdialysis method combined with a sensitive radioimmunoassay was used to monitor extracellular cyclic GMP (cGMP) levels in the frontal cortex and the cerebellum of anesthetized rats in vivo. Basal cGMP release remained constant throughout the perfusion period and was ≈ 2 fmol/30 min in the frontal cortex and ≈ 4 fmol/30 min in the cerebellum. The nitric oxide (NO) donor sodium nitroprusside (SNP) stimulated cGMP release transiently in both regions. However, the maximal response was 3-fold in the frontal cortex (obtained with 5 μM SNP) but 90-fold in the cerebellum (obtained with 1 mM SNP). Perfusion with the NO synthase (NOS) inhibitor N G-nitro- l-arginine methyl ester ( l-NAME) suppressed cerebellar cGMP release by 74% indicating that NO is the major regulator of basal cGMP levels in the cerebellum. Quite opposite, l-NAME exhibited no potency in the frontal cortex suggesting that other activators of guanylyl cyclase may regulate basal cortical cGMP levels in vivo.

Methodological variables during analysis of in vivo cerebellar cyclic GMP, an indirect marker of nitric oxide release

In light of the recent recognition of the physiological significance of nitric oxide, there is considerable interest in the methodological variables that can confound the results of the cerebellar cGMP analysis from in vivo experiments. In this study, using male Swiss Webster mice, the effect of such methodological variables as 1) weight of the animals; 2) tissue extraction procedures used in radioimmunoassay for cGMP; and 3) the commercial source of the assay kit on, harmaline-, pentylenetetrazole- or SNAP-induced increase in cerebellar cGMP in vivo were evaluated. Results indicate that mice in the 15- to 19-g weight range are most sensitive and best suited for in vivo drug effects on cerebellar cGMP. Furthermore, for the extraction of cerebellar cGMP, use of ice-cold 0.5 N hydrochloric acid and subsequent dilution of the sample in assay buffer is the simplest and fastest method. Present data also indicate that the source of the radioimmunoassay kit has a significant effect on the cerebellar cGMP results. Based on the present results, the protocol developed and the guidelines drawn are timely and of high practical significance for research in the area of pharmacology of nitric oxide.

Cerebellar nitric oxide synthase, cGMP and motor function in two lines of cerebellar mutant mice, Staggerer and Wriggle Mouse Sagami

We investigated the hypothesis that nitric oxide (NO) is involved in the cerebellar motor function, by measuring nitric oxide synthase (NOS) activities and cGMP in the cerebellum using two lines of mutant mice having motor dysfunction, Staggerer (SG) and Wriggle Mouse Sagami (WMS). In SG, the NOS activity per cerebellum was reduced to 5.8% of that of the controls, while no significant change was observed in WMS. The cerebellar cGMP in SG was reduced to 3.3% of that of the controls and to 43% in WMS. In contrast with these neurochemical markers of NO, the locomotor dysfunction and the number of falls were greater in WMS than in SG. The reductions of the neurochemical markers of NO are consistent with the results of the previous neuropathological studies in SG and WMS whereas the cerebellar motor dysfunction was independent of these neurochemical and neuropathological changes.

Involvement of granule, basket and stellate neurons but not purkinje or Golgi cells in cerebellar cGMP increases in vivo

Recent immunocytochemical studies of cerebellar nitric oxide synthase (NOS) and cGMP have aided dramatically in defining possible cellular sources of cGMP generation in the signal transduction cascade evoked by excitatory amino acids in the cerebellum. Using a mouse mutant deficient in cerebellar Purkinje cells (“nervous” mouse) and chemical lesions of cerebellar neurons with methylazoxymethanol (MAM), we have examined in vivo generation of cGMP to determine the roles of different cerebellar neuronal populations. In the case of “nervous” mice, our data indicate that cerebellar Purkinje cells are not required for NMDA-dependent increases in cGMP in the cerebellum. In marked contrast, MAM lesions which reduce granule but not Golgi cells in the granule cell layer and reduce basket and stellate cells in the molecular layer, dramatically reduced the ability of NMDA to increase cerebellar cGMP. These data support immunocytochemical data of cerebellar NOS pools and indicate the importance of granule, basket and possibly stellate cells in the generation of nitric oxide, which in turn activates guanylate cyclase, in a diversity of cells, to increase cerebellar cGMP levels.

Chronic administration of a nitric oxide synthase inhibitor, N omega-nitro-L-arginine, and drug-induced increase in cerebellar cyclic GMP in vivo.

N omega-nitro-L-arginine (NG-nitro-L-arginine) is a potent nitric oxide synthase inhibitor which crosses the blood brain barrier and does not undergo extensive metabolism in vivo. In this study, effect of chronic pretreatment of N omega-nitro-L-arginine (75 mg/kg, i.p., twice daily for 7 days) on the harmaline- (100 mg/kg, s.c.), picrotoxin- (4 mg/kg, s.c.), pentylenetetrazole- (50 mg/kg, i.p.), and L-glutamic acid- (400 micrograms/10 microliters/mouse, i.c.v.) induced increase in cerebellar cGMP was assessed. All the four drugs produced significant increase in cerebellar cGMP in vehicle pretreated control animals. Cerebellar cGMP increased induced by harmaline, picrotoxin, and L-glutamic acid was attenuated in N omega-nitro-L-arginine pretreated animals. These results indicate that in vivo cerebellar cGMP levels are increased by the prototype excitatory amino acid receptor agonist, L-glutamic acid and also by the drugs which augment the excitatory amino acid transmission. Furthermore, parenteral chronic administration of N omega-nitro-L-arginine blocks NO synthase in the brain and hence cerebellar cGMP response in chronic N omega-nitro-L-arginine treated animals could be used as a tool to assess the physiological functions of nitric oxide in vivo.

Neurobehavioural effects of SR 27897, a selective cholecystokinin type A (CCK-A) receptor antagonist.

The activity of SR 27897, a potent and selective CCK-A vs CCK-B receptor antagonist (Ki = 0.2 nM on guinea-pig pancreas vs 2000 nM on rat brain) was studied on behavioural, electrophysiological and biochemical effects induced by peripheral or central injection of CCK-8S. For comparative purposes, devazepide, a reference CCK-A receptor antagonist, was investigated in these same models. CCK-induced hypophagia and CCK-induced hypolocomotion in rats, two behavioural changes associated with the stimulation of peripheral CCK-A receptors, were dose-dependently antagonized by SR 27897 (ED50 = 0.003 and 0.002 mg/kg i.p., respectively) and devazepide (ED50 = 0.02 and 0.1 mg/kg i.p., respectively). CCK-induced decrease of cerebellar cGMP levels in mice was also reduced by SR 27897 (ED50 = 0.013 mg/kg) and by devazepide (0.084 mg/kg). The CCK-induced turning behaviour after intrastriatal injection in mice, and the potentiation of the rate suppressant activity of apomorphine on rat DA neurons, were blocked by higher doses of SR 27897 and devazepide, consistent with the probable central origin of these effects. The respective ED50s were 0.2 mg/kg i.p. for SR 27897 and 4.9 mg/kg i.p. for devazepide in the former model, while the respective minimal effective doses were 1.25 and 5 mg/kg i.p. in the latter test. In most tests the i.p./p.o. ratio for SR 27897 was near unity, suggesting a high oral bioavailability of the compound. Taken together, these findings support the notion that SR 27897 behaves as a potent CCK-A antagonist able to cross the blood brain barrier.

NMDA-, but not kainate- or quisqualate-dependent increases in cerebellar cGMP are dependent upon monoaminergic innervation

As previously reported, intracerebellar injectors of D-serine, quiaqualate and kainate elevated mouse cerebellar cGMP levels. Similarly, activation of endogenous excitatory amino acid utilizing neurons with harmaline or pentylenetetrazole also increased cerebellar cGMP. We previously have demonstrated that the harmaline- and pentylenetetrazol-dependent cGMP increases are NMDA receptor mediated. In this report we further demonstrated that NMDA-dependent increases in cGMP are dependent upon monoaminergic innervation of the cerebellum, while kainate- and quisqualate-dependent cGMP increases are independent of such innervation.

Excitatory amino acid signal transduction in the hippocampus: Role of noradrenergic afferents and nitric oxide in cGMP increases [formula omitted

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 α1-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.

Experimental absence seizures: potential role of gamma-hydroxybutyric acid and GABAB receptors.

We have investigated whether the pathogenesis of spontaneous generalized non-convulsive seizures in rats with genetic absence epilepsy is due to an increase in the brain levels of gamma-hydroxybutyric acid (GHB) or in the rate of its synthesis. Concentrations of GHB or of its precursor gamma-butyrolactone (GBL) were measured with a new GC/MS technique which allows the simultaneous assessment of GHB and GBL. The rate of GHB synthesis was estimated from the increase in GHB levels after inhibition of its catabolism with valproate. The results of this study do not indicate significant differences in GHB or GBL levels, or in their rates of synthesis in rats showing spike-and-wave discharges (SWD) as compared to rats without SWD. Binding data indicate that GHB, but not GBL, has a selective, although weak affinity for GABAB receptors (IC50 = 150 microM). Similar IC50 values were observed in membranes prepared from rats showing SWD and from control rats. The average GHB brain levels of 2.12 +/- 0.23 nmol/g measured in the cortex and of 4.28 +/- 0.90 nmol/g in the thalamus are much lower than the concentrations necessary to occupy a major part of the GABAB receptors. It is unlikely that local accumulations of GHB reach concentrations 30-70-fold higher than the average brain levels. After injection of 3.5 mmol/kg GBL, a dose sufficient to induce SWD, brain concentrations reach 240 +/- 31 nmol/g (Snead, 1991) and GHB could thus stimulate the GABAB receptor. Like the selective and potent GABAB receptor agonist R(-)-baclofen, GHB causes a dose-related decrease in cerebellar cGMP. This decrease and the increase in SWD caused by R(-)-baclofen were completely blocked by the selective and potent GABAB receptor antagonist CGP 35348, whereas only the increase in the duration of SWD induced by GHB was totally antagonized by CGP 35348. The decrease in cerebellar cGMP levels elicited by GHB was only partially antagonized by CGP 35348. These findings suggest that all effects of R(-)-baclofen are mediated by the GABAB receptor, whereas only the induction of SWD by GHB is dependent on GABAB receptor mediation, the decrease in cGMP being only partially so. Taken together with the observations of Marescaux et al. (1992), these results indicate that GABAB receptors are of primary importance in experimental absence epilepsy and that GABAB receptor antagonists may represent a new class of anti-absence drugs.

Role of cGMP in the mechanism of anxiolytic activity of U-78875

The inhibition constant (K i) of U-78875 was investigated without and with mus incubation medium using in vitro ( 3H)-flunitrazepam [( 3H)-FNZ] binding to coritical membrane preparation. Also, the effect of U-78875 on cerebellar cyclic 3′,5′-guanosine monophosphate (cGMP) was studied in control and stressed (electric footshock) mice. The K i of U-78875 was 1.56 nM for inhibition of ( 3H)-FNZ binding. The presence of muscimol (10 −5 M) had no significant effect on the K i of U-78875. U-78875 and diazepam significantly decreased cerebellar cGMP, and this effect was antagonized by flumazenil. Both U-78875 and diazepam dose-dependently antagonized electric footshock-induced increases in cGMP, and U-78875 was two orders of magnitude more potent in stressed animals as compared to control animals. These biochemical investigations indicate that U-78875 is an agonist of benzodiazepine receptors, and cGMP may mediate its anxiolytic activity.

Neurochemical Interactions of Competitive N‐Methyl‐D‐Aspartate Antagonists with Dopaminergic Neurotransmission and the Cerebellar Cyclic GMP System: Functional Evidence for a Phasic Glutamatergic Control of the Nigrostriatal Dopaminergic Pathway

Direct intrastriatal injection of N-methyl-D-aspartate (NMDA; 100 micrograms/rat) increased striatal dopamine (DA) release in vivo. However, parenteral administration of (+/-)-3-(2-carboxypiperizin-4-yl)propyl-1-phosphonic acid (CPP) and cis-4-phosphonomethyl-2-piperidine carboxylic acid (CGS-19755) did not alter DA metabolism and release in several brain regions in the rat and mouse. Intracerebroventricular administration of the competitive NMDA antagonists CPP, CGS-19755, 2-amino-5-phosphonopentanoate, and 2-amino-7-phosphonoheptanoate did not alter rat striatal DA metabolism and release but profoundly reduced cerebellar cyclic GMP (cGMP) levels in the same animals. CPP and CGS-19755 decreased basal cerebellar cGMP levels in the mouse with ED50 values of 6 and 1 mg/kg, i.p., respectively. CPP antagonized the harmaline-induced increases in cGMP levels with an ED50 value of 5.0 mg/kg, i.p. CPP (25 mg/kg, i.p.) also decreased basal cGMP levels in mouse cerebellum for up to 3 h, a result suggesting brain bioavailability and a long duration of NMDA receptor antagonism in vivo. These contrasting patterns suggest that NMDA receptors exert a tonic excitatory tone on the guanine nucleotide signal transduction pathway in the cerebellum while exerting a phasic control over nigrostriatal dopaminergic neurotransmission. These results also indicate that competitive NMDA antagonists, unlike phencyclidine receptor agonists, may not mediate biochemical and behavioral effects via dopaminergic mechanisms.

Opipramol, a potent sigma ligand, is an anti-ischemic agent: Neurochemical evidence for an interaction with the [formula omitted] receptor complex in vivo by cerebellar cGMP measurements

Opipramol, a potent sigma ligand and a tricyclic antidepressant compound, provided significant neuronal protection ( P < 0.0001) against ischemia-induced neuronal cell loss in the hippocampus in Mongolian gerbils, at a dose of 50 mg/kg (30 min pretreatment). However, opipramol did not oner protection when given 60 min after the ischemic insult. Opipramol decreased basal levels of cGMP in the cerebellum of the mouse and harmaline-induced increases in levels of cGMP, with approximate ED 50 values of 4 and 27 mg/kg. Opipramol antagonized methamphetamine- and pentylenetetrazol-induced increases in levels of cGMP. Parenteral administration of opipramol also antagonized the increases in levels of cGMP in the cerebellum of the mouse after the local administration of D-serine, an agonist at the N- methyl- D-aspartate (NMDA)-associated, strychnine-insensitive glycine receptor. These results indicate that opipramol attenuates responses mediated through the NMDA receptor complex. These results further support the functional modulation of the NMDA receptor complex by sigma ligands and provide a neurochemical correlate for the observed anti-ischemic properties of opipramol.

Glycine, glycinamide and d-serine act as positive modulators of signal transduction at the N-Methyl-D-Aspartate (NMDA) receptor [formula omitted]: Differential effects on mouse cerebellar cyclic guanosine monophosphate levels

Direct intracerebellar (icb) administration of glycine, glycinamide and D-serine produced time- and dose-dependent changes in mouse cerebellar cGMP levels, indicating a modulation of ongoing neuronal activity through the NMDA receptor complex. Intracerebro-ventricular administration of glycinamide also produced a timedependent change in cGMP levels, indicating a central mechanism of action. The icb dose-response data indicated a unimolecular interaction for these compounds. D-serine-, glycine-, and glycinamide-mediated increases in cGMP levels were reversed by the competitive NMDA antagonist, CPP and the NMDA-associated glycine receptor antagonist, HA-966, indicating mediation via the NMDA receptor complex. Glycine and D-serine were less effective than glycinamide at increasing cerebellar cGMP levels. In contrast, L-and D-serinamide did not affect cGMP levels. These results indicate that glycine receptor is not saturated under physiological conditions and also suggest possible existence of multiple glycine pools.

Inhibition of Nitric Oxide Synthase Blocks N‐Methyl‐D‐Aspartate‐, Quisqualate‐, Kainate‐, Harmaline‐, and Pentylenetetrazole‐Dependent Increases in Cerebellar Cyclic GMP In Vivo

The synthesis of nitric oxide by brain slices has been demonstrated in several laboratories. In addition, in vitro studies have demonstrated stimulation of nitric oxide synthesis by excitatory amino acid receptor agonists. These data have led to the hypothesis that this readily diffusible "intercellular messenger molecule" acts to generate a cascade effect by activating guanylate cyclase in several cell types and thereby augment levels of the second messenger cyclic GMP (cGMP). Therefore, we evaluated this hypothesis in vivo, by testing the actions of the nitric oxide synthase inhibitor N-mono-methyl-L-arginine (NMMA) on elevations in level of mouse cerebellar cGMP generated by excitatory amino acid receptor agonists. The stimulatory effects of D-serine, quisqualate, and kainate were all found to be antagonized by this enzyme inhibitor. In addition, NMMA antagonized the increases in cerebellar cGMP level elicited by harmaline and pentylenetetrazole, pharmacological agents that augment endogenous excitatory amino acid transmission. Our data are, therefore, the first in vivo demonstration that nitric oxide is an important "messenger molecule" in the cerebellum, mediating the actions of kainate, quisqualate, and N-methyl-D-aspartate receptor agonists on guanylate cyclase. These data are consistent with previous in vitro findings with kainate and N-methyl-D-aspartate.

Neuropharmacological characterization of 1-Aminocyclopropane-1-carboxylate and 1-Aminocyclobutane-1-carboxylate, Ligands of the N-Methyl-D-Aspartate-associated Glycine Receptor

Following intravenous administration, 1-aminocyclobutane-1-carboxylate (ACBC, 100 mg/kg), a N-methyl-D-aspartate (NMDA)-associated glycine receptor antagonist, was eliminated with a T 1/2 of 5 min in mouse brain and 4 min in rat cerebrospinal fluid (CSF). 1-Aminocyclopropane-1-carboxylate (ACC), a NMDA-associated glycine receptor agonist, was found to have a T 1/2 of less than 5 min in mouse brain. ACC and ACBC did not alter basal cerebellar cGMP. Glycine and D-serine increased cGMP, and 1-hydroxy-3-aminopyrrolidone-2 (HA-966), a glycine antagonist, reversed the D-serine-induced increases in cGMP. In contrast, ACBC did not reverse the D-serine-induced increases in cGMP. These data suggest that despite their brain bioavailability and marked potency at the glycine receptor in vitro. ACC and ACBC are rapidly inactivated and thus have limited in vivo utility.

The polyamines, spermine and spermidine, negatively modulate N-methyl-d-aspartate (NMDA) and quisqualate receptor mediated responses in vivo: Cerebellar cyclic GMP measurements

The effects of the polyamines, spermine and spermidine on basal and d-serine-, harmaline- and quisqualate-induced cyclic GMP (cGMP) were measured in mouse cerebellum. Spermine and spermidine at 200 ?g/mouse, intracerebellar injection (icb), did not alter basal cGMP levels. d-Serine (200 ?g/mouse, icb) and quisqualic acid (5 ?g/mouse, icb) caused 5- and 15-fold increases in cGMP. These increases were significantly reversed by co-injected spermine and spermidine (200 ?g/mouse, icb). Furthermore, direct intracerebellar spermidine (200 ?g) completely reversed harmaline (100 mg/kg, sc) induced increases in cGMP. These data indicate that the polyamines, spermine and spermidine attenuate responses mediated through the N-methyl-D-aspartate (NMDA), NMDA-associated glycine receptor and quisqualate receptors. These results provide an in vivo neurochemical evidence for polyamine modulation of excitatory amino acid receptors. Due to their putative abilities to increase mitochondrial uptake of [Ca(+2)], spermine and spermidine are likely to modulate the responses of several excitatory amino acid agonists. The polyamines spermine and spermidine are widely distributed in neural and non-neural tissues and have been shown to play a key role in cell differentiation and growth (Kremzner et al ., 1970; Harik and Snyder, 1974; Raina and Janne, 1975; Seiler, 1981; Pegg and McCann, 1982). Extensive studies have indicated an important role of the polyamines, spermine and spermidine, in the regulation of intracellular [Ca(+2)] levels (Nicchitta and Williamson, 1984; Iqbal and Koenig, 1985; Jensen Lynch and Baudrey, 1987; 1989 a, b). The precise regulation of intracellular [Ca(+2)] levels and maintenance of [Ca(+2)] gradients across the cell membranes are essential in cell survival as prolonged and excessive exposure to calcium can result in neuronal injury and death (Farber, 1981; Rasmussen and Barnett, 1984). The excitatory amino acid glutamate is known to interact with three subclasses of receptors, N-methyl-D-aspartate (NMDA), quisqualate and kainate (Cotman and Iversen, 1987). The role of excitatory amino acids in the etiology of ischemic neuronal death is also well established (Cotman and Iversen, 1987). Although the precise mechanisms of ischemic injury are unknown, excessive release of glutamate and/or other endogenous excitatory amino acid(s) which result(s) in massive influx of [Ca(+2)] into neurons (Garthwhite et al ., 1986; Choi et al ., 1987) is considered in important event. Due to the pivotal role played by polyamines in the regulation of intracellular [Ca(+2)] levels, it is likely that polyamines may have an important role in the pathophysiology of ischemic injury. Recent in vitro evidence suggests that the NMDA receptor complex has recognition sites for the polyamines, spermine and spermidine, besides the putative sites for glycine, phencyclidine (PCP), and divalent ions such as [Mg(+2)] and [Zn(+2)] (Ransom and Stec, 1988). Recently, ifenprodil, a novel NMDA receptor antagonist, was shown to interact with the polyamines (Reynolds and Miller, 1989) which may explain its unique pharmacological profile. However, the precise functional interactions of polyamines with NMDA and other excitatory amino acids are unknown at this time. The present investigation was aimed at elucidating the in vivo functional interrelationships between polyamines and the excitatory amino acids in general, and with the NMDA receptor complex in particular, by examining the effects of spermine and spermidine on mouse cerebellar cGMP levels, a well characterized excitatory amino acid receptor-mediated second messenger response (Wood et al ., 1982; 1987; 1989; Rao et al ., 1989).

Increased response of cerebellar cGMP to kainate but not NMDA or quisqualate following barbital withdrawal from dependent rats

Female Sprague-Dawley rats were maintained on a diet of powdered food containing barbital for 8 weeks before the drug was abruptly withdrawn. Twenty-four hours later both barbital-dependent and control rats were injected intracerebroventricular (i.c.v.) with saline or one of four doses of kainic acid (KA) or in a separate experiment with saline or one of three doses of N-methyl-D-aspartic acid (NMDA) or of quisqualic acid (QA). After 4.5 min, the animals were killed by focused microwave irradiation, and the cerebella were collected. The levels of cyclic guanosine 3′,5′ monophosphate (cGMP) were markedly elevated in the cerebella of barbital-withdrawn rats when compared to controls. When compared to saline treatment, KA, at all dosages, resulted in a significantly greater elevation of cerebellar cGMP in the barbital-withdrawn rats than was induced by drug withdrawal alone. Only the two higher dosages of KA produced a significant elevation of this parameter in the control rats. Unlike KA, neither QA or NMDA produced any greater elevations of cGMP in barbital withdrawn rats than were induced by drug withdrawal alone. These collective results suggest that there is an increase in the response to KA but not QA or NMDA following the withdrawal of barbital from dependent rats.