Modulation of GABA release by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-D-aspartate receptors in matrix-enriched areas of the rat striatum.

Using a new in vitro superfusion device, the release of preloaded [3H]GABA was examined in microdiscs of tissues taken from sagittal slices in matrix-enriched areas of the rat striatum. Potassium (9 mM, 15 mM) stimulated the release of [3H]GABA in a concentration- and calcium-dependent manner and the veratridine (1 microM)-evoked release of [3H]GABA was completely abolished in the presence of tetrodotoxin (1 microM). The selective glutamatergic agonist alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (1 mM) enhanced the potassium-evoked release of [3H]GABA as well as the basal outflow of [3H]GABA. This latter effect was found to be calcium-dependent, partially diminished by tetrodotoxin (1 microM), completely blocked by 6,7-dinitro-quinoxaline-2,3-dione (0.1 mM), which is generally used as an antagonist of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors, but not affected by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK801, 10 microM), a specific antagonist of N-methyl-D-aspartate receptors. Similarly, N-methyl-D-aspartate (1 mM) enhanced both the potassium (9 mM) and the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (1 mM)-evoked release of [3H]GABA but when used alone, due to the presence of magnesium in the superfusion medium, was ineffective on the basal efflux of [3H]GABA. A stimulatory effect of N-methyl-D-aspartate (1 mM) on the basal outflow of [3H]GABA was observed, however, when magnesium was omitted from the superfusion medium. The stimulatory effect of N-methyl-D-aspartate (1 mM) observed in the presence of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate was not potentiated by glycine (1 microM, in the presence of strychnine 1 microM) and the N-methyl-D-aspartate-evoked response seen in the absence of magnesium was not enhanced by D-serine (1 mM), suggesting that endogenous glycine is already acting on N-methyl-D-aspartate receptors. In fact, in the absence of magnesium, 7-chloro-kynurenate (1 mM) completely abolished the stimulatory effect of N-methyl-D-aspartate on the release of [3H]GABA confirming that under our conditions, the glycine site of the N-methyl-D-aspartate receptor is saturated. N-methyl-D-aspartate-evoked responses were all blocked by MK801 (10 microM). Finally, the N-methyl-D-aspartate-evoked response seen in the absence of magnesium was markedly reduced in the presence of tetrodotoxin (1 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Complex involvement of nitric oxide and cGMP at N-methyl- -aspartic acid receptors regulating γ-[H]aminobutyric acid release from striatal slices

Whilst the depolarization of postsynaptic N-methyl- -aspartic acid (NMDA) receptors leads to an influx of Ca²⁺ and subsequent synthesis of nitric oxide (NO), we examined roles for NO at striatal NMDA receptors regulating transmitter release. In superfused rat striatal slices, NMDA-evoked release of γ-[³H]aminobutyric acid ([³H]GABA) was investigated in the presence of nitrergic drugs. NMDA-induced release of [³H]GABA was attenuated by -2-aminophosphonopentanoate, tetrodotoxin and omission of Ca²⁺. -Arginine enhanced NMDA-evoked release of [³H]GABA, but exogenous NO donors were ineffective. Inhibitors of NO synthase (N^G-nitro- and N^G-amino- -arginine) and guanylate cyclase (LY83583) elevated release. Since NMDA-evoked release of [³H]GABA was partially tetrodotoxin-sensitive, nitrergic-linked NMDA receptors regulating the release are both pre- and extrasynaptic. Thus not only does NO arise from multiple sites, and involve NMDA receptors with their redox site insensitive to exogenous NO donors, but the NMDA receptors are under the influence of nitrergic and cGMP-linked negative feedback mechanisms.     

Complex involvement of nitric oxide and cGMP at N-methyl-d-aspartic acid receptors regulating γ-[H]aminobutyric acid release from striatal slices

Whilst the depolarization of postsynaptic N-methyl-d-aspartic acid (NMDA) receptors leads to an influx of Ca²⁺ and subsequent synthesis of nitric oxide (NO), we examined roles for NO at striatal NMDA receptors regulating transmitter release. In superfused rat striatal slices, NMDA-evoked release of γ-[³H]aminobutyric acid ([³H]GABA) was investigated in the presence of nitrergic drugs. NMDA-induced release of [³H]GABA was attenuated by d-2-aminophosphonopentanoate, tetrodotoxin and omission of Ca²⁺. l-Arginine enhanced NMDA-evoked release of [³H]GABA, but exogenous NO donors were ineffective. Inhibitors of NO synthase (N^G-nitro- and N^G-amino-l-arginine) and guanylate cyclase (LY83583) elevated release. Since NMDA-evoked release of [³H]GABA was partially tetrodotoxin-sensitive, nitrergic-linked NMDA receptors regulating the release are both pre- and extrasynaptic. Thus not only does NO arise from multiple sites, and involve NMDA receptors with their redox site insensitive to exogenous NO donors, but the NMDA receptors are under the influence of nitrergic and cGMP-linked negative feedback mechanisms.     

Differential involvement of μ1-opioid receptors in endomorphin- and β-endorphin-induced G-protein activation in the mouse pons/medulla

Several genetic mouse models of differential sensitivity to opioids have been used to investigate the mechanisms underlying individual variation in responses to opioids. The CXBK mice are inbred recombinant mice which have a lower level of μ₁-opioid receptors than their parental strain. Endomorphin-1 and endomorphin-2 are endogenous opioid peptides that are highly selective for μ-opioid receptors, while β-endorphin, which is also an endogenous opioid peptide, is non-selective for μ-, δ- and putative -opioid receptors. The present study was designed to investigate the effects of these endogenous opioid peptides on G-protein activation by monitoring guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding to pons/medulla membranes of CXBK mice and their parental strain C57BL/6ByJ mice. Endomorphin-1 (0.1–10 μM), endomorphin-2 (0.1–10 μM) and β-endorphin (0.1–10 μM) increased guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding to the pons/medulla membranes from C57BL/6ByJ and CXBK mice in a concentration-dependent manner. However, the increases of guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding induced by either endomorphin-1 or endomorphin-2 in CXBK mice were significantly much lower than those in C57BL/6ByJ mice. However, no significant difference was found in the increases of the guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding induced by β-endorphin in C57BL/6ByJ and CXBK mice. Moreover, whereas the increase of guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding induced by 10 μM endomorphin-1 or endomorphin-2 were almost completely blocked by a μ-opioid receptor antagonist β-funaltrexamine (10 μM) in both strains, the increase of guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding induced by 10 μM β-endorphin was attenuated to approximately 70% of stimulation by co-incubation with 10 μM β-funaltrexamine in both strains. The residual stimulation of [³⁵S]guanosine-5′-o-(3-thio)triphosphate binding by 10 μM β-endorphin in the presence of 10 μM β-funaltrexamine was further attenuated by the addition of putative -opioid receptor partial agonist β-endorphin (1–27) (1 μM) in both strains. Like the endomorphins, the synthetic μ-opioid receptor agonist [ -Ala²,N-MePhe⁴,Gly-ol⁵]enkephalin at 10 μM showed lower increases of guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding in CXBK mice than those in C57BL/6ByJ mice. However, there was no strain difference in the stimulation of guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding induced by 10 μM of the selective δ₁-opioid receptor agonist [ -Pen^2,5]enkephalin, δ₂-opioid receptor agonist [ -Ala²]deltorphin II or κ-opioid receptor agonist U50,488H.The results indicate that the G-protein activation by endomorphin-1 and endomorphin-2 in the mouse pons/medulla is mediated by both μ₁- and μ₂-opioid receptors. Moreover, β-endorphin-induced G-protein activation in the mouse pons/medulla is, in part, mediated by μ₂- and putative -, but not by μ₁-opioid receptors.     

Apoptosis induced by β-N-oxalylamino-l-alanine on a motoneuron hybrid cell line

It has been suggested that β-N-oxalylamino-l-alanine, a non-protein amino acid present in the Lathyrus Sativus seeds, may play a role in the etiopathogenesis of neurolathyrism, a toxic form of motor neuron disease clinically characterized by a severe spastic paraparesis. In order to investigate the mechanisms of β-N-oxalylamino-l-alanine-mediated cell death, we studied the effect of this neurotoxin as well as other excitatory amino acids agonists on the growth and survival of motoneuron hybrid ventral spinal cord 4.1 cells. β-N-oxalylamino-l-alanine was toxic to ventral spinal cord 4.1 cells in a concentration-dependent fashion (0.5–10 mM). Among the excitatory amino acids tested, only glutamate (1–10 mM), quisqualate (1 mM) and, with less extent, β-N-methylamino-l-alanine (10 mM) induced a significant reduction of cell survival. The effect of Lathyrus Sativus neurotoxin was a slow process, becoming apparent only after 24–48 h of incubation. Interestingly, a mathematical analysis applied to the time course and dose curve of β-N-oxalylamino-l-alanine toxicity suggested that even for very low concentrations of the amino acid it is theoretically possible to predict a time-dependent effect. The cell death was not blocked by antagonists of N-methyl-d-aspartate or non-N-methyl-d-aspartate receptors; aurintricarboxylic acid and α-tocopherol gave a partial protection; cysteine (1 mM) prevented the toxic effect of both Lathyrus Sativus neurotoxin and glutamate as well as quisqualate. Morphologically, in the presence of either β-N-oxalylamino-l-alanine, glutamate or quisqualate, ventral spinal cord 4.1 cells showed apoptotic features also confirmed by ISEL technique and agarose gel electrophoresis of genomic DNA.Thus, our results suggest that in ventral spinal cord 4.1 motoneuron hybrid cells, in the absence of functional synaptic excitatory amino acid receptors, β-N-oxalylamino-l-alanine induces cell degeneration through an apoptotic mechanism, possibly mediated by a block of cystine/glutamate X⁻_c antiporter.     

Oocytes from Xenopus laevis contain an intrinsic σ2-like binding site

In preparation for expression studies for rat brain σ-binding sites, Xenopus oocytes were tested for the presence of [³H]di-o-tolylguanidine (DTG)-binding sites. Native oocytes were found to contain two intrinsic [³H]DTG-binding sites, a high-affinity site (K_d = 32 ± 6 nM, B_max of 45.7 ± 19 pmol/mg protein) and a low-affinity binding site (K_d = 1.3 ± 0.7 μM, B_max of 3.2 ± 0.7 nmol/mg protein). In a series of radioligand-binding-displacement studies, the high-affinity binding sites were found to have a binding profile which has a similar K_d to that of the mammalian σ₂-binding site (32 vs. 38 nM). Comparison of the IC₅₀ values for inhibition of [³H]DTG binding in rat liver and oocytes for DTG, haloperidol (HAL), (−)-pentazocine, (+)-3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride ((+)-3-PPP), (+(-pentazocine and Zn²⁺, showed similarity in rank (r² = 0.913) but a 7-fold lower potency in oocytes. These results suggest that the high-affinity [³H]DTG-binding site in oocytes represents a σ₂-like binding site.     

Sulphur-containing excitatory amino acid-stimulated inositol phosphate formation in primary cultures of cerebellar granule cells is mediated predominantly by N-methyl-d-aspartate receptors

The stimulatory effect of excitatory sulphur-containing amino acids on inositol phosphate formation was investigated in primary cultures of cerebellar granule cells. l-Cysteine sulphinate (CSA), l-cysteate (CA), l-homocysteine sulphinate (HCSA), l-homocysteate (HCA) and S-sulpho-l-cysteine (SSC) dose-dependently stimulated the formation of [^u3H]inositol phosphates exhibiting EC₅₀ values in the range 60–200 μM and maximal effects of six- to 17-fold that of basal [³H]inositol phosphate levels. Endogenous l-glutamate spontaneously released into the extracellular medium or following exposure of cells to HCSA, HCA or SSC did not contribute significantly to formation of [³H]inositol phosphates, whereas 10% of the total [³H]inositol phosphates accumulated following exposure to CSA and CA was due to released l-glutamate. The selective N-methyl-d-aspartate receptor antagonist, d,l-2-amino-5-phosphonopentanoic acid (APV, 500 μM) attenuated by 20% (HCSA) to between 80 and 100% (CSA, CA, SSC, HCA) the formation of [³H]inositol phosphates induced by 1 mM sulphur-containing amino acids. When, however, HCSA was used at 100 μM (a concentration near to its EC₅₀ for phosphoinositide hydrolysis), APV inhibited induced responses by 70%. Sulphur-containing amino acid-stimulated [³H]inositol phosphate formation was unaffected by the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μM). Inhibition of sulphur-containing amino acid-stimulated [³H]inositol phosphate formation by co-administration of APV and CNQX was similar to that obtained in the presence of APV alone. CSA-, CA-, SSC- and HCA-stimulated [³H]inositol phosphate formation was markedly reduced by removal of Ca²⁺ from the extracellular medium whereas that stimulated by HCSA was less affected. A similar inhibitory profile was observed when the levels of sulphur-containing amino acid-induced increases in intracellular free calcium ([Ca²⁺₁) were measured in the presence of 500 μM APV; 1 mM HCSA-induced responses being inhibited by only 30% whereas responses to the remaining sulphur-containing amino acid (also at 1 mM) were inhibited by >45%. When the sulphur-containing amino acids were used at concentrations approximating their EC₅₀ values for phosphoinositide hydrolysis, APV inhibited the induced increases in [Ca²⁺]_i by 70–100%. HCA and SSC co-administered with the less efficacious but selective metabotropic glutamate receptor agonist, ( ± )-1-aminocyclopentane-trans-1,3-dicar☐ylic acid (trans-ACPD) at maximally effective concentrations (1 mM) of each agonist stimulated [³H]inositol phosphate formation in an additive manner. In the case of CSA, CA and HCSA, however, co-administration with trans-ACPD caused no increase in level of [³H]inositol phosphate formation compared to that observed in the presence of either sulphur-containing amino acid alone. In cells pre-treated with 1 μg/ml pertussis toxin, HCA-stimulated [³H]inositol phosphate formation was not significantly affected, however CSA-, CA- and HCSA-stimulated [³H]inositol phosphate formation was significantly but only partially (by 40%) inhibited. Taken together, these results indicate that in cerebellar granule cells sulphur-containing amino acid-stimulated [³H]inositol phosphate formation is mediated predominantly by N-methyl-d-aspartate receptor activation. There is evidence however to support an additional role for CSA, CA and particularly HCSA as agonists of the metabotropic glutamate receptor.The data are consistent with the sulphur-containing amino acids possibly having a physiological role as endogenous activators of both ionotropic and metabotropic excitatory amino acid receptors. The present findings may, however, be of more significance in suggesting possible mechanisms by which the sulphur-containing amino acids or disturbances in sulphur-containing amino acid metabolism are linked to the aetiology of a number of neuropathologies.     

Cerebellar excitatory amino acid binding sites in normal, granuloprival, and Purkinje cell-deficient mice.

Using quantitative autoradiography, the cellular localization and characterization of cerebellar excitatory amino acid binding sites in normal, Purkinje cell-deficient and granuloprival (granule cell-deficient) mouse cerebella were investigated. In the molecular layer of normal mouse cerebellum, the quisqualate subtype of excitatory amino acid receptor (assayed by [3H](RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate, quisqualate-sensitive L-[3H]glutamate, and [3H]6-cyano-7-nitroquinoxaline-2,3-dione binding) predominated. In the granule cell layer of the cerebellum, N-methyl-D-aspartate-sensitive L-[3H]glutamate and [3H]glycine binding sites were predominant. In the molecular layer of Purkinje cell-deficient mutant mice, [3H](RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate binding sites and [3H]6-cyano-7-nitro-quinoxaline-2,3-dione binding were reduced to 24% (P less than 0.01) and 36% (P less than 0.001) of control, respectively, while quisqualate-sensitive [3H]glutamate binding sites were reduced to 54% of control (P less than 0.01). N-Methyl-D-aspartate-sensitive [3H]glutamate and [3H]glycine binding were unchanged. In the granule cell layer of these mouse cerebella, there was no change in excitatory amino acid receptor binding. In the molecular layer of granuloprival mouse cerebella, [3H](RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate binding was increased to 205% of control (P less than 0.01), [3H]6-cyano-7-nitro-quinoxaline-2,3-dione binding was increased to 136% of control (P less than 0.02), and quisqualate-sensitive [3H]glutamate binding was increased to 152% of control (P less than 0.01). N-Methyl-D-aspartate-sensitive [3H]glutamate and [3H]glycine binding were unchanged. In areas of granule cell depletion N-methyl-D-aspartate-sensitive [3H]glutamate and [3H]glycine binding were reduced to 68% (P less than 0.01) and 59% (P less than 0.01) of control, respectively. In the granule cell layer, binding to quisqualate receptors was not significantly different from binding in controls with any of the ligands tested. These results suggest that three different receptor assays: [3H](RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate, quisqualate-sensitive L-[3H]glutamate, and [3H]6-cyano-7-nitro-quinoxaline-2,3-dione binding can be used to demonstrate that quisqualate receptor specific binding sites are located on Purkinje cell dendrites in the molecular layer of cerebellum, and that these binding sites apparently up-regulate in response to granule cell ablation and Purkinje cell deafferentation.     

Potassium-evoked release of [14C]GABA and [3H]taurine from rat olfactory bulb slices

Rat olfactory bulb slices were preloaded with [³H] taurine or with [¹⁴C] GABA. Upon stimulation of the slices with increasing concentrations of KCl, we observed release of [³H] taurine or [¹⁴C] GABA. Superfusion of the slices with high concentrations of K⁺ in the absence of Ca²⁺ in the perfusion medium, led to a marked decrease in the stimulated release of both [³H] taurine and [¹⁴C] GABA.     

Glufosinate ammonium stimulates nitric oxide production through N-methyl -aspartate receptors in rat cerebellum

Glufosinate ammonium, a structural analogue of glutamate, is an active herbicidal ingredient. The neuronal activities of this compound were investigated by use of a microdialysis system that allowed us to measure nitric oxide production in the rat cerebellum in vivo. Kainate (0.3–30 nmol/10 μl), N-methyl- -aspartate (NMDA) (3–300 nmol/10 μl) and glufosinate ammonium (30–3000 nmol/10 μl), which were administered through the microdialysis probe at a rate of 1 μl/min for 10 min, stimulated nitric oxide production. The glufosinate ammonium-elicited increase in nitric oxide production was suppressed by an inhibitor of nitric oxide synthase and was antagonized by NMDA receptor antagonists, but not by a kainate/(±)-αamino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist. These results suggest that glufosinate ammonium stimulates nitric oxide production through NMDA receptors.     

K+-evoked Ca2+-dependent release of d-[3H]aspartate from terminals of the cortico-pontine pathway

Pontine nuclei disseted from rat brain slices released previously accumulated d-[³H]aspartate (d-Asp) and [¹⁴C]γ-aminobutyrate (GABA) Ca-depentlytly exposed to 50 mM K. These efflux rates were substantially increased by including 0.5 mg/ml bovine serum albumin in the superfusion fluid. Degeneration of the cortico-pontine fibres 5 days after cutting the crus cerebri caused an 80% reduction in the fractional rate of Ca-dependent d-Asp release and a 60% reduction in uptake. The fractional rate of GABA efflux was significantly less reduced than that of d-Asp efflux, and GABA uptake was nearly unchanged.     

Effects of nigral application of muscimol on release of [3H]γ-aminobutyrate and on multi-unit activity in various cat thalamic nuclei

The release of [³H]γ-aminobutyrate (GABA) neosynthesized from [³H]glutamine was estimated in one substantia nigra and in the ipsilateral thalamus of halothane-anesthetized cats by perfusing a [³H]glutamine-enriched physiological medium through a push-pull cannula implanted in the two structures under investigation. After two hours of superfusion, muscimol (10^?6 M) was delivered through the nigral push-pull cannula for 50–60 min and local- and distal-evoked changes of [³H]GABA release were analyzed. In some experiments, changes of global neuronal activity induced by muscimol application were recorded in different thalamic nuclei, using a bipolar electrode. In a few of the above experiments, biochemical and electrophysiological determinations were simultaneously performed in the substantia nigra and the thalamus. The nigral application of muscimol (10^?6 M) induced locally an activation of the substantia nigra reticulata cells, as well as an increase in release of [³H]GABA.Distally, in the thalamus, two types of biochemical and electrophysiological responses were observed according to the localization of the tip of the push-pull cannula or the electrode. (1) An increased release of [³H]GABA and a depression of the global multi-unit cellular activity were obtained in the ventralis medialis-ventralis lateralis, the centralis lateralis and the paracentralis nuclei. These effects could reflect an activation of the GABAergic nigrothalamic neurons projecting to these different thalamic nuclei. (2) In contrast, in the medialis dorsalis paralamellar zone adjacent to the intralaminar nuclei of the thalamus, a decrease of [³H]GABA release and an activation of the multi-unit activity were obtained. These latter results may suggest either a polysynaptic response or the non-GABAergic nature of the nigrothalamic neurons afferent to the medialis dorsalis paralamellar zone.     

Dissociation between changes in glutamate receptor binding sites and their coupling to phosphatidylinositol metabolism following intrahippocampal colchicine injection

Intrahippocampal colchicine injection produces a rapid death of granule cells and pyramidal neurons in the hippocampus in the rat. Under the appropriate assay conditions, [3H]glutamate labels the N-methyl-D-aspartate type of glutamate receptors while [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate labels the quisqualate type. Unilateral injection of colchicine (15 micrograms) in the dorsal hippocampus did not produce any change in [3H]glutamate and [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate binding in membrane fractions from the dentate gyrus or CA1 field contralateral to the injection side, at least up to 12 days after the injection. However, it produced a progressive decrease in the binding of both ligands in dentate gyrus and CA1 of the injected hippocampus. In the dentate gyrus the changes in binding as a function of time after the injection were biphasic with a rapid exponential decrease (t1/2 about 8 days for both [3H]glutamate and [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) until 12 days after the injection followed by a much slower decrease afterwards. A similar pattern was observed in CA1 although the changes in binding were smaller and delayed by about three days as compared to the dentate gyrus. Kinetic analyses of the binding at equilibrium were performed seven days after the injection and indicated that the changes in [3H]glutamate binding were due to a change in the maximum number of sites but not in affinity for the ligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

The phosphodiesterase III inhibitor olprinone inhibits hippocampal glutamate release via a cGMP/PKG pathway

Olprinone, an inhibitor of cyclic nucleotide phosphodiesterase III, inhibited an increase in intracellular Ca²⁺ concentrations for acutely dissociated rat hippocampal pyramidal neurons induced by extracellular high K⁺ (35 mM) depolarization. Olprinone (100 μM) significantly reduced spontaneous glutamate release from rat hippocampal slices. Furthermore, olprinone significantly decreased the rate of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated miniature excitatory postsynaptic currents (AMPA-mEPSCs) monitored from CA1 pyramidal neurons of rat hippocampal slices, and the effect was blocked by KT5823, an inhibitor of protein kinase G (PKG), but not by H-89, an inhibitor of protein kinase A (PKA). In the PKA assay using PC-12 cells, olprinone did not activate PKA. Taken together, the results of the present study show that olprinone attenuates intracellular Ca²⁺ rise through voltage-sensitive Ca²⁺ channels and inhibits presynaptic glutamate release via a cGMP/PKG pathway.     

Selectivity of a series of clonidine-like drugs for α1 and α2 adrenoceptors in rat brain

The relative selectivity of a series of imidazoline derivatives and substituted guanidines structurally related to clonidine for α₁ and α₂ adrenoceptors has been examined in binding studies using membranes prepared from rat cerebral cortex and the radioligands [³H]prazosin and [³H]clonidine. There was a 150-fold difference between the most and least selective clonidine-like drugs in their relative affinity for α₁ and α₂ adrenoceptors.     

Effects of N-methyl-d-aspartate (dizocilpine) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (LY215490) receptor antagonists on the voiding reflex induced by perineal stimulation in the neonatal rat

The present study was undertaken to examine the role of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and N-methyl-d-aspartate glutamate receptors in the regulation of voiding reflexes induced by perineal stimulation in the neonatal rat. Four-, six- and 10-day-old awake rats were used in the experiments and perineal stimulation was applied using the tip of a 1-ml syringe to evoke voiding. Voided volume and residual volume were measured. In 10-day-old rats, LY215490 (3–10 mg/kg, i.p.), a competitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor antagonist, significantly inhibited reflex voiding, increasing the residual volume 34–53-fold. A 3 mg/kg dose decreased the urine release by 55%, whereas 10 mg/kg totally suppressed the voiding reflex induced by the perineal stimulation. LY215490 (10 mg/kg, i.p.) produced similar effects in four- and six-day-old rats. Dizocilpine (1–3 mg/kg, i.p.), a non-competitive N-methyl-d-aspartate receptor antagonist, also significantly decreased the urine release (62–82%) and increased residual volume (180–230-fold). Combined administration of LY215490 (1 mg/kg, i.p.) and dizocilpine (0.3 mg/kg, i.p.) to 10-day-old rats, in doses that individually had no effect on perineal stimulation-evoked voiding, depressed voided volume by 65%.These results indicate that, in neonatal rats, glutamatergic transmission in the spinal cord has an essential role in reflex micturition induced by perineal stimulation, and that facilitatory interactions between α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and N-methyl-d-aspartate glutamatergic mechanisms are important for voiding, as noted previously in adult rats.     

Relation of exocytotic release of γ-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the -aminobutyric acid (GABA) carrier, NNC-711, {1-[(2-diphenylmethylene) amino]oxyethyl}-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride, blocks the Ca²⁺-independent release of [³H]GABA from rat brain synaptosomes induced by 50 mM K⁺ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca²⁺-dependent release of [³H]GABA in the total absence of carrier-mediated release. Reversal of the Na⁺/Ca²⁺ exchanger was used to increase the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) to test whether an increase in [Ca²⁺]_i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca²⁺]_i may rise to values above 400 nM, as a result of Na⁺/Ca²⁺ exchange, without inducing release of [³H]GABA, but subsequent K⁺ depolarization immediately induced [³H]GABA release. Thus, a rise of only a few nanomolar Ca²⁺ in the cytoplasm induced by 50 mM K⁺ depolarization, after loading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange, induced exocytotic [³H]GABA release, whereas the rise in cytoplasmic [Ca²⁺] caused by reversal of the Na⁺/Ca²⁺ exchanger was insufficient to induce exocytosis, although the value for [Ca²⁺]_i attained was higher than that required for exocytosis induced by K⁺ depolarization. The voltage-dependent Ca²⁺ entry due to K⁺ depolarization, after maximal Ca²⁺ loading of the synaptosomes by Na⁺/Ca²⁺ exchange, and the consequent [³H]GABA release could be blocked by 50 M verapamil. Although preloading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange did not cause [³H]GABA release under any conditions studied, the rise in cytoplasmic [Ca²⁺] due to Na⁺/Ca²⁺ exchange increased the sensitivity to external Ca²⁺ of the exocytotic release of [³H]GABA induced by subsequent K⁺ depolarization. Thus, our results show that the vesicular release of [³H]GABA is rather insensitive to bulk cytoplasmic [Ca²⁺] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca²⁺ entry through Ca²⁺ channels near the active zones.     

Neurosteroid regulation of inhibitory synaptic transmission in the rat hippocampus in vitro

The effect of the neurosteroid dehydroepiandrosterone sulfate on inhibitory synaptic transmission was studied in area CA1 of the rat hippocampus using an in vitro hippocampal slice preparation. Synaptic responses elicited by stimulation of Schaffer collateral fibers were recorded extracellularly as population spikes in the somatic region and as synaptic field potentials in the dendritic region. Bath application of dehydroepiandrosterone sulfate (10 μM) enhanced the synaptically evoked somatic population spike with no effect on the dendritic synaptic potential. Isolation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor-mediated component of the synaptic response by addition of antagonists of N-methyl- -aspartate and GABA receptors to the perfusion saline demonstrated that dehydroepiandrosterone sulfate had no effect on this component of the dendritic synaptic potential. In contrast, dehydroepiandrosterone sulfate antagonized GABA receptor-mediated inhibitory effects in the somatic region, resulting in an augmentation of the somatic population spike amplitude. Paired-pulse facilitation was unaltered by dehydroepiandrosterone sulfate, thus arguing against possible presynaptic sites of dehydroepiandrosterone sulfate's actions.These results indicate that dehydroepiandrosterone sulfate can alter synaptic transmission in the hippocampus through selective postsynaptic actions on inhibitory synaptic transmission. A synaptic effect of dehydroepiandrosterone sulfate is consistent with a neuromodulatory role for this neurosteroid in the central nervous system, and may contribute to the reported effects of dehydroepiandrosterone sulfate on cognitive processes such as learning and memory.     

Cholecystokinin releases [3H]GABA from the perfused subarachnoid space of the anaesthetized rat spinal cord

The neuropeptide cholecystokinin(26-33) (CCK) is widely distributed in the mammalian central nervous system, including the spinal cord. We have studied the possible interaction of CCK with GABA release mechanisms. Low doses of CCK-8 (1 nM) have been found to evoke calcium-dependent [3H]GABA release from an in vivo perfused spinal cord preparation in the anaesthetized rat. Tachyphylaxis was seen to the [3H]GABA releasing action of CCK-8. The injection of proglumide (150 mg/kg i.p.) totally blocked the [3H]GABA release produced by CCK-8 or by a medium containing 50 mM potassium. Substance P (10 microM) did not produce release of [3H]GABA, although in the same animals 50 mM potassium containing solutions could be shown to evoke release of [3H]GABA.