Pharmacological heterogeneity of NMDA receptors in cerebellar granule cells in immature rat slices. A microfluorimetric study with the [Ca2+]i sensitive dye Indo-1.

We have developed a technique for monitoring the internal calcium concentration--[Ca2+]i--in a single selected cell in cerebellar slices of 8-day-old rats. In post-migratory granule cells NMDA induced a rapid, reversible and concentration-dependent increase in [Ca2+]i (+22% at 10 microM and +210% at 100 microM). This effect was dependent on the presence of Ca2+ in the extracellular medium. When Mg2+ was absent from the perfusion buffer the sensitivity to NMDA was greatly increased (+108% at 10 microM). The NMDA response was not affected by glycine site agonists or by tetrodotoxin, a sodium channel blocker. In the absence of magnesium, the NMDA (10 microM)-induced increase in [Ca2+]i was prevented in a monophasic manner by the recognition site antagonist 2-amino-phosphonovalerate (2-APV; IC50 = 13 microM), and in a biphasic manner by the glycine site antagonist 7-chlorokynurenate (IC50s = 25 nM and 5.9 microM) and by the channel blocker dizocilpine (IC50s = 5 nM and 3 microM). In contrast, this NMDA response was only partially antagonized by the polyamine site antagonists ifenprodil and eliprodil (maximal inhibition approximately 50% at concentrations > or = 10 microM, IC50 1 and 2 microM, respectively). These results demonstrate the presence in granule cells from immature rat cerebellum of at least two NMDA receptor populations which can be differentiated by their sensitivity to inhibitors acting upon the different sites of the NMDA receptor complex.

Pharmacology of excitatory amino acid receptors coupled to inositol phosphate metabolism in neonatal rat striatum.

The production of inositol phosphate from tritiated inositol was studied in striatal slices of neonatal rats. The excitatory amino acids glutamic, aspartic, quisqualic and ibotenic acids directly stimulated the formation of inositol phosphate in this system. The receptor mediating the response to quisqualic acid bore important pharmacological differences to the quisqualate-preferring receptor described electrophysiologically, notably in its insensitivity to antagonists. The receptor mediating the response to ibotenic acid bears no resemblance to any receptor subtype defined electrophysiologically; l -serine -O- phosphate is an antagonist of this response. N-methylaspartic acid selectively depressed the stimulation of inositol phosphate formation evoked by carbachol in a calcium dependent manner. The receptor mediating the inhibitory response to N-methylaspartate appears pharmacologically similar to the NMDA-preferring receptor defined electrophysiologically.

Peripheral benzodiazepine binding sites: effect of PK 11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide. I. In vitro studies.

[3H] RO5-4864 binding sites have been characterized in kidney, heart, brain, adrenals and platelets in the rat. In all these organs the following order of potency in the RO5-4864 displacement was found: RO5-4864 greater than diazepam greater than clonazepam indicating that they correspond to the "peripheral type" of benzodiazepine binding sites. PK 11195, an isoquinoline carboxamide derivative, displaces [3H] RO5-4864 from its binding sites in all the organs. PK 11195 was as potent as RO5-4864 in the platelets, heart, adrenals, kidney and several brain regions (midbrain, hypothalamus, medulla + pons and hippocampus. However it was 5 to 10 times more effective in cortex and striatum. In conclusion PK 11195 might represent a new tool to elucidate the physiological relevance of "peripheral type" benzodiazepine binding sites and might help to discriminate the hypothetical subclasses of these binding sites.

Differentiation between two ligands for peripheral benzodiazepine binding sites, [3H]RO5-4864 and [3H]PK 11195, by thermodynamic studies.

The [3H]PK 11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinolinecarboxamide, binding sites in rat cardiac membranes are saturable, with high affinity, specific GABA-independent and correspond to the peripheral type of benzodiazepine. The order of potency of displacing agents was: PK 11195 greater than RO5-4864 greater than dipyridamole greater than diazepam greater than clonazepam. The Bmax obtained with [3H]PK 11195 was equivalent of the Bmax obtained with [3H]RO5-4864 in the same experimental conditions. However thermodynamic analysis indicates that the [3H]PK 11195 binding was entropy driven whereas the [3H]RO5-4864 binding was enthalpy driven. Consequently PK 11195 might be an antagonist of these binding sites and RO5-4864 an agonist or a partial agonist. The simultaneous use of both drugs might help to elucidate the physiological relevance of peripheral benzodiazepine binding sites.

Dihydropyridine- and omega-conotoxin-resistant, neomycin-sensitive calcium channels mediate the depolarization-induced increase in internal calcium levels in cortical slices from immature rat brain.

In order to characterize pharmacologically voltage-operated calcium channels in the rat brain, we have developed a technique to measure intracellular calcium levels ([Ca++]i) in immature rat cortical slices loaded with the fluorescent calcium probe Fura-2. KCl depolarization caused a rapid and reversible increase in cortical [Ca++]i. A significant increase was already observed at 20 mM KCl and the maximal effect was obtained at 77 mM. This response was not modified when extracellular Na+ was substituted by the nonpermeant cation bis(2-hydroxyethyl)-dimethylammonium chloride and was insensitive to the Na+ channel blocker tetrodotoxin (1 microM). In the absence of extracellular Ca++, KCl (50 mM) failed to increase [Ca++]i. The KCl (50 mM)-induced increase in [Ca++]i was not affected by the L-type calcium channel blockers nifedipine and isradipine and was only partially inhibited (by less than 30% at 50 microM) by verapamil and diltiazem. In contrast, nimodipine prevented this response by 41% at 50 microM. Flunarizine (a nonselective T channel blocker) inhibited the KCl response by 47% at 30 microM, whereas nicergoline (another nonselective T channel blocker) reduced this entry by 74% at 300 microM (IC50 = 120 microM). Cyclandelate, an atypical calcium antagonist, inhibited KCl-induced increase in [Ca++]i with a maximal effect of 41% at 30 microM, whereas perhexiline was inactive. The KCl-induced rise in [Ca++]i was only marginally inhibited by omega-conotoxin with a maximal effect of 20% from 1 nM to 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)