The effects of the cyclic nucleotide cAMP on r-aminobutyric acid-gated chloride channel function were investigated. The membrane-permeant cAMP analog N',02'-dibutyryladenosine 3',5'-cycic monophosphate inhibited muscimol-induced 36C-uptake into rat cerebral cortical synaptoneurosomes in a concentration-dependent manner (IC5. = 1.3 mM). The inhibition was due to a decrease in the maximal effect of muscimol, with no change in potency. Similar effects were observed with 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, 8-bromoadenosine 3',5'-cyclic monophosphate, and the phosphodiesterase inhibitor isobutylmethylxan- ATP (9,10). Although both studies (9,10) suggested that an ATP-dependent process, possibly phosphorylation, is involved in the maintenance of GABAA receptor function, no evidence for the role ofcAMP or cGMP in that regulatory mechanism was provided. Indirect evidence for the regulation of the GABAA receptor complex by phosphorylation was obtained in studies in which the activation of protein kinase C decreased GABA-induced Clconductance in Xenopus oocytes injected with chicken forebrain mRNA (11).In the absence of evidence that cyclic nucleotides, such as cAMP, regulate the function ofthe GABA-gated Cl-channel, we have investigated the effects of (i) membrane-permeant cAMP analogs and (il) forskolin-induced endogenous cAMP accumulation on muscimol-stimulated 36Cl-uptake in an intact vesicular preparation (synaptoneurosomes) from rat cerebral cortex. Our findings indicate that cAMP analogs and forskolin decrease the functional activity of the GABAA receptor complex. However, we suggest that the main actions of forskolin on the GABA-gated chloride channel are independent of its ability to generate cAMP by activation of adenylate cyclase. These findings have been reported in preliminary form (12). MATERIALS AND METHODSPreparation of Synaptoneurosomes. Synaptoneurosomes (13) were prepared from adult male Sprague-Dawley rats (150-225 g; Zivic Miller) as described by Schwartz et al. (14) with minor modifications. Brains were removed rapidly after decapitation and placed immediately in ice-cold buffer (pH 7.4) containing 10 mM glucose, 20 mM Hepes, 9 mM Tris, 118 mM NaCl, 4.7 mM KCl, 1.18 mM MgSO4, and 1.0 mM CaCl2.Cerebral cortices were dissected free of white matter and homogenized (five strokes by hand) in 7 vol (wt/vol) of buffer by using a glass-glass homogenizer. The homogenate was diluted with 30 ml of buffer and filtered by gravity through three layers of nylon mesh (160 ,um, Tetko, Elmsford, NY).Abbreviations: GABA, y-aminobutyric acid; IBMX, 3-isobutyl-1-methylxanthine; Bt2cAMP, N6,02'-dibutyryladenosine 3',5'-cyclic monophosphate.
The Mongolian gerbil, with its spontaneous epileptiform seizures, was chosen as an experimental model of human epilepsy. Neurochemical parameters possibly related to the seizure process were studied. In the immediate seizure process amino acid profiles of cortex, hippocampus, and striatum were not different in seizuring animals when compared to seizure-resistance controls. Of two peptides analyzed, only somatostatin appeared elevated in the cortex 2 hr postictal (143 fmol/mg protein; controls, 123 fmol/mg protein); neuropeptide Y was not affected. A follow up of the time course of cyclic AMP and cyclic GMP showed significant elevations of both substances as a consequence of seizures. Most prominent was a 5.5-fold increase in cyclic GMP in the cerebellum 30 sec after seizure onset.
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