Glutamate-Stimulated, Guanine Nucleotide-Mediated Phosphoinositide Turnover in Astrocytes Is Inhibited by Cyclic AMP

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Abstract
The potential for cross-talk between the adenyl cyclase and phosphoinositide (PPI) lipid second messenger system was investigated in astrocytes cultured from neonatal rat brain. Glutamate-stimulated PPI turnover, measured by the formation of total inositol phosphates from myo-[3H]inositol-labeled lipids, was inhibited in a concentration-dependent manner by the elevation of intracellular cyclic AMP levels produced either by stimulation of the isoproterenol receptor linked to adenyl cyclase or by its direct activation by forskolin. N6,2''-O-Dibutyryl cyclic AMP, an analogue that can also acivate cyclic AMP-dependent kinase, inhibited glutamate-stimulated PPI turnover in a concentration-dependent manner as well, a result suggesting that cyclic AMP-dependent kinase is involved in mediating the inhibition. Inclusion of an inhibitor of cyclic AMP-dependent kinase, 1-(5-isoquinolinesulfonyl)-2 methylpiperazine dihydrochloride or N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride, blocked the cyclic AMP-mediated inhibition in a concentration-dependent manner, a finding further supporting this hypothesis. The site of inhibition of the phosphoinositol lipid pathway by cyclic AMP was probed using a digitonin-permeabilized cell system. Guanosine 5''-O-(3-thiotriphosphate), a nonhydrolyzable analogue of GTP, stimulated PPI turnover and potential glutamate-stimulated PPI turnover, and guanosine 5''-O-(3-thiodiphosphate) inhibited glutamate-stimulated PPI turnover in the cells, results providing evidence that glutamate receptors are coupled to phospholipase C by a guanine nucleotide binding protein in astrocytes. N6,-2''-O-Dibutyryl cyclic AMP and agents that elevate cyclic AMP levels inhibited the PPI turnover stimulated by guanosine 5''-O-(3-thiotriphosphate), as well as that potentiated by guanosine 5''-O-(3-thiotriphosphate) in the presence of glutamate, results suggesting that the cyclic AMP-dependent inhibition occurs at or distal to the putative guanine nucleotide binding protein. Because basal PPI turnover was not altered by elevation of cyclic AMP levels, direct inhibition of phospholipase C is unlikely.