INHIBITION OF CARBACHOL-STIMULATED PHOSPHOINOSITIDE TURNOVER BY U-50,488H IN RAT HIPPOCAMPUS - INVOLVEMENT OF GTP-BINDING PROTEIN

The effect of U-50,488H, a selective kappa-opioid agonist, on carbachol-stimulated phosphoinositide (PI) turnover response in rat hippocampal slices was examined. U-50,488H which stimulates PI turnover response in this preparation (Periyasamy and Hoss, 1990, Life Sci. 47, 219), inhibited carbachol-stimulated PI turnover in a concentration-dependent manner with an IC50 value of 33 +/- 9.0-mu-M. The inhibitory effect of U-50,488H was not blocked by the kappa-selective antagonists, e.g., nor-binaltorphimine (10-mu-M), and MR2266 (10-mu-M), or tetrodotoxin (1-mu-M) suggesting that the effect of U-50,488H was mediated neither through the kappa-receptors nor through the release of an endogenous neurotransmitter(s). A Lineweaver-Burke plot of the stimulation of PI turnover by carbachol in the presence and absence of U-50,488H showed that the K(m) was not changed (11.4 +/- 3.4 and 11.5 +/- 2.6-mu-M) whereas the V(max) was reduced from 3849 +/- 460 to 1534 +/- 31 cpm indicating that the inhibition was non-competitive. U-50,488H also inhibited guanosine 5'-[beta,gamma-imido]triphosphate (Gpp[NH]p)-stimulated PI turnover in rat hippocampal membranes in a concentration-dependent manner with an IC50 value of 33 +/- 12-mu-M. However, U-50,488H had no effect on exogenously added phospholipase-C-stimulated PI turnover. In receptor binding studies, U-50,488H displaced the non-selective muscarinic antagonist [H-3]quinuclidinyl benzilate ([H-3]QNB) binding in a concentration-dependent manner with an IC50 value of 4.5 +/- 1.5-mu-M. A Scatchard plot of [H-3]QNB binding in the presence and absence of U-50,488H showed that the K(d) was changed from 883 +/- 250 to 364 +/- 147 pM whereas the B(max) remained the same (1.66 +/- 0.16 and 1.65 +/- 0.18 pmol/mg protein) suggesting that the inhibition was competitive. These data indicate that the inhibitory effect of U-50,488H on carbachol-stimulated PI turnover was mediated through the interaction with the G-protein but not through kappa-receptors or release of neurotransmitter(s).