INHIBITION OF MUSCARINIC RECEPTOR-INDUCED INOSITOL PHOSPHOLIPID HYDROLYSIS BY CAFFEINE, BETA-ADRENOCEPTORS AND PROTEIN-KINASE-C IN INTESTINAL SMOOTH-MUSCLE

1 The effects of caffeine, isoprenaline, dibutyryl cyclic AMP, isobutylmethylxanthine (IBMX), 12-O-tetradecanoylphorbol-13-acetate (TPA) or 1-oleoyl-2-acetylglycerol (OAG), (protein kinase C (PKC) activators), 2-methoxy verapamil (D600), thapsigargin and ryanodine on muscarinic acetylcholine receptor (AChR)-stimulated inositol phospholipid hydrolysis were studied in smooth muscle fragments from the longitudinal layer of the small intestine of the guinea-pig. 2 Incubation of the fragments with the muscarinic agonist, carbachol (CCh) (100 mu M) resulted in rapid increases in the levels of all the inositol phosphate isomers with maximal increases in the [H-3]-inositol (1,4,5) trisphosphate ([H-3]-Ins(1,4,5)P-3) isomer occurring 10 s following incubation. 3 The beta-adrenoceptor agonist, isoprenaline (10 mu M) and dibutyryl cyclic AMP (10 mu M), a membrane permeant analogue of cyclic AMP both reduced the CCh stimulation, but not the basal levels of [H-3]-inositol phosphates. This inhibition by dibutyryl cyclic AMP was enhanced in the presence of the phosphodiesterase inhibitor, IBMX. CCh inhibited the isoprenaline-induced increases in the levels of cyclic AMP and this was via a pertussi toxin (PTX)-sensitive G-protein mechanism. 4 TPA (1 mu M) and OAG (100 mu M) a 1,2-diacylglycerol (DAG) analogue both reduced the CCh-induced increases in [H-3]-inositol phosphates levels but neither affected basal values nor the basal levels of cyclic AMP. 5 D600 (10 mu M), which blocks voltage-dependent Ca2+ channels, also reduced the CCh-stimulated levels of [H-3]-inositol phosphates suggesting that some of the agonist-induced increases are due to a potentiating effect of Ca2+ entering the cell. 6 Caffeine (0.5-30 mM) significantly inhibited both the basal and CCh-induced increases in all the [H-3]-inositol phosphate isomers. Its inhibitory action was not due to increases in cyclic AMP since caffeine had no effect on the levels of cyclic AMP at concentrations up to 30 mM. 7 Incubation with thapsigargin (1 mu M) and ryanodine (10 mu M) had no effect on either basal or CCh-induced inositol phospholipid hydrolysis or cyclic AMP levels. 8 The results indicate a reciprocal inhibition by beta-adrenoceptors and muscarinic AChRs of their effects on cyclic AMP and inositol phosphate levels respectively. Ca2+ entering the cell (but not the action of ryanodine or thapsigargin) potentiates while caffeine inhibits muscarinic AChR-induced rises in inositol phosphate levels. Diacylglycerols may exert a negative feedback inhibition on inositol phosphate production.