Activation of diacylglycerol kinase by carbachol in guinea pig taenia coli.
Changes in diacylglycerol kinase (DG kinase) activity in carbachol (CCh)-stimulated guinea pig taenia coli were investigated. In a mixed micellar assay system, added 1,2-dioctanoyl-sn-glycerol (diC8) and endogenous DG were competitively bound to common DG kinase isozymes from guinea pig taenia coli and phosphorylated, suggesting that diC8 is useful as a probe of agonist effects on DG kinase activity. In phosphorus-32 ([32P]Pi)- and diC8-prelabeled guinea pig taenia coli, diC8 was phosphorylated by DG kinase to [32P]dioctanoyl-phosphatidic acid ([32P]diC8-PA). CCh increased the accumulation of both [32P]diC8-PA and endogenous [32P]phosphatidic acid ([32P]PA) in a time- and dose-dependent manner (0.1-100 microM CCh). CCh-induced increases in [32P]diC8-PA and [32P]PA were inhibited by 1 microM atropine and 3 microM DG kinase inhibitor (R59022). These findings indicated the activation of DG kinase by muscarinic receptor stimulation in guinea pig taenia coli. Therefore, DG kinase activation may play an important role in CCh-induced PA formation. CCh-induced [32P]diC8-PA and [32P]PA accumulation was dependent on intracellular calcium concentrations. However, a KCl-induced increase in intracellular calcium, without receptor stimulation, was ineffective. Moreover, treatment with phorbol ester also increased accumulation of both PA species in KCl-treated tissues. These findings suggest that muscarinic receptor mediated activation of DG kinase may require both an increase in intracellular calcium and PKC activation in guinea pig taenia coli.
Nobe
K
K
Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
Ohata
H
H
Momose
K
K
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology*; D003106:Colon / Q000201:enzymology*; D019852:Diacylglycerol Kinase; D004075:Diglycerides / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D006168:Guinea Pigs; D010712:Phosphatidic Acids / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000302:isolation & purification / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D011744:Pyrimidinones / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology
D004075:Diglycerides; D010712:Phosphatidic Acids; D011744:Pyrimidinones; D013844:Thiazoles; C084527:dioctanoylphosphatidic acid; C037149:stearoylarachidonylglycerol; D002217:Carbachol; C047505:R 59022; D017853:Phosphotransferases (Alcohol Group Acceptor); D019852:Diacylglycerol Kinase; D011493:Protein Kinase C; D002118:Calcium