Contribution of different phospholipases and arachidonic acid metabolites in the response of gallbladder smooth muscle to cholecystokinin

Guinea pig gallbladder muscle strips were used to investigate the contribution of different sources of diacylglicerol (DAG) in the cholecystokinin (CCK)-induced contraction. The involvement of arachidonic acid (AA) in this response was also investigated. Three distinct pathways for DAG production were investigated with specific phospholipase (PL) inhibitors. U-73122 (10 muM) was used for inhibition of phosphomositide-specific-PLC (PC-PLC), D-609 (100 muM) for phosphatidylcholine specific-PLC (PC-PLC), and propranolol (100 muM) for phospholipase D (PLD). Separate or combined inhibition of each of these enzymes showed that the CCK-induced output of DAG involves the parallel activation of each of these phospholipases. Thus, after inhibition of a PL subtype, the remaining subtypes were able to functionally compensate in mediating CCK-induced contraction. Inhibition of AA production via DAG-lipase or phospholipase A(2) (PLA(2)) was accomplished using RHC-80267 (40 muM), mepacrine (100 muM) and 4-BPB (100 muM). These inhibitors diminished contractile response, indicating that AA is an important modulator of CCK-induced contraction. Indomethacin (10 muM) and nordihydroguaiaretic acid (NDGA, 100 M), which inhibit subsequent steps in AA metabolism through the cyclooxygenase and 5-lipooxygenase pathways, also inhibited contractions. Taken together, these results show that CCK redundantly activates PC-PLC, PI-PLC and PLD, to produce DAG, which in turn stimulates PKC and provides a substrate for the generation of AA. sPLA(2) is also a source of AA, whose metabolites are, in part, responsible for determining the magnitude of the CCK-evoked contraction. (C) 2002 Elsevier Science Inc. All rights reserved.