Receptor-linked early events induced by vasoactive intestinal contractor (VIC) on neuroblastoma and vascular smooth-muscle cells.
Vasoactive intestinal contractor (VIC) caused a series of biochemical events, including the temporal biphasic accumulation of 1,2-diacylglycerol (DAG), transient formation of Ins(1,4,5)P3, and increase in intracellular free Ca2+ [( Ca2+]i) in neuroblastoma NG108-15 cells. In these cellular responses, VIC was found to be much more potent in NG108-15 cells than in cultured rat vascular smooth-muscle cells. The single cell [Ca2+]i assay revealed that in the presence of nifedipine (1 microM) or EGTA (1 mM), the peak [Ca2+]i declined more rapidly to the resting level in VIC-stimulated NG108-15 cells, indicating that the receptor-mediated intracellular Ca2+ mobilization is followed by Ca2+ influx through the nifedipine-sensitive Ca2+ channel. Pretreatment with pertussis toxin only partially decreased Ins(1,4,5)P3 generation as well as the [Ca2+]i transient induced by VIC, whereas these events induced by endothelin-1 were not affected by the toxin, suggesting involvement of distinct GTP-binding proteins. The VIC-induced transient Ins(1,4,5)P3 formation coincident with the first early peak of DAG formation suggested that PtdIns(4,5)P2 is a principal source of the first DAG increase. Labelling studies with [3H]myristate, [14C]palmitate and [3H]choline indicated that in neuroblastoma cells phosphatidylcholine (PtdCho) was hydrolysed by a phospholipase C to cause the second sustained DAG increase. Down-regulation of protein kinase C (PKC) by prolonged pretreatment with phorbol ester markedly prevented the VIC-induced delayed DAG accumulation. Furthermore, chelation of intracellular CA2+ completely abolished the second sustained phase of DAG production. These findings suggest that PtdCho hydrolysis is responsible for the sustained production of DAG and is dependent on both Ca2+ and PKC.
Fu
T
T
Department of Biochemistry, Gifu University School of Medicine, Japan.
Okano
Y
Y
Zhang
W
W
Ozeki
T
T
Mitsui
Y
Y
Nozawa
Y
Y
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D002794:Choline / Q000378:metabolism; D004075:Diglycerides / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D016257:Fura-2; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D009447:Neuroblastoma; D009543:Nifedipine / Q000494:pharmacology; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology*
D004075:Diglycerides; D036341:Intercellular Signaling Peptides and Proteins; D009227:Myristic Acids; D010169:Palmitic Acids; D010455:Peptides; C059568:vasoactive intestinal constrictor; D019814:Myristic Acid; D019308:Palmitic Acid; D004533:Egtazic Acid; D015544:Inositol 1,4,5-Trisphosphate; D009543:Nifedipine; D002794:Choline; D002118:Calcium; D016257:Fura-2