Concerted action of cytosolic Ca2+ and protein kinase C in receptor-mediated phospholipase D activation in Chinese hamster ovary cells expressing the cholecystokinin-A receptor.

摘要: Receptor-mediated activation of phosphatidylcholine phosphatidohydrolase or phospholipase D (PLD) was studied in Chinese hamster ovary (CHO) cells expressing the cholecystokinin-A (CCK-A) receptor. Cells were labelled with [3H]myristic acid for 24 h and PLD-catalysed [3H]phosphatidylethanol formation measured presence 1% (v/v) ethanol. Cholecystokinin-(26-33)-peptide amide (CCK8) increased PLD activity both time- dose-dependently. Maximal protein kinase C (PKC) 1 microM PMA sustained elevation cytosolic free Ca2+ concentration ([Ca2+]i) thapsigargin to 50% 70% maximal value obtained CCK8 respectively. The stimulatory effects CCK8, abolished which PKC downregulated inhibited by chelerythrine. PMA/Ca2+-stimulated absent a homogenate PKC-downregulated but could be restored upon addition purified rat brain PKC. CCK8-induced 90% absence external Ca2+, demonstrating that receptor-mediated itself does not significantly add requires increase [Ca2+]i. Taken together, results presented demonstrate that, CHO-CCK-A cells, is completely dependent on PKC, extent becomes activated depends largely, if entirely, magnitude duration agonist-induced