Intracellular Ca2+ and protein kinase C interact to regulate alpha 1-adrenergic- and bradykinin receptor-stimulated phospholipase A2 activation in Madin-Darby canine kidney cells.
Alpha 1-Adrenergic receptors and bradykinin receptors are two distinct membrane receptors that stimulate phospholipid breakdown and arachidonic acid and arachidonic acid metabolite release. In the current studies, we have examined several mechanisms to assess their possible contribution to arachidonic acid release in the Madin-Darby canine kidney cell line by agonist stimulation of these receptors: 1) activation of phospholipase A2 (PLA2); 2) sequential activation of phospholipase C, diacylglycerol lipase, and monoacylglycerol lipase; and 3) inhibition of the sequential action of fatty acyl-CoA synthetase and lysophosphatide acyltransferase. Experiments were conducted to measure the stimulation of lysophospholipid production by epinephrine and bradykinin, the rate of incorporation of [3H]arachidonic acid into stimulated and unstimulated cells, and the effect on [3H]arachidonic acid release of treating cells with exogenous phospholipase C. The data indicate that stimulation of PLA2 activity is regulated by alpha 1-adrenergic and bradykinin receptors and that this stimulation is mediated, at least in part, by the activation of protein kinase C. We find that the role of diacylglycerol in arachidonic acid release is as an activator of protein kinase C and not as a substrate for a lipase. Moreover, the hormonal agonists do not appear to inhibit fatty acid reacylation. Experiments using the Ca2(+)-sensitive dye fura-2 and the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid suggest that bradykinin activates PLA2 by a transient elevation of intracellular Ca2+. This action appears to be less important for activation of PLA2 by epinephrine. Taken together, these data are consistent with the following conclusions. 1) Hormone-stimulated arachidonic acid release in Madin-Darby canine kidney-D1 cells occurs as a consequence of PLA2 activation. 2) The ability of an agonist both to mobilize Ca2+ and to activate protein kinase C contributes to its efficacy as a stimulator of PLA2-mediated arachidonic acid release.
Weiss
B A
BA
Department of Pharmacology, University of California, San Diego, La Jolla 92093.
Insel
P A
PA
D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism; D001920:Bradykinin / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D004075:Diglycerides / Q000378:metabolism; D004285:Dogs; D004789:Enzyme Activation; D007668:Kidney; D007700:Kinetics; D008563:Membrane Lipids / Q000378:metabolism; D008954:Models, Biological; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism*; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter / Q000378:metabolism*
D001095:Arachidonic Acids; D004075:Diglycerides; D008563:Membrane Lipids; D010743:Phospholipids; D011942:Receptors, Adrenergic, alpha; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter; D011493:Protein Kinase C; D010741:Phospholipases A; D054467:Phospholipases A2; D001920:Bradykinin; D002118:Calcium