Defining the role of protein kinase C in epinephrine- and bradykinin-stimulated arachidonic acid metabolism in Madin-Darby canine kidney cells.
Madin-Darby canine kidney cells (MDCK) are known to release free arachidonic acid and arachidonic acid metabolites (AA) in response to tumor-promoting phorbol esters, such as tetradecanoyl phorbol-13-acetate, and to agonists active at alpha 1-adrenergic and bradykinin B2 receptors. These experiments were conducted to define the role of Ca2+/phospholipid-dependent protein kinase (protein kinase C) activation in the stimulation of AA release, in the clonal isolate cell line MDCK-D1, by use of three inhibitors of protein kinase C, sphingosine, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and staurosporine. We found that alpha 1-adrenergic- and bradykinin-stimulated [3H]AA release can be distinguished by differential dependence on protein kinase C; epinephrine-stimulated release was more dependent on protein kinase C activation than was bradykinin-stimulated release. The inhibition of bradykinin-stimulated AA release by sphingosine (20.2 +/- 6.1%) was substantially less than the inhibition observed for tetradecanoyl phorbol-13-acetate- (67.2 +/- 5.5%) and epinephrine-stimulated release (50.2 +/- 9.2%). These findings were confirmed by results using H-7 and staurosporine. The relative independence of bradykinin-stimulated AA release of protein kinase C was also demonstrated by the inability of phorbol ester-induced down-regulation of protein kinase C to eliminate bradykinin-stimulated AA release. The inhibition of alpha 1-adrenergic receptor-mediated AA release by sphingosine, H-7, and staurosporine was not due to a change in receptor number or affinity. Analysis of the products comprising [3H]AA release indicated that treatment with sphingosine did not change the composition of the released AA (34-48% prostaglandin E2, 17-27% free arachidonic acid, and 25-51% unidentified metabolites). These results indicate that two different types of hormone receptors in the same cell type can promote AA release by mechanisms that differ in their dependence on protein kinase C. The protein kinase C-dependent mechanism may represent protein kinase C-mediated activation of phospholipase A2.
Weiss
B A
BA
Department of Pharmacology, University of California, San Diego, La Jolla 92093.
Slivka
S R
SR
Insel
P A
PA
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism*; D001920:Bradykinin / Q000494:pharmacology*; D002460:Cell Line; D015232:Dinoprostone / Q000096:biosynthesis; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004837:Epinephrine / Q000494:pharmacology*; D007546:Isoquinolines / Q000494:pharmacology; D007668:Kidney / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D011942:Receptors, Adrenergic, alpha / Q000502:physiology*; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter / Q000502:physiology*; D013110:Sphingosine / Q000494:pharmacology; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000470:Alkaloids; D001095:Arachidonic Acids; D007546:Isoquinolines; D010879:Piperazines; D011942:Receptors, Adrenergic, alpha; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter; D016718:Arachidonic Acid; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; D019311:Staurosporine; D015232:Dinoprostone; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin; D004837:Epinephrine