Activation of phospholipase C and phospholipase D by stimulation of adenosine A₁, bradykinin or P_2U receptors does not correlate well with protein kinase C activation

Abstract

Activation of adenosine A₁-, bradykinin- or P_2U-receptors on DDT₁ MF-2 smooth muscle cells all increased the formation of inositol 1,4,5-trisphosphate and the mobilization of intracellular calcium. All three types of agents could increase [Ca²⁺] in the same cell. Activation of the P_2U receptor with ATP or UTP produced larger responses than activation of bradykinin- and adenosine A₁-receptors, with bradykinin and N⁶-cyclopentyladenosine. When agonist-stimulated levels of diacylglycerol were determined, all agonists caused biphasic changes of similar magnitudes. If anything, ATP and UTP tended to give larger increases in the second phase of stimulation. Phospholipase D, measured as the formation of phosphatidylethanol in cells labeled with [³H]palmitic acid and activated in the presence of ethanol, was activated similarly as phospholipase C, i.e. ATP or UTP caused the largest increase in phosphatidylethanol formation, followed by N⁶-cyclopentyladenosine and bradykinin which caused weaker responses. Activation of PLD by P_2U receptors was pertussis toxin insensitive. The activation of PLD by the agonists was only weakly affected by a PKC inhibitor, Ro 31-7549 (3-[1-(3-aminopropanyl)-3-indolyl]-4-(1-methyl-3-indolyl)-1 H pyrrole-2,5-dione). In contrast, ATP or UTP did not activate protein kinase C, determined in a permeabilized cell assay using two specific protein kinase C substrates, whereas N⁶-cyclopentyladenosine and bradykinin caused a substantial activation.

In summary, the present study shows that the magnitude of the activation of protein kinase C by receptor agonists cannot be predicted from the degree of phospholipase C and phospholipase D activation, accumulation of diacylglycerol or rise in intracellular calcium, and suggests that additional factors are important in the activation of protein kinase C.