Regulation of phosphatidylinositol kinases by arachidonic acid in rat submandibular gland cells

Abstract

Phosphoinositide kinases were characterized in membrane extracts of rat submandibular gland cells. Both phosphatidylinositol (PI) 4-kinase and phosphatidylinositol-4-phosphate (PI(4)P) 5-kinase phosphorylated endogenous substrates in reactions that were linear for up to 5 min, were activated by Mg2+ and showed maximal activity around neutral pH. PI 4-kinase was stimulated by Triton X-100 at an optimal concentration of 0.22%, but the detergent had an inhibitory effect on PI(4)P 5-kinase. Arachidonic acid (AA), at concentrations greater than 100 microM, inhibited the activity of both enzymes in a dose-dependent manner. The inhibitory effect was replicated by other unsaturated fatty acids, but not by a saturated fatty acid of the sn-20 series. The nature of AA inhibition of the kinases was examined in enzyme kinetic studies with exogenous phosphoinositide and adenosine 5'-triphosphate (ATP) substrates. Lineweaver-Burk plots of PI 4-kinase activity showed that AA had no effect on the apparent Km for either PI or ATP, but that the fatty acid significantly reduced Vmax (PI) from 331 to 177 pmol.mg-1.min-1 and Vmax (ATP) from 173 to 59 pmol.mg-1.min-1. This inhibitory action was consistent for PI(4)P 5-kinase kinetics, where again, AA did not alter apparent Km values, but lowered Vmax for both PI(4)P and ATP by around 50%. Since the combination of a reduced Vmax and an unchanged Km value indicates noncompetitive enzyme inhibition, it is proposed that AA regulates phosphoinositide cycle activity in submandibular gland cells by acting as a noncompetitive inhibitor of PI 4-kinase and PI(4)P 5-kinase.

Arachidonic acid-induced activation of large-conductance potassium channels and membrane hyperpolarization in mouse B cells

“…While the positive effects of AA on LK bg were apparent at micromolar ranges of concentration, previous studies showed that the inhibition of PIkinase and the activation of PLCγ by AA occur at tens and hundreds of micromolar concentrations, respectively [6,15]. Also, the activation of PLCs in human platelets by AA is mediated by the metabolites of cyclooxygenase [29].…”

Accumulation of Unsaturated Acylcarnitine Molecular Species During Acute Myocardial Ischemia: Metabolic Compartmentalization of Products of Fatty Acyl Chain Elongation in the Acylcarnitine Pool

“…Since myocardial ischemia is accompanied by dramatic increases in the mass of the acylcarnitine pool and since the biologic sequelae of amphiphilic constituents containing unsaturated aliphatic chains can be quite different from their saturated counterparts in some systems [e.g., Okamura and Yamashita (1994), Cao and Hatch (1994), and Chung and Fleming (1995)], we sought to identify the distribution of individual molecular species of long-chain acylcarnitines in ischemic myocardium. Historically, the mass of carnitines in biologic tissues has been quantitated by measuring the carnitine acetyltransferase catalyzed conversion of tissue carnitine to [ 3 H]acetyl carnitine (McGarry & Foster, 1976).…”