Activation of BK Channels in GH3 Cells by ac-PLA2-Dependent G-Protein Signaling Pathway

Abstract

BK-channels in GH3 cells are activated by arachidonic acid produced by c-PLA2. β-adrenergic agonists also activate BK channels and were presumed to do so via production of cAMP. We, however, show for the first time in GH3 cells that a β-adrenergic agonist activates a pertussis-toxin-sensitive G protein that activates c-PLA2. The arachidonic acid produced by c-PLA2then activates BK channels. We examined BK channels in cell-attached patches and in excised patches from untreated GH3 cells and from GH3 cells exposed to c-PLA2antisense oligonucleotides. For the cell-attached patch experiments, physiologic pipette and bath solutions were used. For the excised patches, 150 mM KCl was used in both the pipette and bath solutions, and the cytosolic surface contained 1 μM free Ca2+(buffered with 5 mM K2EGTA). Treatment of GH3 cells with the G protein activator, fluoroaluminate, (AlF4−) produced an increase in the Poof BK channels of 177 ± 41% (mean ± SD) in cell-attached patches. Because G proteins are membrane associated, we also added an activator of G proteins, 100 μM GTP-γ-S, to the cytosolic surface of excised patches. This treatment leads to an increase in Poof 50 ± 9%. Similar treatment of excised patches with GDP-β-S had no effect on Po. Isoproterenol (1 μM), an activator of β-adrenergic receptors and, consequently, some G proteins, increased BK channel activity 229 ± 37% in cell-attached patches from cultured GH3 cells. Western blot analysis showed that GH3 cells have β-adrenergic receptor protein and that isoproterenol acts through these receptors because the β-adrenergic receptor antagonist, propanolol, blocks the action of isoproterenol. To test whether G protein activation of BK channels involves c-PLA2, we studied the effects of GTP-γ-S on excised patches and isoproterenol on cell attached patches from GH3 cells previously treated with c-PLA2antisense oligonucleotides or pharmacological inhibitors of c-PLA2. Neither isoproterenol nor GTP-γ-S had any effect on Poin these patches. Similarly, neither isoproterenol nor GTP-γ-S had any effect on Poin cultured GH3 cells pretreated with pertussis toxin. Isoproterenol also significantly increased the rate of arachidonic production in GH3 cells. These results show that some receptor-linked, pertussis-toxin-sensitive G protein in GH3 cells can activate c-PLA2to increase the amount of arachidonic acid present and ultimately increase BK-channel activity.