Differential potentiation of arachidonic acid release by rat α2 adrenergic receptor subtypes

CHO transfectants expressing the three subtypes of rat alpha(2) adrenergic receptors (alpha(2)AR): alpha(2D), alpha(2B), alpha(2c) were studied to compare the transduction pathways leading to the receptor-mediated stimulation of phospholipase A(2) (PLA(2)) in the corresponding cell lines CHO-2D, CHO-2B, CHO-2C. The alpha(2B) subtype stimulated the arachidonic acid (AA) release after incubation of the cells with 1 mu M epinephrine, whereas alpha(2D) and alpha(2C) gave no stimulation. Calcium ionophore A23187 (1 mu M) increased the release by a factor of 2-4 in the three strains. When cells were incubated with both epinephrine and Ca2+ ionophore, the AA release differed greatly between cell lines with strong potentiation in CHO-2B (2-3 times greater than Ca2+ ionophore alone), moderate potentiation in CHO-2D, and no potentiation in CHO-2C. The three cell lines each inhibited adenylylcyclase with similar efficiencies when 1 mu M epinephrine was used as the agonist. The potentiation depended on both a2AR and G(i) proteins since yohimbine and pertussis toxin inhibited the process. Pretreatment of CHO-2B cells with MAFP which inhibits both cytosolic and Ca2+-independent PLA?, reduced the release of AA induced by epinephrine+Ca2+ ionophore to basal value, whereas bromoenol lactone, a specific Ca2+-independent PLA2 inhibitor, had no effect. Preincubation of the cells with the intracellular calcium chelator BAPTA gave a dose-dependent inhibition of the arachidonic acid (AA) release. In CHO cells expressing the angiotensin II type 1 receptor, coupled to a G(q) protein, the agonist (10(-7) M) produced maximal AA release: there was no extra increase when angiotensin and Ca2+ ionophore were added together. There was no increase in the amount of inositol 1,4,5-triphosphate following stimulation of CHO-2B, -2C, -2D cells with 1 mu M epinephrine. Epinephrine led to greater phosphorylation of cPLA(2), resulting in an electrophoretic mobility shift for all three cell lines, so inadequate p42/44 MAPKs stimulation was not responsible for the weaker stimulation of cPLA2 in CHO-2C cells. Therefore, the stimulation of cPLA(2) by G(i) proteins presumably involves another unknown mechanism. The differential stimulation of cPLA2 in these transfectants will be of value to study the actual involvement of the transduction pathways leading to maximal cPLA2 stimulation. (C) 1999 Elsevier Science B.V. All rights reserved.