Differential potentiation of arachidonic acid release by rat alpha(2) 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 com pare 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) rel ease after incubation of the cells with 1 mu M epinephrine, whereas alpha(2 D) and alpha(2C) gave no stimulation. Calcium ionophore A23187 (1 mu M) inc reased 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 differe d 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 adenylylcyclas e 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 wit h MAFP which inhibits both cytosolic and Ca2+-independent PLA?, reduced the release of AA induced by epinephrine+Ca2+ ionophore to basal value, wherea s bromoenol lactone, a specific Ca2+-independent PLA2 inhibitor, had no eff ect. Preincubation of the cells with the intracellular calcium chelator BAP TA gave a dose-dependent inhibition of the arachidonic acid (AA) release. I n 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. Th ere 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 stimula tion was not responsible for the weaker stimulation of cPLA2 in CHO-2C cell s. Therefore, the stimulation of cPLA(2) by G(i) proteins presumably involv es another unknown mechanism. The differential stimulation of cPLA2 in thes e transfectants will be of value to study the actual involvement of the tra nsduction pathways leading to maximal cPLA2 stimulation. (C) 1999 Elsevier Science B.V. All rights reserved.