alpha 2A-adrenergic receptor stimulated calcium release is transduced by G(i)-associated G beta gamma-mediated activation of phospholipase C

Proposed mechanisms by which alpha 2-adrenergic receptors (alpha 2AR) regulate intracellular calcium ([Ca2+](i)) include stimulation and inhibition of cell surface calcium channels, stimulation of calcium release via receptor coupling to G(q) with subsequent activation of phospholipase C and release of IP3, or stimulation of calcium release via coupling to G(i) in an IP3-independent manner. These potential mechanisms were explored in cells that expressed alpha(2A)AR endogenously (HEL cells), permanently transfected CHO cells, and transiently transfected COS-7 cells. Each cell type displayed agonist (UK14304)-dependent increases in [Ca2+](i) that were blocked by yohimbine, ablated by pertussis toxin, and largely unaffected by chelation of extracellular calcium. Furthermore, calcium release was associated with IP3 accumulation and was blocked by an inhibitor of phospholipase C (PLC). When expressed in CHO cells, a mutated alpha(2A)AR which has the amino and carboxyl termini of the third intracellular loop substituted with beta(2)AR sequence poorly coupled to G(i) in adenylyl cyclase assays, and likewise displayed virtually no coupling to increased [Ca2+](i). These results all point toward a G(i)- versus a G(q)-mediated coupling pathway triggering release of intracellular calcium stores. The possibility that G(beta gamma) subunits released from alpha(2A)AR-G(i) coupling is the mechanism of PLC activation was explored in COS-7 cells by coexpressing alpha(2A)AR With the G(beta gamma) inhibitors transducin or a carboxy-terminal portion of the PAR kinase. Both beta gamma inhibitors markedly inhibited or alpha(2A)AR modulation of [Ca2+](i) while not affecting thromboxane A(2) receptor mediated stimulation of [Ca2+](i) via G(q) coupling. Thus, alpha(2A)AR couple to calcium release via G(i)-associated G(beta gamma) subunits. This coupling is present in multiple cell types and should be considered a major signal transduction pathway of this receptor.