Differential desensitization of thromboxane A(2) receptor subtypes

Two subtypes of the thromboxane A(2) (TxA(2)) receptor (TxA(2)R-E and TxA(2)R-P), which differ in their alternatively spliced cytoplasmic tails, have been identified. The initial concentration of the TxA(2) mimetic IBOP required to reduce peak intracellular Ca2+ concentration ([Ca2+](i)) induced by a second addition of IBOP (100 nmol/L) was similar (IC50 for TxA(2)R-E and TxA(2)R-P, 0.46+/-0.16 and 0.46+/-0.07 nnol/L) in fibroblasts overexpressing either the TxA(2)R-E or -P subtype. Although the number of TxA(2) binding sites decreased in TxA(2)R-P cells after prolonged stimulation with a TxA(2) mimetic, those in the TxA(2)R-E cells increased markedly. To determine whether the mechanism for desensitization differs between subtypes, the effect of activation of protein kinase C (PKC) or cAMP-dependent kinase on TxA(2)-induced [Ca2+](i) mobilization was measured. Forskolin reduced the IBOP-induced peak [Ca2+](i) in neither TxA(2)R-E nor TxA(2)R-P cells; however, treatment with phorbol esters (IC50, 0.5+/-0.70 nmol/L) strongly prevented IBOP-mediated [Ca2+](i) rise in TxA(2)R-E but not in TxA(2)R-P cells. Desensitization of TxA(2)R-E by phorbol esters was prevented by the PKC inhibitor calphostin C or by downregulation of PKC-alpha. Thus, the response of TxA(2)R-E to prolonged stimulation differs from that of TxA(2)R-P in both the regulation of the number of binding sites and the mechanism for desensitization; agonists that activate PKC-alpha might interfere with TxA(2)R-E-mediated signaling.