Abstract
The binding of [3H]-iloprost (ZK36374) to NCB-20 membranes revealed a single population of high affinity receptors (KD = 9.55 nM, Bmax = 431 fmol mg-1 protein) and a low affinity, non-saturable binding component. Desensitization of prostacyclin-responsiveness of NCB-20 cells is induced by culture in the presence of the stable prostacyclin analogue carbacyclin. Desensitization is accompanied by an increase in the Kact value for prostacyclin (64.1 nM to 175 nM), and a reduction in the prostacyclin-dependent increase in adenylate cyclase activity (41.2 to 15.1 pmol cyclic AMP min-1 mg-1 protein). Desensitization is not accompanied by changes in the coupling of the catalytic (C) to the regulatory (Ns) subunit of adenylate cyclase. In addition, the physical identity of the receptor molecule (as characterized by its sensitivity to electron bombardment in the beam of a linear accelerator) is not changed by desensitization. Desensitization of prostacyclin-dependent activation of adenylate cyclase may be explained most simply by a loss of prostacyclin receptors. The anomalous increase in the Kact (concentration of prostaglandin giving half-maximum enzyme activation) for prostacyclin-stimulated adenylate cyclase was not accompanied by a substantial change in the KD of [3H]-iloprost binding, and is explained by a loss of spare receptors. Prostacyclin responsiveness in non-dividing cells may be restored after desensitization by prolonged culture (up to 48 h) in the absence of carbacyclin. Resensitization is accompanied by restoration of the high affinity Kact value (143 nM to 45.5 nM), and is dependent on de novo protein synthesis.
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