Separate receptors mediate oxytocin and vasopressin stimulation of cAMP in rat inner medullary collecting duct cells.

The two neurohypophysial hormones arginine vasopressin (AVP) and oxytocin have actions in the inner medullary collecting duct (IMCD) where both peptides induce an increase in cAMP accumulation. The present study has employed a novel IMCD cell line to determine whether these two hormones induce cAMP accumulation via common or separate receptors, and to characterize the potential receptors responsible. Equal volumes of vehicle (150 mM NaCl) or hormone/antagonist solutions were added to aliquots of 10(4) IMCD cells in the presence of 10(-3) M 3-isobutylmethylxanthine (IBMX) and incubated at 37 degrees C for 4 min. cAMP levels were determined by radioimmunoassay and protein concentration by Bradford assay. Both AVP and oxytocin elicited dose-dependent increases in cAMP generation, though oxytocin was less potent than AVP (EC50 = 1.6 x 10(-8) M vs. 7.4 x 10(-10) M). AVP at 10(-8) M and oxytocin at 10(-8) M, concentrations sufficient to elicit near-maximal cAMP accumulation, resulted in cAMP levels of 73.4 +/- 1.7 and 69.0 +/- 3.3 pmol (mg protein)-1 (4 min)-1, respectively (n = 10), compared with the vehicle-treated basal value of 37.7 +/- 2.2 pmol (mg protein)-1 (4 min)-1 (P < 0.001, n = 10). Combined AVP (10(-8) M) and oxytocin 10(-6) M) resulted in cAMP accumulation of 63.8 +/- 3.1 pmol (mg protein)-1 (4 min)-1 (n = 10), which was not significantly different from the effect of oxytocin alone, but slightly less than that for AVP alone (P < 0.05). A submaximal concentration of AVP (10(-10) M) induced cAMP accumulation of 48.6 +/- 2.5 pmol (mg protein)-1 (4 min)-1 (P < 0.01 compared with basal level of 34.9 +/- 2.4 pmol (mg protein)-1 (4 min)-1, n = 10), which was blocked in the presence of a vasopressin V2 receptor antagonist (10(-7) M OPC-31260) but not by the oxytocin receptor antagonist (10(-6) M [Pen1,pMePhe2, Thr4,Orn8]oxytocin) (36.3 +/- 6.1 and 45.1 +/- 1.3 pmol (mg protein)-1 (4 min)-1 respectively, P < 0.05, n = 10). A submaximal concentration of oxytocin (10(-7) M) induced a cAMP accumulation of 45.8 +/- 1.8 pmol (mg protein)-1 (4 min)-1 (n = 10), which was reduced by addition of 10(-6) M oxytocin antagonist (36.3 +/- 2.1 pmol (mg protein)-1 (4 min)-1, P < 0.05, n = 10), whereas co-incubation with 10(-6) M of the V2 receptor antagonist had no effect (43.2 +/- 1.3 pmol (mg protein)-1 (4 min)-1, n = 10). These results indicate that AVP and oxytocin induce cAMP accumulation from a common ATP pool in IMCD cells, and that separate vasopressin V2 and oxytocin receptor systems are involved, perhaps coupled to a common adenylate cyclase system.