DIPHENYLAMINE-2-CARBOXYLATE ANALOGS BLOCK CL- CONDUCTANCES IN A7R5 CELLS BY AFFECTING CELLULAR CA2+ HOMEOSTASIS

We have investigated the cellular signalling pathway by which vasopres sin stimulates a Ca2+-dependent Cl- conductance and the effects of two known Cl- channel blockers in cultured rat A7r5 aortic smooth muscle cells using anion efflux and fluorescent Ca2+ imaging studies. Additio n of vasopressin (100 nM) to A7r5 cells enhanced I-125 (Cl- substitute ) efflux from the cells through a V1 receptor-mediated pathway. Maxima l increases in the rate of efflux were observed 1 min following additi on of vasopressin (4-fold above basal levels). Activation of the V1 pa thway was demonstrated by an increase in inositol trisphosphate (IP3) formation and lack of cAMP accumulation by the cells following the add ition of vasopressin. Fluorescent ratio imaging with fura-2 revealed t hat addition of vasopressin to the cells results in an increase of [Ca 2+]i which peaks within 20 s and does not return to resting levels dur ing the 100 s observation period. The addition of a Ca2+ ionophore mim icked the vasopressin-induced efflux from the cells. 5-Nitro-2-(3-phen ylpropylamino)-benzoic acid (NPPB) and a chloro-substituted compound ( cpd 149) inhibited the vasopressin-stimulated I-125 efflux from the ce lls. The concentrations of NPPB and cpd 149 required to inhibit I-125 efflux from the cells were similar to those which also attenuated vaso pressin-induced Ca2+ transients in the cells. NPPB and cpd 149 had no effects on the ionomycin stimulated efflux. The mechanism(s) by which cpd 149 exerts its effect on stimulated efflux was examined by measuri ng its action on vasopressin-induced changes in IP3. Compound 149 inhi bited IP3 generation in response to vasopressin. In conclusion, these data provide evidence for the presence of a Ca2+-activated chloride ch annel in A7r5 aortic smooth muscle cells. However, the chloride channe l blockers that we used possess ancillary properties that complicate t heir use as selective pharmacological tools.