MECHANISM OF VASOPRESSIN-INDUCED CALCIUM INFLUX IN AORTIC SMOOTH-MUSCLE CELLS - INVOLVEMENT OF CALCIUM-PERMEABLE NONSELECTIVE CATION CHANNEL

The effects of vasopressin on cultured rat aortic smooth muscle cell l ines (A7r5) were investigated using the calcium-sensitive dye Indo-1 t o measure intracellular calcium ([Ca2+](i)) and the patch-clamp techni que in the whole-cell and single-channel configurations. Vasopressin ( 100 nM) evoked an initial peak followed by a smaller sustained rise of [Ca2+](i) in the presence of extracellular calcium ([Ca2+](o)). In th e absence of [Ca2+](o), only the initial peak of [Ca2+](i) was observe d. Therefore, the initial peak of [Ca2+](i) was mainly due to calcium store release, whereas the latter sustained rise of [Ca2+](i) was due to calcium entry from outside. The sustained rise of [Ca2+](i) produce d by vasopressin, which was not significantly affected by nifedipine 1 0 mu M, but was completely abolished by La3+ (1 mM). Under the current -clamp condition with K+-containing solution, vasopressin 100 nM produ ced hyperpolarization followed by depolarization. Under the voltage-cl amp condition, at a holding potential of -40mV, vase first activated t he outward current followed by a long-lasting inward current with a hi gh noise level. The first outward current elicited by vasopressin was abolished by charybdotoxin (100 nM) and Cs+ in the patch pipette. In a ddition, high EGTA (10 mM) in the patch pipette completely abolished t he current. Thus, the vasopressin-activated outward current was consid ered to be the Ca2+-sensitive K+ current (I-K-Ca) The inward current w as still elicited with the patch pipette containing Cs+-internal solut ion, and reversed approximately at 0 mV. The reversal potential was no t significantly altered by the replacement of [Cl-](i) or [Cl-](o), su ggesting that vasopressin-induced inward current should be non-selecti ve cation channel (I-NS) Extracellularly applied La3+ (1 mM), Cd2+ (1 mM) or Mg2+ (10 mM) completely abolished the vasopressin-induced I-NS Nifedipine 10 mu M was totally ineffective. I-NS was also observed eve n when the extracellular cation was Ca2+ or Ba2+. Single-channel activ ities were recorded in the cell-attached configuration when vasopressi n was added to the bathing solution. The unitary conductance of the va sopressin-activated channel was approximately 20 pS with 140 mM Na+, C s+, or K+ in the patch pipette, but was 15 pS with 110 mM Ca2+ in the pipette. Permeability sequence for sodium calculated from the reversal potential was Na+ is-approximately -equal-to C-s+ is-approximately-eq ual-to K+ > Ca2+. These results provide evidence that calcium entry el icited by vasopressin is mediated by the receptor-operated Ca2+-permea ble non-selective cation channel in aortic smooth muscle cells.