The isoquinoline derivative LOE 908 selectively blocks vasopressin-activated nonselective cation currents in A7r5 aortic smooth muscle cells

The effect of (R,S)-(3,4-dihydro6,7-dimethoxyisoquinoline-1-yl)-2-phenyl-N, N-di- [2- (2, 3,4-trimethoxyphenyl)ethyl]-acetamide (LOE 908), a cation channel blocker in HL-60 promyeloblasts, was studied in the A7r5 smooth muscle cell line from rat thoracic aorta, using the whole-cell patch-clamp technique. At a holding potential of -60 mV, application of vasopressin induced a nonselective cation conductance in voltage-clamped A7r5 cells. The current-voltage relation was linear, and currents reversed close to 0 mV regardless of the chloride gradient. The activation of the nonselective cation conductance by vasopressin was not affected by dialysing cells with Ca⁺-free internal solution. LOE 908 blocked this current in a concentration-dependent manner with an IC₅₀ of 560 nM, whereas dihydropyridine-sensitive Ba²⁺ current through voltage-dependent Ca²⁺ channels was blocked with an IC₅₀ of 28 μM. Another organic blocker of receptor-mediated Ca^2- entry, 1-β-[3-(4-methoxyhenyl)-propoxy]-4-methoxyphenethyl-1 H-imidazole hydrochloride (SK&F 96365), blocked both, the vasopressin-induced nonselective conductance and the voltage-activated Ba²⁺ current with similar IC₅₀ values of 13 μM and 8 μM, respectively. The rank order of potency of inorganic blockers on the vasopressin-induced inward current was Gd³⁺>La³⁺>Cd²⁺. Vasopressin-induced nonselective cation current was also observed in pertussis toxin-pretreated A7r5 cells but was completely abolished after infusion of the GDP analogue, guanosine 5′-O-[3-thio]diphosphate, from the patch pipette. Furthermore, vasopressin induced a transient outward current, suggesting a Cau²⁺-activated K⁺-current, which overlapped with the nonselective cation conductance. The outward current was blocked by internal Cs⁺ and external Ba²⁺ or TEA. Our data suggest that the activation of nonselective cation current by vasopressin in A7r5 cells involves a pertussis toxin-insensitive G-protein, is independent of the intracellular Ca²⁺ concentration and that LOE 908 selectively blocks this current.