Effects of SCN^- substitution for Cl^- on tension, [Ca²⁺](i), and ionic currents in vascular smooth muscle

Substitution of thiocyanate ions (SCN^-) for chloride ions (Cl^-) in the extracellular medium of aortic rings and strips causes a biphasic contractile response; initial relaxation followed by sustained contraction. Alterations in these responses are sex-specific, and may elucidate fundamental differences in vascular function between males and females. In order to investigate the role of changes in intracellular Ca²⁺ ([Ca²⁺](i)) in these changes in tension, we investigated effects of SCN^- on [Ca²⁺](i) and ionic currents in vascular smooth muscle cells (VSMC). Extracellular substitution of SCN^- for Cl^- caused a biphasic change in [Ca²⁺](i). Initially, [Ca²⁺](i) decreased, reaching a minimum within 1-2 min, subsequently returned to original levels within 4-5 min, and then increased to a higher plateau over the next 10 minutes. This pattern of change in [Ca²⁺](i) is identical to the pattern of tension changes in aortic rings, but it occurs somewhat faster. Partial substitution of SCN^- for Cl^- elicited increased, but no preceding decrease in [Ca²⁺](i). In the absence of external Ca²⁺, anion substitution elicited the decrease in [Ca²⁺](i) but not the subsequent increase. Verapamil (1 μM) blocked the increased [Ca²⁺](i) phase but not the decreased [Ca²⁺](i) phase; whereas, R+ verapamil (up to 5 μM for 20 min), an inactive enantiomer, caused no change. Ionic current measurements obtained using whole cell patch and current clamp techniques revealed two responses to anion substitution: (a) a rapid, transient outward shift in holding current, and (b) a sustained increase in peak current and a hyperpolarizing shift in voltage sensitivity of Ca²⁺ channels. The calcium channel blocker PN200-110 blocked SCN^enhanced current but had no effect on the changes in holding current. S- verapamil, but not R+ verapamil, reduced SCN^enhanced current. In current clamp mode, SCN^- caused an initial hyperpolarization followed by a slow depolarization punctuated by spikes. Thus, SCN^- causes changes in vascular smooth muscle [Ca²⁺](i) that could underlie both phases of its effects on tension in isolated aortas and may be explained by the following model: an initial outward shift in current causes hyperpolarization with a consequent decrease in cell excitability, and the somewhat slower increase in Ca²⁺ channel excitability eventually leads to enhanced calcium influx and tension. These data shed light on possible mechanisms underlying gender-related differences in VSMC physiology.