Inhibitory effect of nitrovasodilators and cyclic GMP on ET-1-activated Ca2+-permeable nonselective cation channel in rat aortic smooth muscle cells

摘要

class="enumerated" style="list-style-type:decimal">In single vascular smooth muscle cells (VSMCs) isolated from the aortae of male Wistar rats, we examined the effects of nitric oxide (NO) donors such as sodium nitroprusside (SNP) and S-nitroso-N-acetyl-DL-penicillamine (SNAP), and 8-bromo-guanosine-3′:5′-cyclic monophosphate (8-bromo-cyclic GMP) on endothelin-1 (ET-1)-activated Ca²⁺-permeable nonselective cation channel by use of whole-cell recordings of patch-clamp technique and monitoring of intracellular free Ca²⁺ concentration ([Ca²⁺]i) with fura-2 real-time digital microfluorometry.ET-1 evoked an initial transient peak and a subsequent sustained elevation in [Ca²⁺]i. After removal of extracellular Ca²⁺, ET-1 evoked only an initial transient peak without a sustained phase. Nifedipine (1 μM), a specific blocker of the L-type voltage-operated Ca²⁺ channel (VOC), reduced the sustained phase to about 40% of the control level. The remaining part of the sustained phase was abolished by 30 μM SK&F 96365, a blocker of nonselective cation channels.The nifedipine-resistant sustained elevation in [Ca²⁺]i was abolished by 100 μM SNP, 10 μM SNAP and 300 μM 8-bromo-cyclic GMP. Neither SNP, SNAP nor 8-bromo-cyclic GMP significantly affected the basal level of [Ca²⁺]i.In a VSMC clamped at a holding potential of −60 mV with K⁺ in the pipette solution replaced by Cs⁺, application of 10⁻⁸ M ET-1 induced an inward current with an increase in baseline fluctuation. With fluctuation analysis, unit conductance of the ET-1-induced current was calculated to be about 21 pS. The ET-1-induced current was linearly related to the membrane potentials with its reversal potential of −5.5 mV.The ET-1-induced current was reversibly and completely inhibited by 30 μM SK&F 96365 or 500 μM Cd²⁺. The current inhibited by SK&F 96365 or Cd²⁺ was linearly related to membrane potential with a reversal potential of about −5 mV.The ET-1-induced current was reversibly and completely inhibited by 100 μM SNP, 10 μM SNAP and 300 μM 8-bromo-cyclic GMP. The current inhibited by SNP, SNAP or 8-bromo-cyclic GMP showed linear voltage-dependence and reversed at about −5 mV.In a bath solution in which all cations were replaced by 30 mM Ca²⁺ and 100 mM nonpermeant cation N-methyl-D-glucamine (NMDG), ET-1 evoked a current with a reversal potential of −11 mV, from which PCa²⁺/PCs⁺ was calculated to be 2.1. This Ca²⁺ current was also abolished by 100 μM SNP, 10 μ class="small-caps">M SNAP and 300 μ class="small-caps">M 8-bromo-cyclic GMP. The current inhibited by SNP, SNAP or 8-bromo-cyclic GMP showed linear voltage-dependence and reversed at about −11 mV.These results taken together indicate that NO through a cyclic GMP signalling pathway inhibits ET-1-activated Ca²⁺-permeable nonselective cation channels, thereby suppressing the sustained increase in [Ca²⁺]i. Thus, the present study indicates that this Ca²⁺-permeable nonselective cation channel is an important target for nitrovasodilators.