Inhibitory effect of nitrovasodilators and cyclic GMP on ET-1-activated Ca(2+)-permeable nonselective cation channel in rat aortic smooth muscle cells

Abstract

1. 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 Ca(2+)-permeable nonselective cation channel by use of whole-cell recordings of patch-clamp technique and monitoring of intracellular free Ca(2+)-concentration ([Ca2+]i) with fura-2 real-time digital microfluorometry. 2. 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 microM), 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 microM SK&F 96365, a blocker of nonselective cation channels. 3. The nifedipine-resistant sustained elevation in [Ca2+]i was abolished by 100 microM SNP, 10 microM SNAP and 300 microM 8-bromo-cyclic GMP. Neither SNP, SNAP nor 8-bromo-cyclic GMP significantly affected the basal level of [Ca2+]i. 4. 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. 5. The ET-1-induced current was reversibly and completely inhibited by 30 microM SK&F 96365 or 500 microM Cd2+. The current inhibited by SK&F 96365 or Cd2+ was linearly related to membrane potential with a reversal potential of about -5 mV. 6. The ET-1-induced current was reversibly and completely inhibited by 100 microM SNP, 10 microM SNAP and 300 microM 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. 7. 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+/Pcs1 was calculated to be 2.1. This Ca2+ current was also abolished by 100 microM SNP, 10 microM SNAP and 300 microM 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. 8. These results taken together indicate that NO through a cyclic GMP signalling pathway inhibits ET-1-activated Ca(2+)-permeable nonselective cation channels, thereby suppressing the sustained increase in [Ca2+]i. Thus, the present study indicates that this Ca(2+)-permeable nonselective cation channel is an important target for nitrovasodilators.