INHIBITORY EFFECT OF NITROVASODILATORS AND CYCLIC-GMP ON ET-1-ACTIVATED CA2-PERMEABLE NONSELECTIVE CATION CHANNEL IN RAT AORTIC SMOOTH-MUSCLE CELLS()

1 In single vascular smooth muscle cells (VSMCs) isolated from the aor tae of male Wistar rats, we examined the effects of nitric oxide (NO) donors such as sodium nitroprusside(SNP) and S-nitroso-N-acetyl-DL-pen icillamine (SNAP), and 8-bromo-guanosine-3':5'-cyclic monophosphate (X -bromo-cyclic GMP) on endothelin-l (ET-1)-activated Ca2+-permeable non selective cation channel by use of whole-cell recordings of patch-clam p technique and monitoring of intracellular free Ca2+ concentration ([ Ca2+](i)) with fura-2 real-time digital microfluorometry. 2 ET-l evoke d an initial transient peak and a subsequent sustained elevation in [C a2+](i). After removal of extracellular Ca2+, ET-l evoked only an init ial transient peak without a sustained phase. Nifedipine (1 mu M), a s pecific blocker of the L-type voltage-operated Ca2+ channel (VOC), red uced the sustained phase to about 40% of the control level. The remain ing part of the sustained phase was abolished by 30 mu M SK&F 96365, a blocker of nonselective cation channels. 3 The nifedipine-resistant s ustained elevation in [Ca2+](i) was abolished by 100 mu M SNP, 10 mu M SNAP and 300 mu M 8-bromo-cyclic GIMP. 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-I induced an inwa rd current with an increase in baseline fluctuation. With fluctuation analysis, unit conductance of the ET-l-induced current was calculated to be about 21 pS. The ET-1-induced current was linearly related to th e membrane potentials with its reversal potential of -5.5 mV. 5 The ET -l-induced current was reversibly and completely inhibited by 30 mu M SK&F 96365 or 500 mu M Cd2+. The current inhibited by SK&F 96365 or Cd 2+ was linearly related to membrane potential with a reversal potentia l of about -5 mV. 6 The ET-l-induced current was reversibly and comple tely inhibited by 100 mu M SNP, IO mu M SNAP and 300 mu M 8-bromo-cycl ic GMP, The current inhibited by SNP, SNAP or 8-bromo-cyclic GMP showe d linear voltage-dependence and reversed at about -5 mV. 7 In a bath s olution in which all cations were replaced by 30 mM Ca2+ and 100 mM no npermeant cation N-methyl-D-glucamine (NMDG) ET-I evoked a current wit h a reversal potential of -11 mV, from which P-Ca(2+)/P-Cs(+) was calc ulated to be 2.1. This Ca2+ current was also abolished by 100 mu M SNP , 10 mu M SNAP and 300 mu M 8-bromo-cyclic GMP, The current inhibited by SNP, SNAP or S-bromo-cyclic GMP showed linear voltage-dependence an d reversed at about -11 mV. 8 These results taken together indicate th at NO through a cyclic GMP signalling pathway inhibits ET-1-activated Ca2+-permeable nonselective cation channels, thereby suppressing the s ustained increase in [Ca2+](i). Thus, the present study indicates that this Ca2+-permeable nonselective cation channel is an important targe t for nitrovasodilators.