title: 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.
authors.affiliation: Department of Pharmacology, Kyoto University Faculty of Medicine, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Minowa
authors.forename: S
authors.initials: S
authors.lastname: Miwa
authors.forename: S
authors.initials: S
authors.lastname: Kobayashi
authors.forename: T
authors.initials: T
authors.lastname: Enoki
authors.forename: X F
authors.initials: XF
authors.lastname: Zhang
authors.forename: T
authors.initials: T
authors.lastname: Komuro
authors.forename: Y
authors.initials: Y
authors.lastname: Iwamuro
authors.forename: T
authors.initials: T
authors.lastname: Masaki
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000187:drug effects*; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology*; D019332:Endothelin-1 / Q000494:pharmacology*; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009599:Nitroprusside / Q000494:pharmacology; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D026423:S-Nitroso-N-Acetylpenicillamine; D014665:Vasodilator Agents / Q000494:pharmacology*
chemical_list: D015220:Calcium Channels; D019332:Endothelin-1; D014665:Vasodilator Agents; D009599:Nitroprusside; C016276:8-bromocyclic GMP; D026423:S-Nitroso-N-Acetylpenicillamine; D010396:Penicillamine; D006152:Cyclic GMP; D002118:Calcium