Calcium currents in the A7r5 smooth muscle-derived cell line.
We have studied voltage-dependent calcium channels in the A7r5 smooth muscle cell line by measuring the high-affinity binding of radiolabelled dihydropyridines (DHPs), whole-cell and single-channel currents in patch-clamped cells, as well as cytosolic calcium ([Ca2+]i) in fura-2-loaded cell suspensions and monolayers. Intact A7r5 cells express saturable, high-affinity, voltage-sensitive DHP binding sites with pharmacological properties characteristic of L-type calcium channels. When cells were voltage clamped in the whole-cell configuration with near normal intra- and extracellular solutions, a DHP-sensitive inward current resembling the L-type calcium current was dominant. With barium (10 mM) as the charge carrier, peak inward currents were typically recorded at test potentials between 0 and +20 mV. Currents were blocked by extracellular cadmium with a half-maximal inhibitory concentration of approximately 1 microM. Isoproterenol (1 microM) or forskolin (10 microM) increased currents in approximately half of the cells tested. Forskolin (10 microM) increased single-channel activity in five of eight cell-attached patches. After cells had been quiescent for several weeks, cell suspensions showed changes in resting [Ca2+]i in response to DHPs and increased potassium. Most confluent monolayers of cells showed spontaneous transient elevations in [Ca2+]i. Bath application of Bay K 8644 increased the frequency and magnitude of these [Ca2+]i transients, whereas nifedipine abolished the transients. These data suggest that the [Ca2+]i transients were due to synchronous action potentials in electrically coupled cell monolayers.
Marks
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Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106.
Dubyak
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GR
Jones
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D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000818:Animals; D001011:Aorta; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002118:Calcium / Q000032:analysis / Q000378:metabolism / Q000493:pharmacokinetics*; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D004095:Dihydropyridines / Q000378:metabolism / Q000494:pharmacology; D004553:Electric Conductivity / Q000187:drug effects / Q000502:physiology; D005453:Fluorescence; D007545:Isoproterenol / Q000494:pharmacology; D008024:Ligands; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D009543:Nifedipine / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology
D015220:Calcium Channels; D004095:Dihydropyridines; D008024:Ligands; D005576:Colforsin; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; C038806:1,4-dihydropyridine; D009543:Nifedipine; D007545:Isoproterenol; D011188:Potassium; D002118:Calcium