Characteristics of Ca2+ release induced by Ca2+ influx in cultured bullfrog sympathetic neurones.
1. A rise in intracellular Ca2+ ([Ca2+]i) and a Ca2+ current (ICa) induced by a depolarizing pulse were simultaneously recorded by fura-2 or indo-1 fluorescence and whole-cell patch clamp techniques in cultured bullfrog sympathetic ganglion cells. 2. [Ca2+]i (calculated from the ratio of fura-2 fluorescences excited at 380 and 340 nm and recorded with a photomultiplier at > 492 nm) rose regeneratively (in most cells) during a command pulse (from -60 to 0 mV, 100 ms), continued to rise thereafter, peaked at 666 ms (on average) and decayed slowly with a half-decay time of 22.8 s. 3. Scanning a single horizontal line across the cytoplasm with an ultraviolet argon ion laser (351 nm) and recording indo-1 fluorescences at two wavelengths (peaked at 410 and 475 nm) with a confocal microscope demonstrated that [Ca2+]i beneath the cell membrane rose much faster than that in the deeper cytoplasm. The time course of the spatial integral of [Ca2+]i, however, corresponded well with that recorded with fura-2 fluorescence using a photomultiplier. 4. [Ca2+]i measured by fura-2 fluorescence ratio using a photomultiplier did not increase during a strong depolarizing pulse (-60 to +80 mV), but sometimes rose after the pulse. A depolarization-induced rise in [Ca2+]i ([Ca2+]i transient) was blocked in a Ca(2+)-free, EGTA solution, reduced by lowering the extracellular Ca2+ concentration ([Ca2+]o) to 0.45 or 0.9 mM and enhanced by raising [Ca2+]o to 7.2 or 14.4 nM. 5. The extracellular Ca2+ dependence was non-linear when long depolarizing pulses (up to 500 ms) were applied; the amplitude of [Ca2+]i transient/Ca2+ entry (unit [Ca2+]i transient) increased with an increase in Ca2+ entry. 6. Increasing the duration of depolarization (-50 or -60 to 0 mV) from 20 to 500 ms enhanced asymptotically the integral of ICa (due to inactivation), and progressively the magnitude of [Ca2+]i transients, leading to the apparent non-linear dependence of unit [Ca2+]i transient on Ca2+ entry as well as on the duration of membrane depolarization. The peak time of [Ca2+]i transient was unchanged for pulse durations up to 300 ms, but prolonged with an increase in pulse duration to 500 ms. 7. Inhibitors of Ca2+ release from intracellular Ca2+ reservoirs, dantrolene (10 microM) and ryanodine (50 microM), blocked the [Ca2+]i transient to 56 and 30%, respectively, of the control. 8. The higher the basal [Ca2+]i level, the greater was the magnitude of the [Ca2+]i transients.(ABSTRACT TRUNCATED AT 400 WORDS)
Hua
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SY
Department of Physiology, Saga Medical School, Japan.
Nohmi
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Kuba
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D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D004558:Electric Stimulation / Q000379:methods; D005110:Extracellular Space / Q000378:metabolism; D005456:Fluorescent Dyes; D016257:Fura-2; D005728:Ganglia, Sympathetic / Q000166:cytology / Q000378:metabolism*; D007211:Indoles; D007425:Intracellular Membranes / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D009994:Osmolar Concentration; D011892:Rana catesbeiana
D002121:Calcium Channel Blockers; D005456:Fluorescent Dyes; D007211:Indoles; C048960:indo-1; D002118:Calcium; D016257:Fura-2