CHARACTERISTICS OF CA2+ RELEASE INDUCED BY CA2+ INFLUX IN CULTURED BULLFROG SYMPATHETIC NEURONS

1. A rise in intracellular Ca2+ ([Ca2+]i) and a Ca2+ current (I(Ca)) 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 Ca2+-free, EGTA solution, reduced by lowering the extracellular Ca2+ concentration ([Ca2+]i) to 0.45 or 0-9 mm and enhanced by raising [Ca2+]o to 7.2 or 14.4 mm. 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 I(Ca) (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 muM) and ryanodine (50 muM), 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. 9. When two [Ca2+]i transients were induced by a pair of pulses (- 60 to 0 mV, 100 ms), the second transient was facilitated. The facilitation decreased in amplitude as the interval between the two pulses became longer, and was not seen when the second pulse was applied after the tail of the first transient. When the facilitation was induced by triple pulses, however, the facilitation of the third [Ca2+]i transient was greater at a relatively longer preceding interval. The facilitation was completely blocked by dantrolene (10 mum). 10. The amplitudes of both I(Ca) and [Ca2+]i transient showed a bell-shaped voltage dependence. There was a quantitative difference, however, in such a way that the unit [Ca2+]i transient was enhanced by membrane depolarization in the range - 35 to - 10 mV, and constant at more depolarized levels. 11. These results suggest that not only Ca2+ entry but also the resultant Ca2+ release from intracellular calcium stores are involved in a [Ca2+]i transient in cultured bullfrog sympathetic ganglion cells. Possible mechanisms for the graded activation of Ca2+-induced Ca2+ release are discussed.