Characteristics of Ca2+ release induced by Ca2+ influx in cultured bullfrog sympathetic neurones.

Abstract

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)