Intracellular Ca2+ regulation by the leech giant glial cell

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Abstract
We have measured the intracellular Ca2+ concentration, [Ca2+]i, and the intracellular Na+ concentration, [Na+]i, with the fluorescent dyes fura‐2 (for Ca2+) and SBFI (for Na+) in situ in giant glial cells of the central nervous system of the leech Hirudo medicinalis. The basal [Ca2+]i was 79 ± 35 nm (n= 27) in cells voltage clamped at −70 to −80 mV, and 75 ± 29 nm (mean ±s.d., n= 82) in unclamped cells at a mean membrane potential of −67 ± 6 mV. Removal of external Na+ evoked a small reversible [Ca2+]i increase of 29 ± 21 nm (n= 27) in cells voltage clamped at −70 to −80 mV, and of 35 ± 18 nm (n= 37) in unclamped cells. This [Ca2+]i increase, and the time constant of the subsequent [Ca2+]i recovery after Na+ re‐addition, did not change significantly with the holding potential between −110 and −60 mV. The basal [Na+]i was 5.6 ± 1.3 mm (n= 18). Increasing [Na+]i by inhibiting the Na+‐K+ pump with 100 μm ouabain had no effect on the [Ca2+]i rise upon removal of external Na+. The time course of recovery from a [Ca2+]i load mediated by voltage‐dependent Ca2+ influx during depolarization in high K+ was unaffected by the removal of external Na+. Cyclopiazonic acid (10 μm), an inhibitor of the endoplasmic reticulum Ca2+‐ATPase, caused a transient increase in [Ca2+]i of 28 ± 11 nm (n= 5), and significantly slowed the recovery from imposed [Ca2+]i loads. Iontophoretic injection of orthovanadate, an inhibitor of P‐type ATPases including the plasma membrane Ca2+‐ATPase, caused a persistent increase in the basal [Ca2+]i of 163 ± 101 nm (n= 5) in standard saline, and of 427 ± 338 nm in Na+‐free saline (n= 5). Vanadate injection significantly slowed the recovery from [Ca2+]i loads. Removal of external Na+ during vanadate injection induced an additional, reversible [Ca2+]i increase of 254 ± 64 nm (n= 3). The results suggest that the low basal [Ca2+]i in these glial cells is predominantly maintained by a Ca2+‐ATPase in the plasma membrane. This ATPase is also the main Ca2+ extruder after an intracellular Ca2+ load, while intracellular stores appear to contribute little to this recovery. A Na+‐Ca2+ exchanger seems to play a minor role in the maintenance of basal [Ca2+]i in these cells, but becomes prominent when the plasma membrane Ca2+‐ATPase is blocked.