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The effects of intracellular pH changes on resting cytosolic calcium in voltage-clamped snail neurones

Debbie Willoughby, Roger C. Thomas and Christof J. Schwiening

1. We have investigated the effects of changing intracellular pH on intracellular free calcium concentration ([Ca²⁺]_i) in voltage-clamped neurones of the snail Helix aspersa. Intracellular pH (pH_i) was measured using the fluorescent dye 8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS) and changed using weak acids and weak bases. Changes in [Ca²⁺]_i were recorded using either fura-2 or calcium-sensitive microelectrodes.

2. Acidification of the neurones with 5 mM or 20 mM propionate (~0.2 or 0.3 pH units acidification, respectively) caused a small reduction in resting [Ca²⁺]_i of 5 ± 2 nM (n = 4) and 7 ± 16 nM (n = 4), respectively. The removal of the 20 mM propionate after ~40 min superfusion resulted in an alkalinization of ~0.35 pH units and an accompanying rise in resting [Ca²⁺]_i of 31 ± 9 nM (n = 4, P < 0.05). The removal of 5 mM propionate did not significantly affect [Ca²⁺]_i.

3. Alkalinizations of ~0.2-0.4 pH units of Helix neurones induced by superfusion with 3 mM concentrations of the weak bases trimethylamine (TMA), ammonium chloride (NH₄Cl) and procaine were accompanied by significant (P < 0.05) increases in resting [Ca²⁺]_i of 42 ± 4 nM (n = 26), 30 ± 7 nM (n = 5) and 36 ± 4 nM (n = 3), respectively. The effect of TMA (0.5-6 mM) on [Ca²⁺]_i was dose dependent with an increase in [Ca²⁺]_i during pH_i increases of less than 0.1 pH units (0.5 mM TMA).

4. Superfusion of neurones with zero calcium (1 mM EGTA) Ringer solution inhibited depolarization-induced calcium increases but not the calcium increase produced by the first exposure to TMA (3 mM). In the prolonged absence of extracellular calcium (~50 min) TMA-induced calcium rises were decreased by 64 ± 10% compared to those seen in the presence of external calcium (P < 0.05).

5. The calcium rise induced by TMA (3 mM) was reduced by 60 ± 5% following a 10 min period of superfusion with caffeine (10 mM) to deplete the endoplasmic reticulum (ER) stores of calcium (P < 0.05).

6. Cyclopiazonic acid (10-30 M CPA), an inhibitor of the ER calcium pump, inhibited the calcium rise produced by TMA (3 mM) and NH₄Cl (3 mM) by 61 ± 4% compared to controls (P < 0.05).

7. These data are consistent with physiological intracellular alkaline shifts stimulating release of calcium, or inhibiting re-uptake of calcium by an intracellular store. The calcium increase was much reduced following application of caffeine, treatment with CPA or prolonged removal of external calcium. Hence the ER was likely to be the source of mobilized calcium.

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