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

1. We have investigated the effects of changing intracellular pH on intrace llular free calcium concentration ([Ca2+](i)) in voltage-clamped neurones o f 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 e ither fura-2 or calcium-sensitive microelectrodes. 2. Acidification of the neurones with 5 mM or 20 mM propionate ( similar to 0.2 or 0.3 pH units acidification, respectively) caused a small reduction i n resting [Ca2+](i) of 5 +/- 2 nM (n = 4) and 7 +/- 16 nM (n = 4), respecti vely. The removal of the 20 mill propionate after similar to 40 min superfu sion resulted in an alkalinization of similar to0.35 pH units and an accomp anying rise in resting [Ca2+](i) of 31 +/- 9 nM (n = 4, P < 0.05). The remo val of 5 mM propionate did not significantly affect [Ca2+](i). 3. Alkalinizations of <similar to>0.2-0.4 pH units of Helix neurones induce d by superfusion with 3 mM concentrations of the weak bases trimethylamine (TMA), ammonium chloride (NH4Cl) and procaine were accompanied by significa nt (P < 0.05) increases in resting [Ca2+](i) of 42 +/- 4 nM (n = 26), 30 +/ - 7 nM (n = 5) and 36 +/- 4 nh (n = 3), respectively. The effect, of TMA (0 .5-6 mM) on [Ca2+](i) was dose dependent with an increase in [Ca2+](i) duri ng 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 in hibited depolarization-induced calcium increases but not the calcium increa se produced by the first exposure to TMA (3 mM). In the prolonged absence o f extracellular calcium (<similar to>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 +/- 6% followin g a 10 min period of superfusion with caffeine (10 mM) to deplete the endop lasmic reticulum (ER) stores of calcium (P < 0.05). 6. Clyclopiazonic acid (10-30 muM CPA), an inhibitor of the En calcium pump , inhibited the calcium rise produced by TMA (3 mw) and NH,CI (3 mM) by 61 +/- 4% compared to controls (P < 0.05). These data are consistent with phys iological intracellular alkaline shifts stimulating release of calcium, or inhibiting re-uptake of calcium by an intracellular store. The calcium incr ease was much reduced following application of caffeine, treatment with CPA or prolonged removal of external calcium. Hence the ER was likely to he th e source of mobilized calcium.