Intracellular pH changes induced by calcium influx during electrical activity in molluscan neurons

Simultaneous measurements of electrical activity and light absorbance have been made on nerve cell bodies from Archidoris monteryensis injected with indicator dyes. pH indicators, phenol red and bromocresol purple, and arsenazo III, which under normal conditions is primarily a calcium indicator have been employed. Voltage clamp pulses which induced calcium influx caused an absorbance decrease of the pH dyes indicating an internal acidification. The onset of the pH drop lagged the onset of Ca2+ influx by 200-400 ms, and pH continued to decrease for several seconds after pulse termination which shut off Ca2+ influx. Trains of action potentials also produced an internal pH decrease. Recovery of the pH change required periods greater than 10 min. The magnitude of the pH change was largely unaffected by external pH in the range 6.8-8.4. The voltage dependence of the internal p/ change was similar to the voltage dependence of calcium influx determined by arsenazo III, and removal of calcium from the bathing saline eliminated the pH signal. In neurons injected with EGTA (1-5 mM), the activity- induced internal Ca2+ changes were reduced or eliminated, but the internal pH drop was increased severalfold in magnitude. After the injection of EGTA, voltage clamp pulses produced a decrease in arsenazo III absorbance instead of the normal increase. Under these conditions the dye was responding primarily to changes in internal pH. Injection of H+ caused a rise in internal free calcium. The pH buffering capacity of the neurons was measured using three different techniques: H+ injection, depressing intrinsic pH changes with a pH buffer, and a method employing the EGTA-calcium reaction. The first two methods gave similar measurements: 4-9 meq/unit pH per liter for pleural ganglion cells and 13-26 meq/unit pH per liter for pedal ganglion cells. The EGTA method gave significantly higher values (20-60 meq/unit pH per liter) and showed no difference between pleural and pedal neurons.