Intracellular pH of giant salivary gland cells of the leech Haementeria ghilianii: regulation and effects on secretion
Authors: Wuttke Werner A. , Munsch Thomas , Berry Michael S. 
Published: April 2021

ABSTRACT
Intracellular pH (pHi) and membrane potential (Em) of giant salivary gland cells of the leech, Haementeria ghilianii, were measured with double-barrelled, neutral-carrier, pH-sensitive microelectrodes. Em was -51±11.2 mV and pHi was 6.98±0.1 (mean ± S.D., N=41) in Hepes-buffered saline (nominally HCO3−-free; extracellular pH, pHe=7.4). pHi was independent of Em. Amiloride (2 mmol l−1) had no effect on resting pHi or on pHi recovery from an acid load (induced by the NH4+ pre-pulse technique). Removal of external Na+ produced a progressive acidification which was blocked by amiloride, and the drug also slowed the recovery of pHi on reintroduction of Na+. The results indicate the presence of an electroneutral Na+/H+ exchanger whose access to amiloride is competitively blocked by Na+. In certain smaller cells of the gland, which probably form a separate population, removal of external Na+ did not affect pHi, and recovery from an acid load was blocked by amiloride. There may, therefore, be two types of Na+/H+ exchanger, differing in reversibility and sensitivity to amiloride. Recovery of pHi from NH4+-induced acid loading was not affected by bicarbonate-buffered saline (2 % CO2; 11 mmol l−1 HCO3−) or by addition of the anion-exchange blocker SITS (10−4 mol l−1). This suggests that there is no significant contribution of a HCO3−-dependent transport mechanism to pHi regulation in the gland cells. Removal of external Cl− slowly reduced pHi and there was a transient increase (overshoot) in pHi when Cl− was reintroduced. These effects of Cl− are probably explained by changes in the Na+ gradient. Intracellular Na+ and Cl− activities were measured with ion-selective microelectrodes. Acidification with NH4+ was difficult, probably because of the cells’ poor permeability to this ion. Attempts to introduce NH4+ via the Na+ pump or Na+/Cl− transporter were not successful. The H+/K+ ionophore nigericin (1 μg ml−1), however, produced a rapid and reversible acidification. N-methylmaleimide (0.5–1 mmol l−1), which blocks proton-pumping ATPase, produced a prolonged acidification of almost 1 pH unit, well beyond the level expected for simple equilibration with pHe. The results are consistent with the presence of a vesicular proton pump, acidifying the secretory vesicles which pack the cell body. NH4+ (50 mmol l−1) or trimethylamine (50 mmol l−1) increased pHi and stimulated salivary secretion, while propionate (50 mmol l−1) decreased pHi and stopped secretion. The secretory effects of NH4+ were associated with little change in intracellular free Ca2+ concentration (measured with Fura-2) compared with the change produced by high extracellular K+ concentration ([K+]e, 100 mmol l−1) which weakly stimulated secretion. Stimulation of secretion by Ca2+-free saline or high [K+]e had no effect on pHi. We conclude that changes in intracellular pH probably do not represent a causal step in excitation–secretion coupling in the gland cells, but have a potential modulatory role.