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Journal of Experimental Biology, Vol 189, Issue 1 179-198, Copyright © 1994 by Company of Biologists

JOURNAL ARTICLES

Intracellular pH of giant salivary gland cells of the leech Haementeria ghilianii: regulation and effects on secretion

WA Wuttke, T Munsch and MS Berry
Biomedical and Physiological Research Group, School of Biological Sciences, University College of Swansea, Singleton Park, Wales, UK.

1. 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. 2. 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. 3. 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+. 4. 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. 5. 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. 6. 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. 7. 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 microgram ml-1), however, produced a rapid and reversible acidification. 8. 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. 9. 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.(ABSTRACT TRUNCATED AT 400 WORDS)




© The Company of Biologists Ltd 1994