Mechanisms of pH regulation in lamprey (Lampetra fluviatilis) red blood cells

Abstract

Mechanisms regulating the red cell pH in lamprey (Lampetra fluviatilis) were studied using the ammonium chloride prepulse technique. The cells were initially incubated in a physiological saline containing 20 mmol l-1 ammonium chloride, and intracellular pH measured with the DMO technique. Ammonium chloride was then rapidly removed by centrifugation, and the changes in the intracellular pH followed. The intraerythrocytic pH is primarily regulated by an amiloride-sensitive sodium/proton exchange. When sodium is present in the incubation medium, the intracellular pH rapidly recovers from the acidification associated with the removal of ammonium chloride from the incubation. When sodium is removed from the incubation medium, intracellular pH does not recover, and when the cells are treated with 10(−3) mol l-1 amiloride in the presence of sodium, carbon dioxide and bicarbonate, the intracellular pH recovery is drastically reduced. The movements of carbon dioxide, its consecutive catalysed hydration and dissociation to protons and bicarbonate and, possibly, movements of bicarbonate out of the cell acidify the cell contents. This is shown by the observation that the steady-state intracellular pH is higher in a HEPES-buffered medium than in a CO2/HCO3(−)-buffered medium at the same extracellular pH. The acidification is dependent on cellular carbonic anhydrase activity, present in lamprey red cells, which speeds up the hydration reaction. When the action of carbonic anhydrase is inhibited by acetazolamide, removal of ammonium chloride from the incubation medium does not cause intracellular acidification.