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Cytoplasmic pH recovery in acid-loaded haemocytes of squid (Sepioteuthis lessoniana).

Cytoplasmic pH (pHi) of haemocytes of bigfin reef squid (Sepioteuthis lessoniana Lesson) was determined with the fluorescent probe, 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF). The pHi of haemocytes suspended in nominally HCO3(-)-free medium (extracellular pH 7.4) averaged (+/- S.E.) 7.32 +/- 0.02. Intracellular pH was independent of external Na+ concentration and varied only slightly with changes in extracellular pH (pHe) (delta pHi/delta pHe = 0.16 over the pHe range 6.8-7.8). Addition of weak acids (sodium propionate, potassium acetate) to haemocyte suspensions resulted in a rapid decrease in pHi. Haemocyte pHi then recovered with an average half-time of 3-4 min. Recovery of pHi was independent of external Na+ concentration and insensitive to amiloride, but was abolished by N-ethylmaleimide (NEM). These results argue against the involvement of plasma membrane Na+/H+ exchange or other Na(+)-dependent transport mechanisms in the pHi recovery of acid-loaded haemocytes. The results suggest that there is an NEM-sensitive proton extrusion mechanism in the plasma membrane of squid haemocytes.