Regulation of Intracellular pH by Toadfish (Opsanus Beta) Hepatocytes

The BCECF [2’,7’-bis(carboxyethyl)-5(6)-carboxyfluorescein] method for measurement of intracellular pH (pHi) was successfully applied to toadfish (Opsanus beta Goode and Bean) hepatocytes and used for investigating the pHi regulatory properties of the hepatocytes. As in previous studies of fish hepatocytes, toadfish hepatocyte pHi showed a marked dependence on extracellular pH (pHe) with characteristic values of about 0.15–0.2 units below pHe. Measurement of membrane potential (–33.76±3.07mV, N=3) in toadfish hepatocytes by the S¹⁴CN⁻ distribution method allowed calculation of equilibrium pHi values and, despite the close tracking of pHi and pHe, proton equilibration across the membrane was never observed. Furthermore, when toadfish hepatocytes were acidified by the NH4CI prepulse method, recovery to normal pHi values could be inhibited by addition of 0.5 mmol I⁻¹ amiloride or replacement of extracellular Na⁺, indicating that these cells possessed a Na⁺/H⁺ exchanger. These experiments led to the conclusion that pHi is regulated in toadfish hepatocytes. I next exposed toadfish hepatocytes to a physiological range of several extracellular variables that change during exercise (pHe, [lactate], [catecholamine], [glucose]), in the presence and absence of amiloride, in an attempt to determine the importance of Na⁺/H⁺ exchange to hepatocyte pHi regulation in a physiological context. The exchanger plays a significant role in post-exercise recovery from acidosis induced by changes in and pHe. Further experiments employing inhibitors of lactate metabolism (3-mercaptopicolinic acid and amino-oxyacetic acid) and amiloride established that the increased lactate metabolism associated with ‘post-exercise’ recovery creates an intracellular acid load, and that Na⁺/H⁺ exchange protects the cell from changes in pHi caused by lactate metabolism.