Characterization of Signaling Pathways to Na+/H+ Exchanger Activation With Epidermal Growth Factor in Hepatocytes

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Abstract
To investigate the signaling pathways to Na+/H+ exchanger activation with epidermal growth factor in hepatocytes, we measured changes in cytosolic free calcium and intracellular pH levels at the single–cell level using digital imaging fluorescence microscopy of fura–2—or BCECF–loaded hepatocytes in primary culture. Epidermal growth factor induced cytosolic free calcium oscillations consisting of periodic trains of spikes with a latency period of up to several minutes. These calcium responses were inhibited by tyrosine kinase inhibitor genistein (100 μmol/L) and abolished by emptying of intracellular Ca2+ pools with 3 μmol/L thapsigargin, an inhibitor of Ca2+–ATPase on the endoplasmic reticulum. Epidermal growth factor (1 nmol/L) induced an intracellular pH increase of 0.12 ± 0.07 units from the basal level of 7.25 ± 0.09 units after several minutes of latency. This effect was completely abolished by 1 mmol/L amiloride, an inhibitor of the Na+/H+ exchanger. The epidermal growth factor—induced intracellular pH increase was inhibited by pretreatment of hepatocytes with genistein (100 μmol/L), thapsigargin (3 μmol/L) or calmodulin inhibitor W–7 (25 μmol/L), but not with protein kinase C inhibitor H–7 (50 μmol/L) or with cyclic AMP—dependent kinase inhibitor H–8 (60 μmol/L). Phorbol ester PMA (phorbol 12–myristate 13–acetate), a potent activator of protein kinase C, induced a slight intracellular pH increase significantly smaller than that with epidermal growth factor, whereas this effect was completely blocked by pretreatment with H–7, indicating that PMA–induced intracellular pH increase is mediated by protein kinase C pathways, unlike epidermal growth factor. These data indicate that epidermal growth factor—induced intracellular pH increase mediated by activation of Na+/H+ exchanger in primary cultured hepatocytes depends on tyrosine kinase and is downstream from Ca2+ mobilization and Ca2+–calmodulin—dependent pathways. (Hepatology 1994;20:966-974).