Using the fluorescent pH indicator 2′7′-bis(2-carboxyethyl)-5′-(6′)-carboxyfluorescein to monitor intracellular pH (pHi), we have investigated whether transmembrane Na+/H+ exchange, as measured by experimental changes in pHi under bicarbonate-free incubation conditions, may be involved in the early growth-promoting actions of insulin-like growth factor-I (IGF-I) on the rat thyroid cell stain FRTL-5. In initial studies to characterize Na+/H+ exchange in FRTL-5 cell suspensions, the recovery of a resting pHi in acid-loaded cells was shown to be dependent upon the presence of extracellular Na+, was enhanced by the presence of the sodium ionophore monensin and was abolished by amiloride, an antagonist of Na+/H+ antiport activity.
Unlike TSH, which was without effect on the pHi of FRTL-5 cells for up to 15 min after addition, IGF-I (1000 μg/1) caused a rapid and sustained increase within 3 min, which was abolished in medium in which Na+ had been replaced with an iso-osmotic level of choline chloride. The change in pHi in response to IGF-I was mimicked by phorbol 12-myristate 13-acetate (PMA; 100 nmol/1), an activator of thyroid cell proliferation. In the presence of TSH, exposure of cells to IGF-I or PMA had no additional effect on the cytoplasmic alkalinization induced by either of these two agonists alone. However, blockade of transmembrane Na+/H+ exchange with amiloride inhibited both the individual actions of IGF-I and PMA on [methyl-3H]thymidine incorporation, and the synergistic interaction between TSH and IGF-I. These findings are consistent with a differential mode of action of TSH and IGF-I on the early events associated with FRTL-5 cell proliferation, and suggest involvement of an amiloride-sensitive transmembrane Na+/H+ exchange in mediating the early cellular response to the latter. Furthermore, the later actions of PMA, which are also dependent upon early transmembrane Na+/H+ exchange, differ from those of IGF-I with respect to interaction with the growth-promoting actions of TSH.