ABSTRACT

Using the fluorescent pH indicator 2′7′-bis(2-carboxyethyl)-5′-(6′)-carboxyfluorescein to monitor intracellular pH (pH_i), we have investigated whether transmembrane Na⁺/H⁺ exchange, as measured by experimental changes in pH_i 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 pH_i 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 pH_i 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 pH_i 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.

ABSTRACT

Using the fluorescent indicators 2′,7′-bis(2-carboxyethyl)-5′-(6′)-carboxyfluorescein and Oxonol V to monitor intracellular pH (pH_i) and cell membrane potential respectively, we have investigated the involvement of H⁺-dependent ATPase and H⁺-dependent K⁺ channels in the recovery of the rat thyroid cell strain FRTL-5 from experimentally induced cytosolic acidification and membrane hyperpolarization events. Following exposure of cells to the weak acid sodium butyrate (24mmol/l) under bicarbonate-free incubation conditions, cytoplasmic acidification was maximal after 3 min, attaining a pH_i of 6.42. The subsequent recovery of pH_i was unimpaired by the absence of extracellular K⁺, but was reduced in the presence of the Na⁺ antagonist amiloride (1 mmol/l), recovering by 0.11±0.003 units, compared with 0.27±0.02 units under amiloride-free conditions. In the presence of the H⁺-dependent ATPase antagonist N,N′-dicyclohexylcarbodiimide (DCC), the pH_i recovery observed in amiloride-containing, K⁺-free buffer was abolished.

The recovery of pH_i in Na⁺- and K⁺-containing buffer was accompanied by hyperpolarization of the cell membrane, the later stage of which was reduced after blockade of K⁺ channels with BaCl₂, implying a major contribution of transmembrane K⁺ movement to such events. In contrast to its attenuating effect on pH_i recovery, DCC was ineffective in reducing butyrate-dependent membrane hyperpolarization, suggesting that H⁺-dependent ATPase may not be a major contributory factor to this event. However, when K⁺ channels were blocked by addition of BaCl₂, addition of DCC abolished the butyrate-induced membrane depolarization. These findings are consistent with the presence of two independent hyperpolarizing transport processes in the FRTL-5 cell membrane which appear to involve (i) a H⁺-dependent ATPase, activated in response to cytosolic acidification, and allowing partial recovery of pH_i in the absence of extracellular Na⁺ and HCO₃ ⁻, and (ii) H⁺-dependent K⁺ channels which, while contributing to membrane hyperpolarization, may not play a major role in the normal maintenance of pH_i.

ABSTRACT

pH is maintained in cells by plasma membrane exchange mechanisms. In the absence of HCO₃− ions, FRTL-5 cells regulate intracellular pH (pH_i) by an Na⁺/H⁺ antiport but HCO₃−-dependent exchangers cannot operate. We have investigated pH_i regulation (by microfluorimetry and the pH sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6′)-carboxy-fluorescein) in small groups (five to six cells) of FRTL-5 thyroid cell monolayers held in kREBS—Ringer buffer (pH 7·4) with or without HCO₃− ions. The exchangers were investigated with inhibitors (amiloride or its derivative dimethylamiloride for the Na⁺/H⁺ antiporter and the stilbene derivative disodium 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) for HCO₃ −-dependent mechanisms), ionic substitution and by NH₄ ⁺/NH₃ (10mm) acid loading. Basal pH_i was lower in the presence (7·3±0·058, mean±s.d., n= 14) than in the absence (7·59±0·078, n=10) of HCO₃ ions. In HCO₃ −-free media, cells recovered from acid load by 0·34±0·04 pH units in the first 2 min and finally reached a pH_i of 7·35±0·06. This recovery was Na⁺-dependent and blocked by dimethylamiloride during the 15 min following intracellular acidification. In HCO₃ ⁻-containing media, cells recovered from an acid load at a similar rate, but reached 99 ± 10% (n = 9) of the baseline pH; this recovery was also dependent on Na⁺ ions. Moreover, although dimethylamiloride and DIDS reduced the rate of recovery to 0·06±0·02 and 0·18±0·04 pH units respectively during the 2-min period, the cells returned to the basal pHi within 15 min. Removal of Na⁺ from HCO₃ ⁻-containing media acidified the cells (ΔpH=–0·82±0·05, n=10) within 40 min; this acidification was partially blocked by either amiloride or DIDS. Removal of Cl⁻ alkalinized the cells (ΔpH=+0·51 ± 0·06, n=10) after 40 min, and this alkalinization was totally prevented by DIDS. Furthermore, in the absence of Na⁺ and presence of amiloride, alkalinization was still seen on the removal of Cl⁻, albeit at a diminished rate (i.e. ΔpH = +0·25±0·05, n=8) after 40 min. In conclusion, FRTL-5 cells maintain pH_i by two Na⁺-dependent exchangers, one sensitive to amiloride, the other to DIDS, and a Na⁺-independent, Cl⁻/HCO₃− mechanism.