Characterization of Na+/H+ exchange in FRTL-5 thyroid cells. Evidence for dependence on activation of protein kinase C.
Na+/H+ exchange activity was investigated in cultured rat thyroid follicular FRTL-5 cells using the pH sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Basal intracellular pH (pHi) was 7.13 +/- 0.10 in cells incubated in Hepes-buffered saline solution. The intracellular buffering capacity beta i was determined using the NH4Cl-pulse method, yielding a beta i value of 85 +/- 12 mM/pH unit. The relationship between extracellular Na+ and the initial rate of alkalinization of acid-loaded cells showed simple saturation kinetics, with an apparent Km value of 44 +/- 26 mM, and an Vmax value of 0.3 +/- 0.01 pH unit/min. The agonist-induced activation of Na+/H+ exchange was investigated in cells acidified with nigericin. Addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) or ATP induced rapid cytosolic alkalinization in acid-loaded cells. The action of both TPA and ATP was abolished by preincubating the cells with 100 microM amiloride, by substituting extracellular Na+ with equimolar concentrations of choline+, and by pretreating the cells with TPA for 24 h. Chelating extracellular Ca2+, or depleating intracellular Ca2+ pools did not affect the ATP-induced alkalinization. The results indicate, that FRTL-5 cells have a functional Na+/H+ exchange mechanism. Furthermore, stimulation of protein kinase C activity is of importance in activating the antiport.
Törnquist
K
K
Endocrine Research Laboratory, University of Helsinki, Minerva Foundation Institute for Medical Research, Finland.
Alinen
S
S
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002021:Buffers; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation; D005452:Fluoresceins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008985:Monensin / Q000494:pharmacology; D009550:Nigericin / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013961:Thyroid Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D002021:Buffers; D002352:Carrier Proteins; D005452:Fluoresceins; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D000255:Adenosine Triphosphate; D008985:Monensin; D012964:Sodium; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D009550:Nigericin