19F-NMR study of primary human T lymphocyte activation: effects of mitogen on intracellular pH

Intracellular pH of purified human T lymphocytes was studied using nuclear magnetic resonance (NMR) spectroscopy under physiological conditions. In this paper we introduce a new improved 19F-NMR pH probe, 2-amino-3,3'-difluoroisobutyric acid (vic-difluoro-alpha-methylalanine), which has a pKa of approximately 7.0, sensitivity of 0.83 ppm/pH, is noncytotoxic, and provides better signal-to-noise ratio for intracellular pH determinations. Quiescent and stimulated lymphocytes display different homeostatic intracellular pH values. Quiescent cells maintain intracellular pH of 7.04 +/- 0.03 at extracellular pH values between 6.9 and 7.3, and stimulated cells maintain intracellular pH of 7.25 +/- 0.05 at extracellular pH values between 7.0 and 7.5. Stimulation with ionomycin plus phorbol 12-myristate 13-acetate leads to intracellular alkalinization within 90 min, reaching the more alkaline steady-state value of 7.25 within 7-10 h. Proliferation, but not viability, of lymphocytes is dependent on extracellular pH in the range of 6.4-8.0, and this dependence is not due to limiting interleukin-2 elaboration. The mechanisms of pH regulation and the possible implications of a permissive pH for T lymphocyte proliferation are discussed.