F-19-NMR STUDY OF PRIMARY HUMAN T-LYMPHOCYTE ACTIVATION - EFFECTS OF MITOGEN ON INTRACELLULAR PH

Intracellular pH of purified human T lymphocytes was studied using nuc lear magnetic resonance (NMR) spectroscopy under physiological conditi ons. In this paper we introduce a new improved F-19-NMR pH probe, 2-am ino-3,3'-difluoroisobutyric acid (vic-difluoro-alpha-methylalanine nin e), which has a pK(a) of similar to 7.0, sensitivity of 0.83 ppm/pH, i s noncytotoxic, and provides better signal-to-noise ratio for intracel lular pH determinations. Quiescent and stimulated lymphocytes display different homeostatic intracellular pH values. Quiescent cells maintai n 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 wi th ionomycin plus phorbol 12-myristate 13-acetate leads to intracellul ar alkalinization within 90 min, reaching the more alkaline steady-sta te value of 7.25 within 7-10 h. Proliferation, but not viability, of l ymphocytes is dependent on extracellular pH in the range of 6.4-8.0, a nd this dependence is not due to limiting interleukin-2 elaboration. T he mechanisms of pH regulation and the possible implications of a perm issive pH for T lymphocyte proliferation are discussed.