Limits to the early increase in free cytoplasmic calcium concentration during the mitogenic stimulation of lymphocytes

Abstract

Three aspects of the calcium hypothesis we have proposed previously [Metcalfe, Pozzan, Smith & Hesketh (1980) Biochem. Soc. Symp. 45, 1-26] for the control of mitogenic stimulation of lymphocytes are examined in studies on the mitogenic action of the Ca2+ ionophore A23187 and its effect on cap formation. (1) Pig lymphocytes that were mitogenically stimulated by continuous incubation with 3H-labelled A23187 for 48 h contained between 3 and 15 amol of ionophore per cell. Lymphocytes exposed to 3H-labelled A23187 for 2h before washing the cells and resuspending them in ionophore-free medium were only stimulated mitogenically at 48h if the residual ionophore associated with the cells after washing was in the concentration range 3-15 amol per cell. When the cells were washed repeatedly after 2h incubation with ionophore to reduce the cell-associated ionophore below the critical concentration range, no mitogenic stimulation occurred as a result of short-term exposure to any ionophore concentration. Re-addition of ionophore to within the indicated range of cell-associated concentrations restored mitogenic stimulation at 48h. We conclude that large, short-term Ca2+ fluxes into the cells induced by the ionophore cannot generate a mitogenic signal that commits the cells to enter the cell cycle. (2) Further experiments with the ionophore showed that detectable mitogenic stimulation at 48h required a minimum of 3h exposure to optimal ionophore concentrations, and that maximal stimulation required at least 20h exposure. This is consistent with the view that a prolonged increase in the free cytoplasmic calcium concentration is required to stimulate the maximum proportion of the cells into the cell cycle. (3) Mouse splenic lymphocytes treated for short periods with very high ionophore concentrations (30 microM) in the presence of various external Ca2+ concentrations showed significant inhibition of cap formation of surface immunoglobulin receptors in the range 1-10 microM-Ca2+ in normal or depolarizing medium. We conclude that mitogens at optimal concentrations for the stimulation of lymphocytes do not cause any early increase in the free cytoplasmic Ca2+ concentration above 10 microM.

Effects of sulphonylureas and diazoxide on insulin secretion and nucleotide‐sensitive channels in an insulin‐secreting cell line

“…The MgCl2 concentration was increased to 1.1 mm in order to compensate for Mg2+ chelation by ATP. This value was calculated by the programme described by Hesketh et al (1983). Drugs were applied to cells or cell-free membrane patches by superfusing the whole bath using a continuous gravity feed system and continuous expiration by suction.…”

Effects of growth factors on cytosolic free calcium concentration and DNA synthesis in cultured rat aortic smooth muscle cells.

“…Cytosolic calcium has been shown to regulate some important cellular functions such as secretion of biological products, muscle contraction, proliferation and gene expression in various types of cultured cells (Campbell 1983;Hesketh et al 1983). Our results indicate that cytosolic calcium might be involved also in the proliferation of vascular SMCs provoked by EGF, because pretreatment of the cells with calcium antagonist, nifedipine, suppressed both the enhancement of DNA synthesis and the increase in cytosolic calcium concentration provoked by EGF.…”

“…The intensity of fluorescence of calcium free quin 2 (F,,) was calculated according to the equation (Hesketh et al 1983) ; Fm;n =0.16 X (Fmax -background) -background…”

“…The background, which means autofluorescence from cells and coverslips, was obtained by adding 0.5 mM of MnC12 (Hesketh et al 1983). Finally, [Ca++] i was calculated according to the equation ;…”

Hydrolysis of Phosphatidylinositol 4, 5‐Bisphosphate and Increase in Cytosolic Free Ca2+‐Concentration Induced in a Human T Cell Leukemia Line, JURKAT, by Monoclonal Antibodies against the T3 Complex

“…Excitation and emission wavelengths were 339 and 492 nm with 2 and 10 nm slits, respectively. Fluorescence was calibrated in terms of [Ca2li from the fluorescence value at 0.9 mm Ca2+ in the presence of 0.1 % Triton X-100 and the fluorescence value at 0.5 mm MnC12 in the presence of 0.1 % Triton X-100 (8,24). Triton X-100 was added from a 10% (v/v) solution.…”