No abstract

A new, fluorescent, highly selective Ca 2÷ indicator, "quin2", has been trapped inside intact mouse and pig lymphocytes, to measure and manipulate cytoplasmic free Ca 2÷ concentrations, [Ca2+]~. Quin2 is a tetracarboxylic acid which binds Ca 2+ with 1:1 stoichiometry and an effective dissociation constant of 115 nM in a cationic background mimicking cytoplasm. Its fluorescence signal (excitation 339 nm, emission 492 nm) increases about fivefold going from Ca-free to Ca-saturated forms. Cells are loaded with quin2 by incubation with its acetoxymethyl ester, which readily permeates the membrane and is hydrolyzed in the cytoplasm, thus trapping the impermeant quin2 there. The intracellular quin2 appears to be free in cytoplasm, not bound to membranes and not sequestered inside organelles. Cytoplasmic Ca 2+ has been proposed as a key regulator of numerous cellular processes and probably plays one or more important roles in every cell type. However, measurement of the key variable, intracellular free calcium concentration, [Ca2+]i, is always technically demanding, and in small cells has thus far been almost impossible. A novel method for monitoring and manipulating [Ca2+]i in large populations of small cells has now been provided by two recent chemical inventions (29,30): (A) a new family of calcium chelators that shows high affinity for Ca 2+, very low alTmity for Mg 2+ and H +, large absorbance and fluorescence changes resulting from Ca z+-binding, and little or no detectable binding to the membranes; (B) a method for trapping such substances inside intact cells by means of nonpolar ester derivatives which cross the plasma membrane and are hydrolyzed intracellularly back to the parent membrane-impermeant polycarboxylate anions. We have used a fluorescent quinoline Ca2+-indicator (31), "quirt2", whose structure is shown in Fig. 1, to obtain the first measure-

A novel inhibitor of receptor-mediated calcium entry (RMCE) is described. SK&F 96365 (1-(beta-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl)-1H- imidazole hydrochloride) is structurally distinct from the known 'calcium antagonists' and shows selectivity in blocking RMCE compared with receptor-mediated internal Ca2+ release. Human platelets, neutrophils and endothelial cells were loaded with the fluorescent Ca2(+)-indicator dyes quin2 or fura-2, in order to measure Ca2+ or Mn2+ entry through RMCE as well as Ca2+ release from internal stores. The IC50 (concn. producing 50% inhibition) for inhibition of RMCE by SK&F 96365 in platelets stimulated with ADP or thrombin was 8.5 microM or 11.7 microM respectively; these concentrations of SK&F 96365 did not affect internal Ca2+ release. Similar effects of SK&F 96365 were observed in suspensions of neutrophils and in single endothelial cells. SK&F 96365 also inhibited agonist-stimulated Mn2+ entry in platelets and neutrophils. The effects of SK&F 96365 were independent of cell type and of agonist, as would be expected for a compound that modulates post-receptor events. Voltage-gated Ca2+ entry in fura-2-loaded GH3 (pituitary) cells and rabbit ear-artery smooth-muscle cells held under voltage-clamp was also inhibited by SK&F 96365; however, the ATP-gated Ca2(+)-permeable channel of rabbit ear-artery smooth-muscle cells was unaffected by SK&F 96365. Thus SK&F 96365 (unlike the 'organic Ca2+ antagonists') shows no selectivity between voltage-gated Ca2+ entry and RMCE, although the lack of effect on ATP-gated channels indicates that it discriminates between different types of RMCE. The effects of SK&F 96365 on functional responses of cells thought to be dependent on Ca2+ entry via RMCE were also studied. Under conditions where platelet aggregation is dependent on stimulated Ca2+ entry via RMCE, the response was blocked by SK&F 96365 with an IC50 of 15.9 microM, which is similar to the IC50 of 8-12 microM observed for inhibition of RMCE. Adhesion and chemotaxis of neutrophils were also inhibited by SK&F 96365. SK&F 96365 is a useful tool to distinguish RMCE from internal Ca2+ release, and to probe the role of RMCE in mediating functional responses of cells. However, SK&F 96365 is not as potent (IC50 around 10 microM) or selective (also inhibits voltage-gated Ca2+ entry) as would be desirable, so caution must be exercised when using this compound.

Measurements have been made of cytoplasmic pH, (pHi) and free Mg ~÷ concentration, ([Mg2+]~), in pig and mouse lymphocytes, pH~ was measured in four ways: by a digitonin null-point technique; by direct measurement of the pH of freeze-thawed cell pellets; from the 31p nuclear magnetic resonance (NMR) spectrum of intracellular inorganic phosphate; and by the use of a newly synthesized, intracellularly-trappable fluorescent pH indicator. In HEPES buffered physiological saline with pH 7.4 at 37°C, pH~ was close to 7.0. Addition of physiological levels of HCO3 and CO2 transiently acidified the cells by ~0.1 U. Mitogenic concentrations of concanavatin A (Con A) had no measurable effect on pH in the first hour. [Mg2+]~ was assessed in three ways: (a) from the external Mg 2÷ null-point at which the ionophore A23187 produced no net movement of Mg 2÷ or H÷; (b) by Mg-sensitive electrode measurements in freezethawed pellets; and (c) from the 31p nuclear magnetic resonance spectrum of the -},-phosphate of intracellular ATP. Total cell Mg 2÷ was ~12 mmol per liter cell water. The NMR data indicated [Mg2÷], >0.5 mM. The null-point method gave [Mg2+]~ -0.9 nM. The electrode measurements gave 1.35 raM, which was thought to be an overestimate. Exposure to mitogenic doses of Con A for 1 h gave no detectable change in total or free Mg 2÷.The concentration of H ÷ and free Mg 2÷ can powerfully influence many, probably most, intraceltular processes. Knowledge of the normal cytoplasmic pH (pHi) and free [Mg2+]([Mg2+]i) is thus a prerequisite for setting conditions for investigation of intracellular mechanisms, from organelle function to enzyme kinetics. Furthermore, both these ions, though more often H +, have been implicated in various aspects of cell activation, especially cell growth, differentiation, and proliferation (e.g., references 3, 6, 13, 22).One of the most widely studied model systems is mitogenic stimulation of mammalian lymphocytes by iectins such as concanavalin A (Con A). Many transductor systems are proposed as mediators of the effects of these surface ligands, including changes in ion flux and cytoplasmic ion composition (e.g. references 2, 3, 7, 25), and there has been considerable biochemical investigation into the processes of stimulation. Yet, reliable measurements of cytoplasmic pH are not available and there seem to be no data relating to free Mg 2÷. We therefore attempted to fill this gap, spurred on by a specific need for values for phi and [Mg2÷]i to calibrate our fluorescent dye measurements of cytoplasmic free-[Ca 2÷] ([Ca2+]i) (25).Many of the available techniques seemed inappropriate for lymphocytes, which are too small and elusive for microinjection of indicators or impalement with ion-selective microelectrodes.The equilibrium distribution of weak acids and bases gives not cytoplasmic pH, but some average intracellular pH for all the cell compartments (20). This method has given widely varying values in human peripheral lymphocytes, pH 6.8 to 7.4 with pHo 7.4 according to the probe chosen (3, ...