Measurement of pH in tissue has shown that the microenvironment in tumors is generally more acidic than in normal tissues. Major mechanisms which lead to tumor acidity probably include the production of lactic acid and hydrolysis of ATP in hypoxic regions of tumors. Further reduction in pH may be achieved in some tumors by administration of glucose (+/- insulin) and by drugs such as hydralazine which modify the relative blood flow to tumors and normal tissues. Cells have evolved mechanisms for regulating their intracellular pH. The amiloride-sensitive Na+/H+ antiport and the DIDS-sensitive Na+-dependent HCO3-/Cl- exchanger appear to be the major mechanisms for regulating pHi under conditions of acid loading, although additional mechanisms may contribute to acid extrusion. Mitogen-induced initiation of proliferation in some cells is preceded by cytoplasmic alkalinization, usually triggered by stimulation of Na+/H+ exchange; proliferation of other cells can be induced without prior alkalinization. Mutant cells which lack Na+/H+ exchange activity have reduced or absent ability to generate solid tumors; a plausible explanation is the failure of such mutant cells to withstand acidic conditions that are generated during tumor growth. Studies in tissue culture have demonstrated that the combination of hypoxia and acid pHe is toxic to mammalian cells, whereas short exposures to either factor alone are not very toxic. This interaction may contribute to cell death and necrosis in solid tumors. Acidic pH may influence the outcome of tumor therapy. There are rather small effects of pHe on the response of cells to ionizing radiation but acute exposure to acid pHe causes a marked increase in response to hyperthermia; this effect is decreased in cells that are adapted to low pHe. Acidity may have varying effects on the response of cells to conventional anticancer drugs. Ionophores such as nigericin or CCCP cause acid loading of cells in culture and are toxic only at low pHc; this toxicity is enhanced by agents such as amiloride or DIDS which impair mechanisms involved in regulation of pHi. It is suggested that acid conditions in tumors might allow the development of new and relatively specific types of therapy which are directed against mechanisms which regulate pHi under acid conditions.

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Acid pH in Tumors and Its Potential for Therapeutic Exploitation

5. Cyanine-Based pHi Indicators 2717 5.1. Design of pH-Sensitive Cyanine Dyes 2717 5.2. Near-Neutral Cyanine-Based pH Indicators 2718 5.3. Acidic Cyanine-Based pH Indicators 2719 6. Miscellaneous Small Molecule pHi Indicators 2719 6.1. Various Indicators for Near-Neutral pH Values 2719 6.1.1. Europium Complex 2719 6.1.2. Fluorene Derivative 2719 6.1.3. 1,4-Dihydroxyphthalonitrile (1,4-DHPN) 2720 6.1.4. 8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS) 2720

Fluorescent indicators for intracellular pH.

Present methods for measuring or buffering intracellular free calcium concentrations are almost entirely limited to robust and well anchored cells which can tolerate insertion of ion-selective microelectrodes or microinjection of calcium indicators or buffers into one cell at a time. A very few types of small cells can be loaded with buffers or indicators during controlled lysis, but such procedures grossly perturb membrane integrity and soluble cytoplasmic constituents. Liposome fusion releases only trace quantities of the trapped solute into the cytoplasm and incorporates foreign lipid into the target cell membranes. I now describe a simple technique which loads Ca2+-selective chelators into the cytoplasm of intact cells in suspension and avoids the disadvantages of previous methods. The chelators are made temporarily membrane permeable by masking their four carboxylates with special esterifying groups which then hydrolyse inside the cells, regenerating and trapping the original chelators. The method is demonstrated on red cells, mast cells and lymphocytes.

A non-disruptive technique for loading calcium buffers and indicators into cells.

Measurements have been made of cytoplasmic pH, (pHi) and free Mg2+ concentration, ( [Mg2+]i), in pig and mouse lymphocytes. pHi 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 degrees C, pHi was close to 7.0. Addition of physiological levels of HCO3- and CO2 transiently acidified the cells by approximately 0.1 U. Mitogenic concentrations of concanavalin A (Con A) had no measurable effect on pH in the first hour. [Mg2+]i was assessed in three ways: (a) from the external Mg2+ null-point at which the ionophore A23187 produced no net movement of Mg2+ or H+; (b) by Mg-sensitive electrode measurements in freeze-thawed pellets; and (c) from the 31P nuclear magnetic resonance spectrum of the gamma-phosphate of intracellular ATP. Total cell Mg2+ was approximately 12 mmol per liter cell water. The NMR data indicated [Mg2+]i greater than 0.5 mM. The null-point method gave [Mg2+]i approximately 0.9 nM. The electrode measurements gave 1.35 mM, 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 Mg2+.

/pdf/cytoplasmic-ph-and-free-mg2-in-lymphocytes-1563j4foyt.pdf

Cytoplasmic pH and free Mg2+ in lymphocytes.

The present invention provides systems, methods, screens, reagents and kits for optical system analysis of cells to rapidly determine the distribution, environment, or activity of fluorescently labeled reporter molecules in cells for the purpose of screening large numbers of compounds for those that specifically affect particular biological functions.

A system for cell-based screening

Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ.

The synthesis and some of the physical and biological characteristics of a new inhibitor of lactate transport are described. The inhibitor is isobutylcarbonyl lactayl anhydride (iBCLA). It is formed by the condensation of lactic acid and isobutylchloroformate. It inhibits lactate transport 50% at 0.5 microgram/mg of protein in both Ehrlich ascites tumor cells and human erythrocytes. In contrast, 15 microgram of iBCLA/mg of protein is required for 50% inhibition of phosphate transport in erythrocytes, and phosphate transport in Ehrlich ascites tumor cells is unaffected at levels as high as 50 microgram of iBCLA/mg of protein. A time-dependent and concentration-dependent reversal of lactate transport inhibition took place on exposure of iBCLA-treated Ehrlich ascites cells to hydroxylamine or dithiothreitol. These data, along with the observed sensitivity of the lactate transporter to sulfhydryl reagents [Spencer, T. L., & Lehninger, A. L. (1976) Biochem. J. 154, 405-414], suggest that iBCLA acylates an essential sulfhydryl group on the transporter. When glycolyzing Ehrlich ascites tumor cells were treated with concentrations of iBCLA sufficient for complete inhibition of lactate transport, intracellular lactate levels increased, intracellular pH and extra-cellular lactate levels decreased, and overall lactate production was inhibited.

Inhibition of lactate transport in Ehrlich ascites tumor cells and human erythrocytes by a synthetic anhydride of lactic acid

A chloromethyl ketone derivative of lactic acid is a potent inhibitor of glycolysis of Ehrlich ascites tumor cells. It inhibited glycolysis of intact cells by about 50% at 200 microM (100 nmol/mg of protein) while cell-free extracts were inhibited 50% at 50 microM (50 nmol/mg of protein). N alpha-(p-Tosyl)-L-lysine chloromethyl ketone and N alpha-(p-tosyl)-L-phenylalanine chloromethyl ketone inhibited only slightly or not at all at this concentration. The inhibition was localized at the hexokinase and phosphofructokinase steps since these two enzymes added to an inactivated extract restored the glycolytic activity, whereas none of the other glycolytic enzymes did. In fact, addition of pyruvate kinase or lactate dehydrogenase, which stimulated glycolysis, resulted in a more pronounced inhibition. Glycolysis and hexokinase activities in extracts of Rous sarcoma virus transformed cells were considerably more sensitive to the inhibitor than the activities from normal chick embryo fibroblasts. Hexokinase from mouse brain required 50 times higher concentrations for inhibition than the enzyme from mouse Ehrlich ascites tumor cells. Yeast hexokinase was unaffected at all concentrations tested. Since 5,5'-dithiobis(2-nitrobenzoate) protected against the inhibition, the chloromethyl ketone appeared to inhibit by interaction with an essential SH group. A pronounced inhibition of protein kinase activity of plasma membranes of Ehrlich ascites tumor cells was observed in the presence of the chloromethyl ketone. As in the case of glycolysis, the chloromethyl ketone of lactic acid was a more potent inhibitor of protein kinase activity than several other chloromethyl ketones that were tested.

Andrzej Zimniak

Papers

Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ.

Inhibition of lactate transport in Ehrlich ascites tumor cells and human erythrocytes by a synthetic anhydride of lactic acid

Inhibition of hexokinase and protein kinase activities of tumor cells by a chloromethyl ketone derivative of lactic acid.