Quiescent mouse NIH 3T3 cells responded to microinjection of activated ras p21 with a rapid and sustained rise in intracellular pH (-0.17 pH units). The p21-induced pH change was inhibited by amiloride treatment or growth of cells in medium low in sodium, suggesting a role for the Na+/H+ antiporter. Amiloride was found to suppress p21-induced mitosis, also.A small set of cellular genes, termed proto-oncogenes, appear to be frequent targets for genetic alterations leading to cancer. One family of such oncogenes, the ras gene family, has been directly implicated in the malignant process in both animals and humans. The normal ras gene product, a 21,000-molecular-weight protein, exhibits GTP and GDP binding as well as an associated GTPase activity (14,30,34). Mutations that commonly activate the transforming properties of ras genes involve point mutations around codon 12 or 61 (4,13,21,31,35,39). It has been shown that microinjection of activated ras p21 into quiescent fibroblasts results in transient morphological transformation and cell proliferation (6,11,33). Microinjection of the unaltered proto-oncogene p21 failed to produce a response. Thus, microinjection of activated p21 into quiescent cells provides an excellent system for examining the rapid biological effects of this transforming protein on normal cells (1).One of the earliest effects of a variety of growth factors on quiescent cells is a rapid influx of Na+ into the cell. Stimulation of Na+ influx results from activation of a membranebound Na+/H+ antiporter and leads to cytoplasmic alkalinization (16,20,25,32). There is recent evidence that increased intracellular pH (pHj) may be an important determinant for the initiation of DNA synthesis (19). In this study we have developed a novel system for monitoring pHi in single cells following the microinjection of ras p21 to assess the role of pHi changes in mediating the mitogenic effect of p21.Our assay of pHi utilized the pH-sensitive indicator 2',7'bis-(carboxyethyl)-carboxy fluorescein (BCECF) (22). Rodent fibroblasts were labeled by exposure to the permeant ester derivative BCECF-tetraacetoxymethyl ester at 10-5 M for 1 h, and fluorescence was monitored by microspectrofluorometry with a Farrand microscope spectrum analyzer. Emissions were collected from a single cell with a pinhole spotting device. The fluorescence signal was recorded at excitation and emission maxima of 490 and 550 nm, respectively. In these studies fluorescence was translated into pH with a calibration curve generated by the nigericin equilibration method (23, 36). Cells were incubated in buffers containing the K+/H+ ionophore nigericin at various pH values. It was found that BCECF-labeled fibroblasts exhibited a nearly linear relationship between fluorescence and pH over a pH range of 6.4 to 7.4 (Fig. 1A). To test the sensitivity of * Corresponding author. Following an NH4-induced acid load, pH, recovery was rapid and complete in different cells in 3 to 8 min (-70% of cells responded, n = 50) (Fig. 1B). Since phorbol esters have been ...
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