1 Stimulation of chemotaxis of human polymorphonuclear leucocytes (PMNs) with the chemoattractive peptide fMLP (N-formyl-Met-Leu-Phe) is paralleled by profound morphological and metabolic alterations like changes of intracellular pH (pH(i)) and cell shape. The present study was performed to investigate the interrelation of cell volume (CV) regulatory ion transport, pH(i) and migration of fMLP stimulated PMNs.2 Addition of fMLP to PMNs stimulated directed migration in Boyden chamber assays and was accompanied by rapid initial intracellular acidification and cell swelling.3 Inhibition of the Na+/H+ exchanger suppressed fMLP stimulated cell migration, accelerated the intracellular acidification and inhibited the fMLP-induced cell swelling.4 Step omission of extracellular Na+ caused intracellular acidification, which was accelerated by subsequent addition of gastric H+/K+ ATPase inhibitor SCH 28080, or by omission of extracellular K+ ions. In addition Na+ removal caused cell swelling, which was further enhanced by fMLP.5 H+/K+ ATPase inhibitors omeprazole and SCH 28080 inhibited stimulated migration and blunted the fMLP-induced increase in CV.6 Increasing extracellular osmolarity by addition of mannitol to the extracellular solution caused cell shrinkage followed by regulatory volume increase, partially due to activation of the Na+/H(+ )exchanger. In fMLP-stimulated cells the CV increase was counteracted by simultaneous addition of mannitol. Under these conditions the fMLP stimulated migration was inhibited.7 The antibacterial activity of PMNs was not modified by Hoe 694 or omeprazole.8 Western analysis with a monoclonal anti gastric H+/K+-ATPase beta-subunit antibody detected a glycosylated 35 kD core protein in lysates of mouse and human gastric mucosa as well as in human PMNs.9 The results indicate that fMLP leads to cell swelling of PMNs due to activation of the Na+/H+ exchanger and a K+-dependent H+-extruding mechanism, presumably an H+/K+ ATPase. Inhibition of these ion transporters suppresses the increase in CV and precludes PMNs from stimulated migration.

As shown previously, expression of Ha-ras oncogene in NIH 3T3 fibroblasts (+ras cells) increases cellular concentrations of Ins(1,4,5)P-3 and Ins(1,3,4,5)P-4 and enhances bradykinin induced Ca2+ entry [1-3]. These cells respond to low concentrations of serum or bradykinin with sustained oscillations of the cell membrane potential due to pulsatile release of calcium from internal stores and subsequent activation of calcium sensitive K+ channels [1]. Furthermore Ha-ras oncogene expression leads to depolymerization of the actin filament network and delayed increase of cell volume [4-6]. Pretreatment of the same cells not expressing the oncogene (-ras cells) with Li+ similarly increases Ins(1,4,5)P-3 and Ins(1,3,4,5)P-4 [2]. As shown in the present study, -ras cells; pretreated with Li+ similar to Ha-ras oncogene expressing cells respond to bradykinin with sustained oscillations of cell membrane potential, depolymerization of the actin filament network and increase of cell volume. The oscillations of the cell membrane potential and the depolymerization of the actin cytoskeleton can be inhibited by the calcium channel blocker lanthanum and the bradykinin induced increase of cell volume is inhibited by HOE 694, pointing to involvement of Na+/H+ exchange. The data indicate a close functional linkage of the calcium oscillations, cytoskeletal rearrangement and activation of the Na+/H+ exchanger. Thus, Lit pretreatment mimicks crucial cellular events triggered by expression of the Ha-ras oncogene. However, unlike in cells expressing the Ha-ras oncogene, Li+ pretreatment alone does not allow for growth factor-independent proliferation of the cells.

NIH 3T3 fibroblasts expressing the ras oncogene (+ras cells) respond to bradykinin, bombesin or serum with sustained oscillations of cell membrane potential reflecting oscillations of intracellular calcium activity and subsequent activation of calcium-sensitive K+ channels. In contrast, identical cells not expressing the oncogene (-ras cells) respond to bradykinin with a single, transient hyperpolarization of the cell membrane. Furthermore, +ras cells are characterized by a serum-independent proliferation, an increase in cell volume and a marked reorganization of the cytoskeleton. It has been shown previously that the calcium channel blocker nifedipine, but not verapamil and diltiazem, inhibits oscillations of cell membrane potential as well as proliferation. In this study, we have examined the effect of several calcium channel blockers (bepridil, nifedipine, verapamil, diltiazem) on the proliferation, volume and cytoskeletal reorganization of +ras cells. Bepridil (10 mumol/l), which is also shown here to inhibit oscillations of cell membrane potential, and nifedipine (10 mumol/l) caused a decrease in cell number, whereas verapamil and diltiazem (10 mumol/l each) resulted in growth rates which did not differ from untreated +ras cells. The increase in cell volume as observed in untreated +ras cells was also observed for cells treated with verapamil and diltiazem, whereas cell volumes of +ras cells treated with bepridil and nifedipine were markedly reduced and similar to the values obtained for -ras cells. In addition, bepridil and nifedipine markedly inhibited cytoskeletal rearrangement, i.e depolymerization of actin-containing stress fibers. This inhibitory effect was not observed for verapamil and diltiazem.(ABSTRACT TRUNCATED AT 250 WORDS)

In NIH 3T3 fibroblasts expressing the Ha-ras oncogene (+ ras) bradykinin leads to sustained oscillations of cell membrane potential due to oscillations of intracellular Ca2+ with subsequent activation of Ca(2+)-sensitive K+ channels. In cells not expressing the oncogene (-ras), bradykinin leads only to a single transient hyperpolarization of the cell membrane. The present study has been performed to elucidate the possible interaction of cell volume, intracellular pH and bradykinin-induced oscillations of the cell membrane potential. Bradykinin leads to cell shrinkage and intracellular alkalinization of both + ras cells and -ras cells. Inhibition of Na+/H+ exchanger by HOE 694 abolishes the bradykinin-induced alkalinization but does not significantly interfere with the bradykinin-induced oscillations of cell membrane potential. In contrast, prevention of bradykinin-induced cell shrinkage by simultaneous reduction of extracellular osmolarity blunts the oscillations. Thus, cell shrinkage stimulates bradykinin-induced oscillations of cell membrane potential. On the other hand, cell shrinkage alone does not elicit oscillations unless, in addition, Ca2+ entry is stimulated by ionomycin.

Among the sequelae of ras oncogene expression are intracellular alkalinization and increase of cell volume, both phenomena attributed at least in part to activation of the Na+/H+ exchanger. The present study was performed to elucidate the effects of HOE 694--a novel inhibitor of the Na+/H+ exchanger--on intracellular pH, cell volume, cytoarchitecture and cell proliferation of ras oncogene expressing NIH 3T3 fibroblasts. Following transient exposure of the cells to 20 mmol/l NH3/NH4+, intracellular pH decreases sharply. The following slow realkalinization is completely blocked by 10 mumol/1 HOE 694. Half-maximal inhibition is achieved by 100 nmol/l HOE 694. Cell proliferation is inhibited by HOE 694 with similar potency, whereas the increase in cell volume and cytoskeletal transformation are not prevented, even by 10 mumol/l HOE 694.