Cell cycle-related changes in the ability to regulate cell volume following hyposmotic swelling were studied in mouse fibroblasts using videomicroscopy and the whole-cell patch clamp technique. Regulatory volume decrease (RVD) and volume-sensitive Cl- conductance (G(Cl,vol)) were measured: (1) in proliferating cells of different sizes; (2) in cells arrested in defined phases of the cell cycle (G1, G1/S, S, and M phases) using mevastatin, mimosine, hydroxyurea, aphidicolin, cytosine beta-D-arabinofuranoside, and taxol; and (3) in serum-starved cells (G(0) state). Cells in all groups were able to undergo RVD, although the cells approaching mitosis (i.e., the largest cells in proliferating cultures and the taxol-treated cells) had the lowest rates of shrinkage during RVD. In agreement with this finding, the density of G(Cl,vol) was stable in proliferating and cell cycle-arrested cells for most of the cell cycle, with the exception of the cells approaching mitosis and the new daughter cells where the density was decreased to half. The impairment of RVD was greatest in serum-starved cells which also had the lowest density of G(Cl,vol). We conclude that proliferating cells maintain an ability to recover from osmotic swelling as they progress through the cell cycle, although this ability may be compromised during mitosis.

The effects of inhibitors of protein tyrosine kinases (PTKs) on the Cl(-) current (I(Cl(vol))) through volume-regulated anion/chloride (VRAC) channels whilst manipulating cellular ATP have been studied in mouse fibroblasts using the whole-cell patch clamp technique. Removal of ATP from the pipette-filling solution prevented activation of the current during osmotic cell swelling and when the volume of patched cells was increased by the application of positive pressure through the patch pipette to achieve rates exceeding 100%/min. Equimolar substitution of ATP in the pipette solution with its non-hydrolyzable analogs, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) or adenylyl-(beta,gamma-methylene)-diphosphonate (AMP-PCP), not only supported activation of the current but also maintained its amplitude. The PTK inhibitors, tyrphostins A25, B46, 3-amino-2,4-dicyano-5-(4-hydroxyphenyl)penta-2,4-dienonitrile++ + and genistein (all at 100 microM), inhibited I(Cl(vol)) in a time-dependent manner. Tyrphostin A1, which does not inhibit PTK activity, did not affect the current amplitude. The PTK inhibitors also inhibited I(Cl(vol)) under conditions where ATP in the pipette was substituted with ATPgammaS or AMP-PCP. We conclude that in mouse fibroblasts ATP has a dual role in the regulation of the current: it is required for protein phosphorylation to keep VRAC channels operational and, through non-hydrolytic binding, determines the magnitude of I(Cl(vol)). We also suggest that tyrosine-specific protein kinases and phosphatases exhibit an interdependent involvement in the regulation of VRAC channels.

The Cl(-) channels of brown adipocytes electrophysiologically resemble outwardly rectifying Cl(-) channels (ORCC). To study tentative Ca(2+) regulation of these channels, we attempted to control Ca(2+) levels at the cytoplasmic side of the inside-out membrane patches with Ca(2+)-chelating agents. However, we found that the commonly used Ca(2+)-chelators EGTA and BAPTA by themselves influenced the Cl(-) channel currents, unrelated to their calcium chelating effects. Consequently, in this report we delineate effects of Ca(2+)-chelators (acting from the cytoplasmic side) on the single Cl(-) channel currents in patch-clamp experiments. Using fixed (1-2 mM) concentrations of chelators, two types of Cl(-) channels were identified, as discriminated by their reaction to the Ca(2+)-chelators and by their conductance: true-blockage channels (31 pS) and quasi-blockage channels (52 pS). In true-blockage channels, EGTA and BAPTA inhibited channel activity in a classical flickery type manner. In quasi-blockage channels, chelators significantly shortened the duration of individual openings, as in a flickering block, but the overall channel activity tended to increase. This dual effect of mean open time decrease accompanied by a tendency of open probability to increase we termed a quasi-blockage. Despite the complications due to the chelators as such, we could detect a moderate inhibitory effect of Ca(2+). The anionic classical Cl(-) channel blockers DIDS and SITS could mimic the true/quasi blockage of EGTA and BAPTA. It was concluded that at least in this experimental system, standard techniques for Ca(2+) level control in themselves could fundamentally affect the behaviour of Cl(-) channels.

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