Volume regulation in NIH/3T3 cells not expressing P-glycoprotein. I. Regulatory volume decrease

Abstract

Exposure of NIH/3T3 fibroblasts not expressing P-glycoprotein to 50, 30, 20, and 10% hyposmotic solutions led to cell volume increases of 70, 32, 21, and 12%, respectively. After swelling, NIH/3T3 cells exhibited regulatory volume decrease (RVD), attaining complete volume recovery after 30 min except in 50% hyposmotic solution, in which volume recovery was 76%. RVD was accelerated by gramicidin and inhibited by the Cl channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid, 1,9-dideoxyforskolin, dipyridamole, and niflumic acid and by the K channel, blocker quinidine. RVD was reduced 15% by removal of extracellular Ca. The pathway opened by hypotonicity was highly permeable to K and Rb and only partly permeable to other cations. Most anions were able to permeate, with a permeability ranking of nitrate > benzoate = iodide > thiocyanate > chloride > > gluconate. The pathway was permeable to neutral amino acids, with a permeability ranking of glycine > alanine > glutamate > taurine > gamma-aminobutyric acid > glutamine. The pathway was not permeable to basic amino acids. These results show that, despite the absence of P-glycoprotein, NIH/3T3 cells exhibit RVD with properties similar to those expressed in most cell types.

Measurement of the thickness and volume of adherent cells using transmission-through-dye microscopy

“…The kinetics of volume recovery in NIH/3T3 cells transferred into a hypotonic medium was similar to that observed by Pasantes-Morales et al [41] who used Coulter sizing after trypsinizing the cells. In some experiments presented in Supplement, cells were loaded with calcein AM ester (as described in [10]), and confocal fluorescence images were recorded alongside transmission at 633 nm; there was a clear correlation between changes in calcein fluorescence and transmitted intensities in cells that were exposed to solutions of various osmolarities.…”

“…In order to demonstrate both phases of the volume response to hypotonic shock, we performed these experiments with NIH/3T3 cells where RVD has been documented [41]. Following the cited work, the hypotonic solution was prepared with a reduced concentration of NaCl, and the volume recovery phase of the response looked qualitatively similar to that reported in the cited paper (Fig.…”

Characterization of glycerol uptake in bloodstream and procyclic forms of Trypanosoma brucei

“…Statistical analysis was performed using the Student's t-test (* significant inhibition with PϽ 0.05; *** highly significant inhibition with PϽ 0.001; all others were not significant). [14,24,25] such as DIDS and the mercury ions ClHgBzOH and ClHgBzSO 3 H. The inhibitors were added at the beginning of a 5-min incubation. Glycerol uptake was started by transferring cells into the incubation medium which contained [ 14 C]glycerol.…”

“…3). In order to investigate glycerol uptake in procyclic forms in more detail, the effect of various inhibitors known to interfere with channel proteins was tested [14,24,25]. Neither the Cl Ϫ channel inhibitors niflumic acid or 4,4′-diisothiocyanostilbene 2,2′-disulfonate (DIDS), nor the K ϩ channel inhibitor quinidine decreased glycerol uptake significantly even at extremely high concentrations (Fig.…”

Organic Osmolyte Channels: A Comparative View

“…The findings that: (i) the hypoosmotically-activated transport of organic osmolytes in a variety of cell-types is blocked by a range of anion channel blockers (reviewed in [17]), and (ii) in those cases in which there is evidence of hypoosmoticallyactivated transport of solutes of varying charge, the relative rates of transport are, in at least some cells, in the order anions > electroneutral solutes > cations (e.g. [20,21]) point to the channels involved being anion-selective [17].…”