Regulatory volume decrease in cultured kidney cells (A6): role of amino acids.

Abstract

Volume regulation was studied in A6 epithelia grown on permeable supports by measuring cell thickness (To) while simultaneously recording short circuit current (Ix) and tmalsepithelial conductance (Gt). Lowering the tonicity of the basolateral solution (Wb) from 250 or 215 to 140 mOsm/kg elicited a rapid rise in Tc followed by a regulation of the cell volume towards control. This decrease in Tc displays the characteristics of the regulatory volume decrease (RVD). Upon restoring the isoosmotic conditions, T~ decreased rapidly below its control value. A post RVD regulatory volume increase (RVI) as described for other cell types was not observed. The subsequent reduction of the basolateral osmolality increased Tc to the level recorded at the end of the first hypoosmotic pulse. Because cell content was not altered during the isoosmotic period the second hypoosmotic challenge was isotonic with the cell and did therefore not evoke an RVD. However, the cell did not lose its ability to volume regulate since an RVD could be elicited by further reduction of 1rb from 140 to 100 mOsm/kg. The possibility of an involvement of amino acids in the RVD was tested. The amount of amino acids in the cell as well as excreted in the bath was determined by amino acid analysis. Millimolar concentrations of threonine, serine, alanine, glutamate, glycine and aspartate were found in the cell extract. The cellular amino acid concentration was 28.8 -+ 0.4 mM. The amounts of glycine, aspartate and glutamate excreted from the cell during the hypotonic treatment were significantly larger than in control conditions. The excretion of these amino acids during hypotonicity decreased the cellular amino acid concentration by 8.4 -+ 0.2 mM. This quantity cannot completely account for the RVD during the first hypotonic challenge. The addition of glycine, aspartate and glutamate to the bathing solutions, although used at concentrations higher than intracellularly, did not reduce RVD. On the contrary, this maneuver increased the amplitude of the RVD following both hypoosmotic pulses. This result suggests a stimulatory role of the amino acids on the processes responsible for the RVD.

Regulatory volume decrease in a renal distal tubular cell line (A6) I. Role of K+ and Cl−

“…Indeed, the %To l~ec value expected for an ideal osmometer is 54% whereas the experimental values ranged from 55% to 61%. As the Tc u~ values were only recorded 20 min after the solution change in period III, the almost ideal osmotic behaviour of the cells confirms the absence of a post-RVD RVI, as described in a previous paper [7]. Further insight into the nature of the mechanism responsible for CI-excretion could be obtained by the use of C1 -channel blockers.…”

“…In several cell types it has been demonstrated that the RVD is mainly mediated by amino acid and/or KC1 excretion [24]. Our previous study [7] focused on the role of amino acids in the development of the RVD and demonstrated an effect of these compounds which could not fully account for the RVD. In this paper, we test the involvement of K + and C1-.…”

“…The number of beads which could be used from an individual experiment varied between five and ten. 937 exponential function to the averaged Tc curve of the entire population of beads [7]:…”

“…Most cells exposed to hypotonic solutions swell rapidly and decrease their volume towards control, whereas they will increase their volume after shrinkage in hypertonic solutions [20]. In a previous paper [7] we demonstrated that A6 epithelial cells, like most cell types, demonstrate a regulatory volume decrease (RVD) after swelling in diluted basolateral solutions. These cells were unable to develop a post-RVD regulatory volume increase (RVI) and thus restore their initial cell volume.…”

Functional Cftr in crypt epithelium of organotypic enteroid cultures from murine small intestine

“…Crypt epithelial volume and epithelial cell height measured on optical cross sections were used as an index of cell volume, as previously described for polarized epithelium (16,20,61). Enteroids from sex-matched, littermate WT and Cftr KO mouse pairs were grown on glass chamber slides.…”