Measurement of rapid changes in cell volume by forward light scattering

Abstract

Light scattering is an empirical technique employed to measure rapid changes in cell volume. This study describes a new configuration for the method of light scattering and its corroboration by measurements of cell height (as a measure of cell volume). Corneal endothelial cells cultured on glass cover-slips were mounted in a perfusion chamber on the stage of an inverted microscope. A beam of light was focused on the cells from above the stage at an angle of 40 degrees to the plane of the stage. The scattered light intensity (SLI), captured by the objective and referred to as forward light scatter (FLS), increased and decreased in response to hyposmotic and hyperosmotic shocks, respectively. The rapid increase and decrease in SLI corresponded to cell swelling and shrinkage, respectively. Subsequently, SLI decreased and increased as expected for a regulatory volume decrease (RVD) and increase (RVI), respectively. These data are in agreement with measurements of cell height, demonstrating that the method of light scatter in FLS mode is useful for monitoring rapid changes in cell volume of cultured cells. Changes in SLI caused by gramicidin were consistent with cell volume changes induced by equilibration of NaCl and KCl concentrations across the cell membrane. Similarly, an additional decrease in SLI was recorded during RVD upon increasing K+ conductance by valinomycin. Decreasing K+ conductance of the cell membrane with Ba2+ changed the time course of SLI consistent with the effect of the K+ channel blocker on RVD. Bumetanide and dihydro-ouabain inhibited increases in SLI during RVI. In conclusion, FLS is a valid method for qualitative analysis of cell volume changes with a high time resolution.

Fig. 1

Typical 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) fluorescence profile when anisosmotic shocks occur. Confluent layer of endothelial cells was perfused with Cl⁻-free $\mathrm{N}{\mathrm{O}}_3^{-}$ Ringer solution. After initial osmotic equilibrium (i.e., the transitions A to B) and subsequent volume regulation were recorded, cells were returned to isosmotic Ringer at point C. F_A, F_B, F_C, and F_D are the average pixel intensities (in gray levels) at points A, B, C, and D. δF_H = F_B – F_A and δF_I = F_C – F_D represent peak changes in SPQ fluorescence due to immediate volume changes by osmotic equilibrium after exposure to anisosmotic shocks (at point A) and return to isosmotic conditions (at point C), respectively. A: at point A, perfusate was switched to a 33% hyposmotic solution. δF_R = F_B – F_C* = −15 and δF_L = F_C – F_C* = −7 represent changes in SPQ fluorescence due to volume loss by regulatory volume decrease (RVD; also shown as line segment B–C^*) and an estimated dye loss, respectively. Hypo, hyposmotic; Iso, isosmotic. B: at point A, perfusate was switched to a 20% hyperosmotic Ringer (Hyper). There is no observable regulatory volume increase (δF_R = F_B – F_C = 0). Dye loss is relatively small compared with swollen conditions δF_L = F_A – F_D = 2). Note that 1) under isosmotic conditions (Iso), all regions of interest (ROIs) showed different levels of steady-state fluorescence, indicating heterogeneity in dye loading among the cells (data not shown); 2) in response to hypotonic shock, both the rate and extent of fluorescence decreased when RVD was heterogeneous. Therefore, all calculations for determination of dye leakage and RVD should be performed for each ROI separately; 3) profiles in A and B represent typical ROIs from among some 20 ROIs selected in each of the experiments. Results are also typical of >20 experiments on separate coverslips.

Fig. 2

Relationship between SPQ fluorescence and osmolarity. A: confluent monolayer of endothelial cells was perfused with Cl⁻-free gluconate Ringer and then exposed to successive 20 mosM hyperosmotic shocks. Dye leakage curve (dotted line) was calculated assuming an exponential loss. Bracketed number pairs represent values of SPQ fluorescence at each point with and without correction for dye leakage. B: validation of Eqs. 9 and 10 are shown by a plot of [(F – F_B)/(F_I – F_B)] vs. (π/π_I). □, Uncorrected ratios; ■, ratios corrected for dye leakage. All data points are corrected for background. Solid regression line represents a regression curve after correction for dye leakage. Dotted straight line represents Eq. 10. C: segment of response from A is shown with calculation of signal-to-noise ratio (SNR). Results shown are typical of 3 experiments on separate coverslips; 5 experiments with just 2 osmotic shocks on either side of the isosmotic point showed qualitatively similar results.

Fig. 3

Influence of ouabain SPQ fluorescence and cytosolic Na⁺ ([Na⁺]_i) under isosmotic conditions. Cells, being perfused with Cl⁻- free $\mathrm{N}{\mathrm{O}}_3^{-}$ Ringer, were exposed to 200 μM ouabain and 1 μM gramicidin where indicated under isosmotic conditions. A: typical SPQ fluorescence response obtained in at least 5 experiments on separate coverslips at varying levels of ouabain (100-200 μM). B: relative changes in Na⁺-binding benzofuran isophthalate fluorescence ratio (340/380 nm). Increase in fluorescence ratio is indicative of an increase in [Na⁺]_i. Results shown are typical of at least 7 experiments on separate coverslips conducted at varying levels of gramicidin (0.5–2 μM).

Fig. 4

Influence of urea on SPQ fluorescence under isosmotic conditions. Cells were perfused with Cl⁻ free $\mathrm{N}{\mathrm{O}}_3^{-}$ Ringer. Cells were exposed to a 33% hyposmotic shock (i.e., equivalent to 100 mosM shock) and returned to isosmotic conditions after ~5 min. After another ~10 min, cells were exposed to 120 mM urea under isosmotic conditions. Y-axis shows relative change in SPQ fluorescence given by (F_I – F)/F_I, where F_I is SPQ fluorescence under isosmotic conditions. This ratio is equal to the relative change in cell volume given by (V_I – V)/V_I, where V_I is cell volume under isosmotic conditions. This follows from Eq. 8. Results shown are typical of at least 2 experiments on separate coverslips.

Fig 5

Influence of gramicidine (GC) during RVD. Confluent layer of endothelial cells was perfused with Cl⁻ free $\mathrm{N}{\mathrm{O}}_3^{-}$ Ringer. A: SPQ fluorescence after a 33% hyposmotic shock and exposure to gramicidin in presence of Na⁺. B: [Na⁺]_i levels after a 33% hyposmotic shock in presence of Na ⁺. SBFI, Na⁺-binding benzofuran isophthalate. C: SPQ fluorescence after a 33% hyposmotic shock in presence of 4 mM Na⁺. Results shown are typical of at least 6 experiments on separate cover slips.