Regulation of cell volume and [Ca2+]i in attached human fibroblasts responding to anisosmotic buffers

Alteration of the cell volume of attached fibroblasts with anisosmotic buffers was used to examine the relationship between cell membrane perturbation and intracellular Ca2+ concentration ([Ca2+]i) and to study pathways that may be involved in transducing this response. Human periodontal ligament gingival fibroblasts grown on cover slips were loaded with fura 2-acetoxymethyl ester. The relative cell volume change of single fibroblasts was estimated by measurement of fluorescence intensity at the isosbestic wavelength (356 nm), and [Ca2+]i was calculated from ratiometric fura 2 emission with excitation at 345 and 380 nm. Isotonic buffer (300 mosmol/kgH2O) was substituted with either hypertonic (600 mosmol/kgH2O) or hypotonic (150 mosmol/kgH2O) buffer after baseline recordings. Attached cells exhibited a rapid decrease in cell volume and [Ca2+]i after hypertonic buffer treatment, which was associated with an increase in filamentous actin staining. In contrast, cells treated with hypotonic buffers demonstrated an increase in cell volume (mean approximately 10%), a significant decrease in filamentous actin staining, and a rapid transient elevation in [Ca2+]i (mean approximately 280 nM). This [Ca2+]i rise was significantly inhibited by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, gadolinium ions (P < 0.05), and inhibitors of actin assembly. These results indicate that [Ca2+]i fluxes in response to hypotonic cell swelling in attached fibroblasts are mediated by stretch-activated ion channels and are dependent on actin filaments.