Mechanosensitive calcium entry and mobilization in renal A6 cells

Using spectrofluorescence imaging of fura-2 loaded renal A6 cells, we have investigated the generation of the cytosolic Ca2+ signal in response to osm otic shock and localized membrane stretch. Upon hypotonic exposure, the cel ls began to swell prior to a transient increase in [Ca2+](i) and the cells remained swollen after [Ca2+](i) had returned towards basal levels. Exposur e to 2/3rd strength Ringer produced a cell volume increase within 3 min, fo llowed by a slow regulatory volume decrease(RVD). The hypotonic challenge a lso produced a transient increase in [Ca2+] after a delay of 22 sec. Both t he RVD and [Ca2+](i) response to hypotonicity were inhibited in a Ca2+-free bathing solution and by gadolinium (10 mu M), an inhibitor of stretch-acti vated channels. Stretching the membrane by application of subatmospheric pr essure (-2 kPa) inside a cell-attached patch-pipette induced a similar glob al increase in [Ca2+](i) as occurred after hypotonic shock. A stretch-sensi tive [Ca2+](i) increase was also observed in a Ca2+-free bathing solution, provided the patch-pipette contained Ca2+. The mechanosensitive [Ca2+](i) r esponse was by gadolinium (10 mu M) or Ca2+-free pipette solutions, even wh en Ca2+ (2 mM) was present in the bath. Long-term (>10 min) pretreatment of the cells with thapsigargin inhibited the [Ca2+](i) response to hypotonici ty. These results provide evidence that cell swelling or mechanical stimula tion can activate a powerful amplification system linked to intracellular C a2+ release mechanisms.