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 osmotic shock and localized membrane stretch. Upon hypotonic exposure, the cells began to swell prior to a transient increase in [Ca2+]i and the cells remained swollen after [Ca2+]i had returned towards basal levels. Exposure to 2/3rd strength Ringer produced a cell volume increase within 3 min, followed by a slow regulatory volume decrease (RVD). The hypotonic challenge also produced a transient increase in [Ca2+] after a delay of 22 sec. Both the RVD and [Ca2+]i response to hypotonicity were inhibited in a Ca2+-free bathing solution and by gadolinium (10 microm), an inhibitor of stretch-activated channels. Stretching the membrane by application of subatmospheric pressure (-2 kPa) inside a cell-attached patch-pipette induced a similar global increase in [Ca2+]i as occurred after hypotonic shock. A stretch-sensitive [Ca2+]i increase was also observed in a Ca2+-free bathing solution, provided the patch-pipette contained Ca2+. The mechanosensitive [Ca2+]i response was by gadolinium (10 microm) or Ca2+-free pipette solutions, even when Ca2+ (2 mm) was present in the bath. Long-term (>10 min) pretreatment of the cells with thapsigargin inhibited the [Ca2+]i response to hypotonicity. These results provide evidence that cell swelling or mechanical stimulation can activate a powerful amplification system linked to intracellular Ca2+ release mechanisms.