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.
Wellcome Trust Cellular Physiology Research Unit, Department of Physiology, University College, Cork, Ireland.
V
V
Urbach
I
I
Leguen
I
I
O'Kelly
B J
BJ
Harvey
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002460:Cell Line; D048429:Cell Size; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005682:Gadolinium / Q000494:pharmacology; D007038:Hypotonic Solutions / Q000494:pharmacology; D007552:Isotonic Solutions / Q000494:pharmacology; D009399:Nephrons / Q000166:cytology* / Q000187:drug effects; D009997:Osmotic Pressure; D018408:Patch-Clamp Techniques; D000077331:Ringer's Solution; D013050:Spectrometry, Fluorescence; D013314:Stress, Mechanical; D019284:Thapsigargin / Q000494:pharmacology; D014982:Xenopus laevis
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004791:Enzyme Inhibitors; D007038:Hypotonic Solutions; D007552:Isotonic Solutions; D019284:Thapsigargin; D000077331:Ringer's Solution; D005682:Gadolinium; D000252:Calcium-Transporting ATPases; D002118:Calcium