Calcium Waves in Fluid Flow Stimulated Osteoblasts Are G Protein Mediated

Abstract

Calcium (Ca2+) entry upon cell perturbation has been examined in transformed human osteoblast cells (U-2/OS). The cells were deformed by fluid flow from a patch pipette held in proximity to the cell by applying a positive pressure (+50 mm Hg) for the passage of saline over the membrane. Intracellular calcium [Ca2+]iwas examined following loading with 5 μMFura-2 AM. The changes in ratio were determined at 330-ms intervals. Waves of [Ca2+]iwere seen spreading along the length of the individual cell following stimulation (n= 30). The initial change in Ca2+at the site of stimulation occurred within 660 ms after applying the stimulus. Following 1.3 (±0.33) s of raised [Ca2+]i, the values returned to those of predeformation. The Ca2+response following fluid flow stimulation was blocked by 300 μMCd2+, a specific blocker of Ca2+channels, demonstrating an extracellular source of Ca2+. Preincubation with cholera toxin (250 ng/ml for 6 h) prolonged the elevation of Ca2+induced by fluid flow stimulation (n= 20). In contrast, pertussis toxin (250 ng/ml for 6 h) completely eliminated the Ca2+response to fluid flow stimulation (n= 20). Cells maintained in solutions free of Ca2+demonstrated no change in [Ca2+]i. Tetraethylammonium (6 mM) had no effect on the response (n= 10). In addition pretreatment with ryanodine (2 and 10 μM; each groupn= 10) in media showed a reduced wave of Ca2+in response to mechanical deformation. The response to a phospholipase C inhibitor also eliminated the response to the mechanical deformation (n= 10). In addition cells that demonstrated changes in Ca2+-containing media lost the ability to respond when EGTA was added to the media. Following this, 2 μMryanodine was added to the cells, demonstrating a response too small to replicate the fluid flow stimulated wave, but supporting the view that the cells were vital following preincubation.