CALCIUM WAVES IN FLUID-FLOW STIMULATED OSTEOBLASTS ARE G-PROTEIN MEDIATED

Calcium (Ca2+) entry upon cell perturbation has been examined in trans formed human osteoblast cells (U-2/OS). The cells were deformed by flu id 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 me mbrane. Intracellular calcium [Ca2+](i) was examined following loading with 5 mu M Fura-2 AM. The changes in ratio were determined at 330 ms intervals. Waves of [Ca2+](i) were seen spreading along the length of the individual cell following stimulation (n = 30). The initial chang e in Ca2+ at the site of stimulation occurred within 660 ms after appl ying the stimulus. Following 1.3 (+/-0.33) s of raised [Ca2+](i), the values returned to those of predeformation. The Ca2+ response followin g fluid flow stimulation was blocked by 300 mu M Cd2+, a specific bloc ker of Ca2+ channels, demonstrating an extracellular source of Ca2+. P reincubation with cholera toxin (250 ng/ml for 6 h) prolonged the elev ation of Ca2+ induced by fluid flow stimulation (n = 20). In contrast, pertussis toxin (250 ng/ml for 6 h) completely eliminated the Ca2+ re sponse to fluid flow stimulation (n = 20). Cells maintained in solutio ns free of Ca2+ demonstrated no change in [Ca2+](i). Tetraethylammoniu m (6 mM) had no effect on the response (n = 10). In addition pretreatm ent with ryanodine (2 and 10 mu M; each group n = 10) in media showed a reduced wave of Ca2+ in response to mechanical deformation. The resp onse to a phospholipase C inhibitor also eliminated the response to th e mechanical deformation (n = 10). In addition cells that demonstrated changes in Ca2+-containing media lost the ability to respond when EGT A was added to the media. Following this, 2 mu M ryanodine was added t o the cells, demonstrating a response too small to replicate the fluid flow stimulated wave, but supporting the view that the cells were vit al following preincubation. (C) 1996 Academic Press, Inc.