RESUMEN
UNLABELLED: Disruption of the actin cytoskeleton with cytochalasin D enhanced the mechanically induced increase in intracellular Ca(2+) ([Ca(2+)](i)) in osteoblasts in a manner similar to that of PTH. Stabilization of actin with phalloidin prevented the PTH enhanced [Ca(2+)](i) response to shear. Patch-clamp analyses show that the MSCC is directly influenced by alterations in actin integrity. INTRODUCTION: PTH significantly enhances the fluid shear-induced increase in [Ca(2+)](i) in osteoblasts, in part, through increased activation of both the mechanosensitive, cation-selective channel (MSCC) and L-type voltage-sensitive Ca(2+) channel (L-VSCC). Both stimuli have been shown to produce dynamic changes in the organization of the actin cytoskeleton. In this study, we examined the effects of alterations in actin polymerization on [Ca(2+)](i) and MSCC activity in MC3T3-E1 and UMR-106.01 osteoblasts in response to shear +/- PTH pretreatment. MATERIALS AND METHODS: MC3T3-E1 or UMR-106.01 cells were plated onto type I collagen-coated quartz slides, allowed to proliferate to 60% confluency, and mounted on a modified parallel plate chamber and subjected to 12 dynes/cm(2). For patch-clamp studies, cells were plated on collagen-coated glass coverslips, mounted on the patch chamber, and subjected to pipette suction. Modulators of actin cytoskeleton polymerization were added 30 minutes before the experiments, whereas channel inhibitors were added 10 minutes before mechanical stimulation. All drugs were maintained in the flow medium for the duration of the experiment. RESULTS AND CONCLUSIONS: Depolymerization of actin with 1-5 microM cytochalasin D (cyto D) augmented the peak [Ca(2+)](i) response and increased the number of cells responding to shear, similar to the increased responses induced by pretreatment with 50 nM PTH. Stabilization of actin with phalloidin prevented the PTH enhanced [Ca(2+)](i) response to shear. Inhibition of the MSCC with Gd(3+) significantly blocked both the peak Ca(2+) response and the number of cells responding to shear in cells pretreated with either PTH or cyto D. Inhibition of the L-VSCC reduced the peak [Ca(2+)](i) response to shear in cells pretreated with PTH, but not with cyto D. Patch-clamp analyses found that addition of PTH or cyto D significantly increased the MSCC open probability in response to mechanical stimulation, whereas phalloidin significantly attenuated the PTH-enhanced MSCC activation. These data indicate that actin reorganization increases MSCC activity in a manner similar to PTH and may be one mechanism through which PTH may reduce the mechanical threshold of osteoblasts.