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Fluid Shear-Induced ATP Secretion Mediates Prostaglandin Release in MC3T3-E1 Osteoblasts Damian C Genetos, 1 Derik J Geist, 2 Dawei Liu, 2 Henry J Donahue, 1,3 and Randall L Duncan 2 ABSTRACT: ATP is rapidly released from osteoblasts in response to mechanical load. We examined the mechanisms involved in this release and established that shear-induced ATP release was mediated through vesicular fusion and was dependent on Ca 2+ entry into the cell through L-type voltage-sensitive Ca 2+ channels. Degradation of secreted ATP by apyrase prevented shear-induced PGE 2 release. Introduction: Fluid shear induces a rapid rise in intracellular calcium ([Ca 2+ ] i ) in osteoblasts that mediates many of the cellular responses associated with mechanotransduction in bone. A potential mechanism for this increase in [Ca 2+ ] i is the activation of purinergic (P2) receptors resulting from shear-induced extracellular release of ATP. This study was designed to determine the effects of fluid shear on ATP release and the possible mechanisms associated with this release. Materials and Methods: MC3T3-E1 preosteoblasts were plated on type I collagen, allowed to proliferate to 90% confluency, and subjected to 12 dynes/cm 2 laminar fluid flow using a parallel plate flow chamber. ATP release into the flow media was measured using a luciferin/luciferase assay. Inhibitors of channels, gap junctional intercellular communication (GJIC), and vesicular formation were added before shear and main- tained in the flow medium for the duration of the experiment. Results and Conclusions: Fluid shear produced a transient increase in ATP release compared with static MC3T3-E1 cells (59.8 ± 15.7 versus 6.2 ± 1.8 nM, respectively), peaking within 1 minute of onset. Inhibition of calcium entry through the L-type voltage-sensitive Ca 2+ channel (L-VSCC) with nifedipine or verapamil significantly attenuated shear-induced ATP release. Channel inhibition had no effect on basal ATP release in static cells. Ca 2+ -dependent ATP release in response to shear seemed to result from vesicular release and not through gap hemichannels. Vesicle disruption with N -ethylmaleimide, brefeldin A, or monensin prevented increases in flow-induced ATP release, whereas inhibition of gap hemichannels with either 18 -glycyrrhetinic acid or 18 -glycyrrhetinic acid did not. Degradation of extracellular ATP with apyrase prevented shear- induced increases in prostaglandin E 2 (PGE 2 ) release. These data suggest a time line of mechanotransduction wherein fluid shear activates L-VSCCs to promote Ca 2+ entry that, in turn, stimulates vesicular ATP release. Furthermore, these data suggest that P2 receptor activation by secreted ATP mediates flow-induced prosta- glandin release. J Bone Miner Res 2005;20:41–49. Published online on October 18, 2004; doi: 10.1359/JBMR.041009 Key words: ATP release, mechanotransduction, Ca 2+ signaling, osteoblasts, fluid shear INTRODUCTION B ONE IS A dynamic organ, with its architecture constantly changing in accordance with the mechanical usage re- quired of it. As external forces placed on bone decrease,
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