Fluid shear‐induced ATP secretion mediates prostaglandin release in MC3T3‐E1 osteoblasts

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Genetos, Damian C.¹; Geist, Derik J.²; Liu, Dawei²; Donahue, Henry J.^1,3; Duncan, Randall L.²

Fluid shear‐induced ATP secretion mediates prostaglandin release in MC3T3‐E1 osteoblasts

J Bone Miner Res. January 2005;20(1):41-49

©2005 American Society for Bone and Mineral Research

Affiliations

¹Departments of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA

²Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, Indiana, USA

³Orthopaedics and Rehabilitation, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA
Abstract and keywords

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 Ca2+ entry into the cell through L‐type voltage‐sensitive Ca2+ channels. Degradation of secreted ATP by apyrase prevented shear‐induced PGE2 release.

Introduction Fluid shear induces a rapid rise in intracellular calcium ([Ca2+]i) in osteoblasts that mediates many of the cellular responses associated with mechanotransduction in bone. A potential mechanism for this increase in [Ca2+]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/cm2 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 maintained 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 Ca2+ 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. Ca2+‐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 18α‐glycyrrhetinic acid or 18β‐glycyrrhetinic acid did not. Degradation of extracellular ATP with apyrase prevented shear‐induced increases in prostaglandin E2 (PGE2) release. These data suggest a time line of mechanotransduction wherein fluid shear activates L‐VSCCs to promote Ca2+ entry that, in turn, stimulates vesicular ATP release. Furthermore, these data suggest that P2 receptor activation by secreted ATP mediates flow‐induced prostaglandin release.

Keywords: ATP release; mechanotransduction; Ca2+ signaling; osteoblasts; fluid shear
Links

DOI: 10.1359/JBMR.041009

PubMed: 15619668

WoS: 000225986900009
History

Received: 2004-03-4

Revised: 2004-07-20

Accepted: 2004-08-11

Online: 2004-10-18

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