Fluid Shear-Induced ATP Secretion Mediates Prostaglandin Release in MC3T3-E1 Osteoblasts

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 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 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 shearinduced 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 prostaglandin release.

The P2X7 Nucleotide Receptor Mediates Skeletal Mechanotransduction

“…3). We have shown that FSS produces a 10-fold increase in ATP release in MC3T3-E1 osteoblasts within 1 min of the onset of FSS (36). When primary osteoblasts from WT mice were subjected to steady laminar fluid flow at 12 dynes/cm 2 FSS, release of ATP was also observed within 1 min.…”

“…ATP is an early mediator of mechanotransduction in osteoblasts that is released from cells within 1 min after applying mechanical loading (36). ATP initiates intracellular calcium release in osteoblasts through P2Y receptor pathways, which has been the main focus of several previous studies (15)(16).…”

Cell–cell communication in the osteoblast/osteocyte lineage

“…Interestingly, work on fluid flow has disclosed a prominent role of gap junction hemichannels in bone cells. Cx43 hemichannels are active in osteocytic cells, where they mediate fluid flow-induced PGE 2 [10] and ATP [59] release, although dependence of fluid flow stimulated release of ATP from osteoblastic cells has not been universally linked to Cx43 hemichannels [60]. One of the major challenges in this area is represented by the inherent difficulties in rigorously distinguishing hemichannel-mediated diffusion from exocytosis or other related events.…”

Phosphodiesterase Activity of Alkaline Phosphatase in ATP-initiated Ca2+ and Phosphate Deposition in Isolated Chicken Matrix Vesicles

“…The nucleotide concentration of the extracellular fluid aspirated from the hypertrophic zone of rat calcifying epiphyseal cartilage was reported to reach 1-2 mM (34). Nucleotides (ATP/UTP) are also released both by non-stimulated (35,36) or stimulated osteoblasts (37,38) in response to mechanical stimulus. The measured amount of secreted ATP in solution by murine osteoblast-like cell in response to shear was in the order of 100 nM, however this does not accurately show the local ATP concentration at the cell surface immediately on its release (38).…”

“…Nucleotides (ATP/UTP) are also released both by non-stimulated (35,36) or stimulated osteoblasts (37,38) in response to mechanical stimulus. The measured amount of secreted ATP in solution by murine osteoblast-like cell in response to shear was in the order of 100 nM, however this does not accurately show the local ATP concentration at the cell surface immediately on its release (38). In addition, the osteoblasts have been shown to express an membrane-bound ecto-nucleoside diphosphokinase (ecto-NDPK), which allows the ADPuATP conversion in extracellular calcification medium (35).…”