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ATP induces Ca2+signaling in human chondrons cultured in three-dimensional agarose films

https://doi.org/10.1053/joca.2000.0435Get rights and content
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Abstract

ObjectiveIn vivo, chondrocytes are surrounded by an extracellular matrix, preventing direct cell-to-cell contact. Consequently, intercellular communication through gap junctions is unlikely. However, signaling at a distance is possible through extracellular messengers such as nitric oxide (NO) and nucleotides and nucleosides, adenosine triphosphate (ATP), uridine triphosphate (UTP), or adenosine diphosphate (ADP). We hypothesized that chondrons, chondrocytes surrounded by their native pericellular matrix, increase their intracellular calcium concentration ([Ca2+]ic) in response to ATP and other signaling molecules and that the source of Ca2+is from intracellular stores. The objectives of this study were to determine if chondrons in a 3-D gel respond to ATP by increasing [Ca2+]icthrough a purinoceptor mechanism and to test whether chondrons in whole tissue samples would respond to ATP in a similar fashion.

Design Human chondrons, cultured in a three-dimensional agarose gel or in whole cartilage loaded with Fura-2AM, a calcium sensitive dye, were stimulated with 1, 5 and 10 μM ATP. A ratio-imaging fluorescence technique was used to quantitate the [Ca2+]ic.

Results ATP-stimulated chondrons increased their [Ca2+]icfrom a basal level of 60 nM to over 1000 nM. Chondrons incubated in calcium-free medium also increased their [Ca2+]icin response to ATP, indicating the source of Ca2+was not extracellular. ATP-induced calcium signaling was inhibited in chondrons pre-treated with suramin, a generic purinoceptor blocker. In addition, UTP and adenosine 5′-O-(3-thiotriphosphate) (ATPγs) induced a calcium response, but 2-methylthio-ATP (2-MeSATP), ADP, and adenosine did not induce a significant increase in [Ca2+]ic, substantiating that the P2Y2purinoceptor was dominant. Chondrons in whole cartilage increased [Ca2+]icin response to ATP.

Conclusions We conclude that chondrons in 3-D culture respond to ATP by increasing [Ca2+]icvia P2Y2receptor activation. Thus, ATP can pass through the agarose gel and the pericellular matrix, bind purinoceptors and increase intracellular Ca2+in a signaling response.

Keywords

Chondrocytes, Intracellular Calcium, Signal Transduction, ATP

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Address correspondence to: Albert J. Banes, Department of Orthopaedics, 253 Burnett-Womack Bldg, CB#7055, University of North Carolina, Chapel Hill, NC 27599-7055, U.S.A. Tel: (919) 966-2566; Fax: (919) 966-6730; E-mail:[email protected]