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

Objective: In vivo, chondrocytes are surrounded by an extracellular matrix, preventing direct cell-to-cell contact. Consequently, intercellular commun ication 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 triphos phate (UTP), or adenosine diphosphate (ADP). We hypothesized that chondrons , chondrocytes surrounded by their native pericellular matrix, increase the ir 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+](ic) through a purinoceptor mecha nism 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 stimula ted with 1, 5 and 10 muM ATP. A ratio-imaging fluorescence technique was us ed to quantitate the [Ca2+](ic). Results: ATP-stimulated chondrons increased their [Ca2+], from a basal leve l of 60 nM to over 1000 nM. Chondrons incubated in calcium-free medium also increased their [Ca2+](ic) in response to ATP, indicating the source of Ca 2+ was not extracellular. ATP-induced calcium signaling was inhibited in ch ondrons pre-treated with suramin, a generic purinoceptor blocker. In additi on, UTP and adenosine 5'-O- (3-thiotriphosphate) (ATP gammas) induced a cal cium response, but 2-methylthio-ATP (2-MeSATP), ADP, and adenosine did not induce a significant increase in [Ca2+](ic), substantiating that the P2Y(2) purinoceptor was dominant. Chondrons in whole cartilage increased [Ca2+](i c) in response to ATP. Conclusions: We conclude that chondrons in 3-D culture respond to ATP by in creasing [Ca2+](ic) via P2Y(2) receptor activation. Thus, ATP can pass thro ugh the agarose gel and the pericellular matrix, bind purinoceptors; and in crease intracellular Ca2+ in a signaling response. (C) 2001 OsteoArthritis Research Society International.