title: ATP induces Ca(2+) signaling in human chondrons cultured in three-dimensional agarose films.
abstract: OBJECTIVE
  In 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 ([Ca(2+)]ic) in response to ATP and other signaling molecules and that the source of Ca(2+) is from intracellular stores. The objectives of this study were to determine if chondrons in a 3-D gel respond to ATP by increasing [Ca(2+)]ic through a purinoceptor mechanism and to test whether chondrons in whole tissue samples would respond to ATP in a similar fashion.
  
  
  METHODS
  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 microM ATP. A ratio-imaging fluorescence technique was used to quantitate the [Ca(2+)]ic.
  
  
  RESULTS
  ATP-stimulated chondrons increased their [Ca(2+)]ic from a basal level of 60 nM to over 1000 nM. Chondrons incubated in calcium-free medium also increased their [Ca(2+)]ic in response to ATP, indicating the source of Ca(2+) 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) (ATPgammas) induced a calcium response, but 2-methylthio-ATP (2-MeSATP), ADP, and adenosine did not induce a significant increase in [Ca(2+)]ic, substantiating that the P2Y2 purinoceptor was dominant. Chondrons in whole cartilage increased [Ca(2+)]ic in response to ATP.
  
  
  CONCLUSIONS
  We conclude that chondrons in 3-D culture respond to ATP by increasing [Ca(2+)]ic via P2Y2 receptor activation. Thus, ATP can pass through the agarose gel and the pericellular matrix, bind purinoceptors and increase intracellular Ca(2+) in a signaling response.
authors.affiliation: Department of Biomedical Engineering, University of North Carolina, Chapel Hill, North Carolina 27599-7055, USA.
authors.forename: M K
authors.initials: MK
authors.lastname: Elfervig
authors.forename: R D
authors.initials: RD
authors.lastname: Graff
authors.forename: G M
authors.initials: GM
authors.lastname: Lee
authors.forename: S S
authors.initials: SS
authors.lastname: Kelley
authors.forename: A
authors.initials: A
authors.lastname: Sood
authors.forename: A J
authors.initials: AJ
authors.lastname: Banes
mesh_terms: D000244:Adenosine Diphosphate / Q000502:physiology; D000255:Adenosine Triphosphate / Q000502:physiology*; D000970:Antineoplastic Agents / Q000494:pharmacology; D020013:Calcium Signaling / Q000502:physiology*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D016257:Fura-2; D006801:Humans; D012685:Sepharose; D013498:Suramin / Q000494:pharmacology; D014544:Uridine Triphosphate / Q000502:physiology
chemical_list: D000970:Antineoplastic Agents; D013498:Suramin; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D012685:Sepharose; D016257:Fura-2; D014544:Uridine Triphosphate