Abstract

Extracellular nucleotides interact with specific cell surface receptors to mediate a variety of biological responses, including elevation of the cytosolic free Ca2+ concentration ([Ca2+]i) in a number of cell types. Although extracellular ATP has been shown to affect chondrocyte function, the underlying mechanisms are poorly understood. In the present study, we investigated whether Ca2+-mobilizing purinoceptors are present on sheep chondrocytes. Chondrocytes were isolated from the proximal tibial growth plate of day 120-130 sheep fetuses. Early passage cells were loaded with indo-1 or fluo-3, and [Ca2+]i was monitored by fluorescence spectrophotometry. ATP (0.3-100 microM) induced transient elevation of [Ca2+]i, lasting approximately 1 min. Half-maximal elevation of [Ca2+]i was observed at an ATP concentration of 5.0 +/- 0.2 microM. Responses were still observed in the absence of extracellular Ca2+, and were abolished by pretreatment with thapsigargin, consistent with the release of Ca2+ from intracellular stores. Several nucleotides were tested for their ability to elevate [Ca2+]i. In order of potency, these were UTP approximately ATP >> ADP approximately 2-methylthio-ATP. No responses were elicited by benzoylbenzoic-ATP, a P2Z-selective agonist; alpha,beta-methylene-ATP, an agonist selective for certain P2X purinoceptors; AMP; adenosine; or pyrophosphate (all at 100 microM), demonstrating specificity. Taken together, these data indicate that nucleotides elevate [Ca2+]i in chondrocytes through interaction with the P2U purinoceptor subtype. Although pretreatment with pertussis toxin virtually abolished the Ca2+ response to lysophosphatidic acid, the response to UTP was relatively insensitive, suggesting that P2U purinoceptors are not linked to a pertussis toxin-sensitive G protein in chondrocytes. In contrast, the Ca2+ response to UTP was markedly inhibited by the biologically active phorbol ester 12-O-tetradecanoyl-beta-phorbol 13-acetate, but not by the inactive control compound 4 alpha-phorbol 12,13-didecanoate, suggesting that a 12-O-tetradecanoyl-beta-phorbol 13-acetate-sensitive isoform of protein kinase C regulates P2U purinoceptor signaling in these cells. UTP (10 microM) enhanced the proliferative response to basic fibroblast growth factor. The response to basic fibroblast growth factor was also enhanced by ATP, but not by 2-methylthio-ATP, consistent with involvement of P2U purinoceptors. Nucleotides released during trauma, inflammation, or cell death may act through P2U purinoceptors to regulate chondrocyte function in an autocrine or paracrine manner.

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call

Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.