EXTRACELLULAR NUCLEOTIDES ACT THROUGH P-2U PURINOCEPTORS TO ELEVATE [CA2-INDUCED PROLIFERATION IN SHEEP CHONDROCYTES(](I) AND ENHANCE BASICFIBROBLAST GROWTH FACTOR)

Extracellular nucleotides interact with specific cell surface receptor s 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 pre sent study, we investigated whether Ca2+-mobilizing purinoceptors are present on sheep chondrocytes. Chondrocytes were isolated from the pro ximal 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 b y fluorescence spectrophotometry. ATP (0.3-100 mu M) induced transient elevation of [Ca2+](i), lasting approximately 1 min. Half-maximal ele vation of [Ca2+](i), was observed at an ATP concentration of 5.0 +/- 0 .2 mu M. Responses were still observed in the absence of extracellular Ca2+, and were abolished by pretreatment with thapsigargin, consisten t with the release of Ca2+ from intracellular stores. Several nucleoti des were tested for their ability to elevate [Ca2+](i). In order of po tency, these were UTP approximate to ATP >> ADP approximate to 2-methy lthio-ATP. No responses were elicited by benzoylbenzoic-ATP, a P-2z-se lective agonist; alpha,beta-methylene-ATP, an agonist selective for ce rtain P-2X purinoceptors; AMP; adenosine; or pyrophosphate (all at 100 mu M), demonstrating specificity. Taken together, these data indicate that nucleotides elevate [Ca2+](i) in chondrocytes through interactio n with the P-2U purinoceptor subtype. Although pretreatment with pertu ssis toxin virtually abolished the Ca2+ response to lysophosphatidic a cid, the response to UTP was relatively insensitive, suggesting that P -2U purinoceptors are not linked to a pertussis toxin-sensitive G prot ein in chondrocytes. In contrast, the Ca2+ response to UTP was markedl y inhibited by the biologically active phorbol ester 12-O-tetradecanoy l-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 regulate s P-2U purinoceptor signaling in these cells. UTP (10 mu M) enhanced t he proliferative response to basic fibroblast growth factor. The respo nse to basic fibroblast growth factor was also enhanced by ATP, but no t by 2-methylthio-ATP, consistent with involvement of P-2U purinocepto rs. Nucleotides released during trauma, inflammation, or cell death ma y act through P-2U purinoceptors to regulate chondrocyte function in a n autocrine or paracrine manner.