Extracellular nucleotides elevate [Ca2+]i in rat osteoblastic cells by interaction with two receptor subtypes

Abstract

Extracellular nucleotides, through interaction with specific cell-surface receptors, mediate a variety of biological responses, including elevation of cytosolic free Ca2+ concentration ([Ca2+]i) in a number of cell types. The effects of nucleotides on [Ca2+]i in the rat osteoblastic cell line UMR-106 were studied by fluorescence spectrophotometry of indo-1-loaded cells. In response to ATP (100 microM), [Ca2+]i rose to peaks 228 +/- 16 nM (n = 59) above baseline (85 +/- 3 nM) before returning to near basal levels. Half-maximal elevation of [Ca2+]i was observed at an ATP concentration of 3 +/- 1 microM, consistent with a high-affinity interaction. The response arose primarily by release of Ca2+ from internal stores. UTP, ADP, and 2-methylthioadenosine 5'-triphosphate also induced Ca2+ transients, whereas adenosine, AMP, CTP, and TTP did not, demonstrating specificity. Responsiveness to adenosine 5'-O-(3-thiotriphosphate) and inhibition by Mg2+ of the response to ATP indicated that signaling was not dependent on nucleotide hydrolysis. Ca2+ responses to ADP, ATP, and UTP, added sequentially or simultaneously, were consistent with the presence of two distinct P2-purinoceptor subtypes, both linked to Ca2+ mobilization. ADP appeared to interact selectively with one receptor, whereas ATP and UTP interacted selectively with the other. After maximal stimulation with ATP, subsequent responses to ATP were abolished. However, removal of ATP from the extracellular medium rapidly restored responsiveness, suggesting that, with continued receptor occupation, there is time-dependent inactivation of the Ca2+ signaling pathway. Our findings indicate that extracellular nucleotides elevate [Ca2+]i in osteoblastic cells through interaction with two receptor subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence of ectokinase-mediated phosphorylation of osteopontin and bone sialoprotein by osteoblasts during bone formation in vitro

“…Although the extracellular concentrations of nucleoside triphosphates such as ATP are generally low in comparison with their intracellular concentrations, many cell types can release ATP in a controlled fashion through exocytosis or via cell membrane transport, generating extracellular concentrations in excess of 1 mM [26][27][28][29]. Notably, extracellular nucleoside triphosphates at micromolar concentrations are ligands for cell surface P2 purinergic receptors that have been implicated in a variety of biological processes including neuronal [30,31] and smooth muscle activity [32], platelet aggregation [25] and bone cell regulation [33][34][35][36][37]. Although the extracellular ATP concentration near active osteoblasts in i o is not known, it is conceivable that sufficiently high levels of extracellular ATP can be produced to provide substrate for ectokinases that have K m values that range from 10 mM down to 0.04 mM [32].…”

The role of purinergic signalling in the musculoskeletal system

“…Subsequent pharmacological studies revealed that extracellular nucleotides interacted with at least two P2 receptors (P2Y1 and P2Y2) on rat osteoblastlike cells (Reimer et al, 1992;Yu et al, 1993b). The first molecular evidence for P2Y receptor expression was shown in 1995 by Bowler et al, who used RT-PCR and in situ hybridisation to demonstrate P2Y2 receptor expression in human osteoblasts (Bowler et al, 1995).…”

Caveolin-1 regulates P2X7receptor signaling in osteoblasts

“…We and others have shown that ATP and other nucleotides are released in response to fluid shear stress in vitro and activate purinergic (P2) receptors on the membrane of the osteoblasts (13,20,29,38). Although many subtypes of P2 receptors are present in osteoblasts (4), knockout of the purinergic receptor P2X 7 R, a ligand-gated ion channel, resulted in an osteopenic phenotype (22), suggesting that this receptor is important in skeletal maintenance.…”