nitrophenols and alpha-beta-methyleneadenosine-5--triphosphate

Extracellular nucleotides, signalling through P2 receptors, regulate the function of both osteoblasts and osteoclasts. Osteoblasts are known to express multiple P2 receptor subtypes (P2X2,5,7 and P2Y(1),(2,4,6)), levels of which change during differentiation. ATP and UTP potently inhibit bone mineralisation in vitro, an effect mediated, at least in part, via the P2Y(2) receptor. We report here that primary rat osteoblasts express additional, functional P2 receptors (P2X1, P2X3, P2X4, P2X6, P2Y(12), P2Y(13) and P2Y(14)). Receptor expression changed with cellular differentiation: e.g., P2X4 receptor mRNA levels were 5-fold higher in mature, bone-forming osteoblasts, relative to immature, proliferating cells. The rank order of expression of P2 receptor mRNAs in mature osteoblasts was P2X4>>P2Y(1)>P2X2>P2Y(6)>P2X1>P2Y(2)>P2Y(4)>P2X6>P2X5>P2X7>P2X3>P2Y(14)>P2Y(13)>P2Y(12). Increased intracellular Ca(2+) levels following stimulation with P2X-selective agonists indicated the presence of functional receptors. To investigate whether P2X receptors might also regulate bone formation, osteoblasts were cultured for 14days with P2X receptor agonists. The P2X1 and P2X3 receptor agonists, α,β-meATP and β,γ-meATP inhibited bone mineralisation by 70% and 90%, respectively at 1μM, with complete abolition at ≥25μM; collagen production was unaffected. Bz-ATP, a P2X7 receptor agonist, reduced bone mineralisation by 70% and 99% at 10μM and 100μM, respectively. Osteoblast alkaline phosphatase activity was similarly inhibited by these agonists, whilst ecto-nucleotide pyrophosphatase/phosphodiesterase activity was increased. The effects of α,β-meATP and Bz-ATP were attenuated by antagonists selective for the P2X1 and P2X7 receptors, respectively. Our results show that normal osteoblasts express functional P2X receptors and that the P2X1 and P2X7 receptors negatively regulate bone mineralisation.

Topics: Adenosine Triphosphate; Alkaline Phosphatase; Animals; Calcification, Physiologic; Calcium; Cell Count; Collagen Type I; Extracellular Space; Gene Expression Regulation; Intracellular Space; Nitrophenols; Nucleotides; Organophosphorus Compounds; Osteoblasts; Purinergic P2X Receptor Agonists; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2X; RNA, Messenger; Solubility

Glutaraldehyde treatment of sarcoplasmic reticulum vesicles results in formation of cross-linked Ca2+-ATPase oligomers. Under limiting reaction conditions, where minimal interpolypeptide cross-linking occurs, hydrodynamic properties of the monomer are altered, such that, on sodium dodecyl sulfate-polyacrylamide electrophoresis, the enzyme migrates with an apparent molecular weight of 125,000 (E(125], as compared to the native enzyme (E(110]. The E(125) species was also formed following reaction with other cross-linking bis-aldehydes, with formaldehyde and with a bissuccinimidyl ester. Derivitization resulted in inactivation of ATPase activity and of phosphoprotein formation from Pi. E(125) formation was inhibited by ATP, ADP, AMPPCP, and orthovanadate, and by specific modification of active site Lys-514 with fluorescein-5'-isothiocyanate. Tryptic cleavage patterns of the glutaraldehyde-modified enzyme were consistent with covalent linkage of A1 and B fragments that have been postulated to comprise the phosphorylation and nucleotide-binding domains (MacLennan, D. H., Brandt, C. J., Korczak, B., and Green, N. M. (1985) Nature 316, 696-700). The denaturing detergent, sodium dodecyl sulfate, prevented cross-link formation. Interdomain cross-linking was inhibited by prior modification with either 2,4,6-trinitrobenzene sulfonate, phenylglyoxal, or pyridoxal-5'-phosphate but was unaffected by thiol group modification with iodoacetate or N-ethylmaleimide, suggesting involvement of lysine residues. These findings indicate that intramolecular cross-linking at the active site of the Ca2+-ATPase involves phosphorylation- and ATP-binding domains that are widely separated in the linear sequence.

Topics: Adenosine Triphosphate; Animals; Binding Sites; Calcium-Transporting ATPases; Electrophoresis, Polyacrylamide Gel; Fluorescein-5-isothiocyanate; Fluoresceins; Glutaral; Lysine; Macromolecular Substances; Mannose; Molecular Weight; Nitrophenols; Organophosphorus Compounds; Peptide Fragments; Phosphorylation; Rabbits; Sarcoplasmic Reticulum; Thiocyanates; Trypsin; Vanadates; Vanadium