kn-62 and thiazolyl-blue

Mechanical stress is an important factor in maintaining homeostasis of the periodontium. Interleukin-1beta (IL-1β) and adenosine triphosphate (ATP) are considered potent inflammatory mediators. In macrophages, ATP-activated P2X7 receptor is involved in IL-1β processing and release. Our previous works demonstrated mechanical stress-induced expression of osteopontin and RANKL through the ATP/P2Y1 receptor in human periodontal ligament (HPDL) cells. This study was designed to examine the effect of mechanical stress on IL-1β expression in HPDL cells, as well as the mechanism and involvement of ATP and the P2 purinergic receptor.. Cultured HPDL cells were treated with continuous compressive loading. IL-1β expression was analyzed at both mRNA and protein levels, using RT-PCR and ELISA, respectively. Cell viability was examined using the MTT assay. ATP was also used to stimulate HPDL cells. Inhibitors, antagonists and the small interfering RNA (siRNA) technique were used to investigate the role of ATP and the specific P2 subtypes responsible for IL-1β induction along with the intracellular mechanism.. Mechanical stress could up-regulate IL-1β expression through the release of ATP in HPDL cells. ATP alone was also capable of increasing IL-1β expression. The induction of IL-1β was markedly inhibited by inhibitors and by siRNA targeting the P2X7 receptor. ATP-stimulated IL-1β expression was also diminished by intracellular calcium inhibitors.. Our work clearly indicates the capability of HPDL cells to respond directly to mechanical stimulation. The results signified the important roles of ATP/P2 purinergic receptors, as well as intracellular calcium signaling, in mechanical stress-induced inflammation via up-regulation of the proinflammatory cytokine, IL-1β, in HPDL cells.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Diphosphate; Adenosine Triphosphate; Apyrase; Benzenesulfonates; Calcium Channel Blockers; Calcium Signaling; Cell Culture Techniques; Cell Survival; Cells, Cultured; Coloring Agents; Dactinomycin; Homeostasis; Humans; Interleukin-1beta; Luminescent Measurements; Periodontal Ligament; Purinergic P2X Receptor Antagonists; Receptors, Purinergic P2X7; RNA, Small Interfering; Signal Transduction; Stress, Mechanical; Suramin; Tetrazolium Salts; Thiazoles; Time Factors; Up-Regulation

The P2X7 nucleotide receptor is an ATP-gated ion channel that plays an important role in bone cell function. Here, we investigated the effects of L: -tyrosine derivatives 1-3 as potent P2X7 antagonists on human primary osteoclasts. We found that the level of expression of P2X7 receptor increased after treatment with the derivatives 1-3, together with the induction of high levels of apoptosis. This effect is associated with activation of caspase-3 and inhibition of expression of IL-6. Interestingly, no pro-apoptotic effect of compounds 1-3 was found on human osteoblasts. Our results suggest that the development of specific P2X7 receptor antagonists may be considered a useful tool to modulate apoptosis of human osteoclasts. Since bone loss due to osteoclast-mediated resorption represents one of the major unsolved problem in osteopenic disorders, the identification of molecules able to induce apoptosis of osteoclasts is of great interest for the development of novel therapeutic strategies.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acid Phosphatase; Apoptosis; Bone and Bones; Bone Diseases, Metabolic; Caspase 3; Caspases; Cell Nucleus; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Interleukin-6; Isoenzymes; Models, Chemical; NF-kappa B; Osteoblasts; Osteoclasts; Piperazines; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2X7; Tartrate-Resistant Acid Phosphatase; Tetrazolium Salts; Thiazoles; Time Factors