We investigated the direct effects of changes in free ionized extracellular calcium concentrations ([Ca 2؉ ]o) on osteoblast function and the involvement of the calcium-sensing receptor (CaR) in mediating these responses. CaR mRNA and protein were detected in osteoblast models, freshly isolated fetal rat calvarial cells and murine clonal osteoblastic 2T3 cells, and in freshly frozen, undecalcified preparations of human mandible and rat femur. In fetal rat calvarial cells, elevating [Ca 2؉ ]o and treatment with gadolinium, a nonpermeant CaR agonist, resulted in phosphorylation of the extracellular signal-regulated kinases 1 and 2, Akt, and glycogensynthase kinase 3␤, consistent with signals of cell survival and proliferation. In agreement, cell number was increased under these conditions. Expression of the osteoblast differentiation markers core binding factor ␣1, osteocalcin, osteopontin, and collagen I mRNAs was increased by high [ (2, 3) and alter the levels of expression of some differentiation markers (4, 5). During mineralization, decreases in [Ca 2ϩ ] o are also likely to occur (6), but the effect of lowering [Ca 2ϩ ] o in bone cells has not been extensively addressed.The mechanism of [Ca 2ϩ ] o -sensing by osteoblasts is unclear. The parathyroid extracellular calcium-sensing receptor (CaR) is a key player in the maintenance of a constant systemic [Ca 2ϩ ] o , predominantly through regulation of parathyroid hormone (PTH) secretion and urinary calcium excretion (7,8). CaR is also present in osteoblasts (9 and references therein), where a functional role is currently debated. Recently two studies have shown that CaRdeficient mice exhibit an essentially normal skeletal phenotype when the hyperparathyroidism resulting from the lack of the parathyroid CaR is prevented (10, 11). Thus, it remains unclear whether the osteoblast CaR is a true regulator of bone function or whether its expression is vestigial (12).In this study, we investigated the effects of both decreasing and increasing [Ca 2ϩ ] o on osteoblast proliferation and intracellular signaling events, the expression of several osteoblast differentiation markers [core binding factor ␣1 (Cbfa1, also termed Runx2 and Osf2), osteocalcin (OC), osteopontin (OP), and type I collagen (collaI)], the activity of alkaline phosphatase (AlP), and mineralized nodule formation in the absence of systemic calciotropic factors, namely PTH and vitamin D. We further investigated the role played by the CaR in these events using an alternative, nonpermeant CaR agonist, gadolinium (Gd 3ϩ ) and a CaR inhibitor, NPS 89636 (a ''calcilytic''). We used well characterized osteoblast models, freshly isolated fetal rat calvarial cells (FRC) (13) and the clonal murine osteoblast cell line, 2T3 cells (14). The expression of CaR in freshly frozen sections of rat and human bone was also determined. Materials and MethodsAnimals. Sprague-Dawley rats (Charles River Breeding Laboratories) were killed by cervical dislocation and used in accordance to the U.K. Animals Scientific Procedures...

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In humans peripheral administration of a cannabinoid receptor agonist attenuates histamine-induced itch. The observation that protein extravasation was not decreased demonstrates that the alleviation of itch is not due to an anti-histaminergic property of HU210. The reduced neurogenic flare reaction indicates an attenuated antidromic nerve fibre activation and neuropeptide release.

The induction of hyperalgesia upon capsaicin administration requires activation of specific sub-classes of nociceptive afferent C-fibres providing nociceptive input to the central nervous system. It has been demonstrated in animal models that the endocannabinoid anandamide has anti-hyperalgesic properties upon capsaicin stimulation, albeit it also binds to vanilloid receptors. In the present study we topically administered the cannabinoid receptor ligand HU210 to human skin and investigated its effects on capsaicin-induced pain and hyperalgesia.We demonstrated that pre-treatment with HU210 significantly reduced the perception of pain following the administration of capsaicin. Heat pain thresholds were significantly reduced by capsaicin application measured 5 and 30min after administration. In contrast, at the HU210 pre-treated skin sites capsaicin failed to induce heat hyperalgesia during the fifth minute of administration. Secondary mechanical hyperalgesia to touch (allodynia) was measured during the fifth, 15th and 30th minute after capsaicin administration. In comparison to the ethanol control site, the area of touch-evoked allodynia was significantly reduced at the HU210 skin site during the first two measures. However, 30min after the administration of capsaicin no significant differences of allodynia were observed between the HU210 and ethanol pre-treated skin. The present study provided evidence for analgesic and anti-hyperalgesic properties of a topically applied cannabinoid receptor ligand, which might have important therapeutic implications in humans.