Mm Dvorak

Histamine induced responses are attenuated by a cannabinoid receptor agonist in human skin

Abstract:Objective and Design: In the present study we examined the effects of the cannabinoid receptor agonist HU210 on histamine-evoked somatosensory and vascular responses in humans. Subjects: Two sets of experiments were performed, in which twelve (Study 1, iontophoresis) and six participants (Study 2, microdialysis) were recruited. Treatment: HU210 was administered peripherally by skin patch (50 mM) or dermal microdialysis (5 mM), whereas histamine was applied by iontophoresis (50 μAmps) or dermal microdialysis (5 μM). Methods: Skin blood flow was monitored by laser Doppler, widespread flare reaction was evaluated planimetrically, extravasation of plasma proteins was measured in the dialysate and perceived itch was recorded using a visual analogue scale. Data were evaluated by analysis of variance. Results: Experimentally induced itch was significantly reduced by peripheral administration of HU210 (p < 0.05). Additionally, skin blood flow and neurogenic mediated flare responses were attenuated (p < 0.003 and p < 0.03, respectively), whereas protein extravasation due to histamine was enhanced by co-administration of HU210, as investigated by dermal microdialysis. Conclusions: 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.

Cannabinoid agonists attenuate capsaicin-induced responses in human skin.

&NA; 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 30 min 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, 30 min 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.

Thiazide Diuretics Directly Induce Osteoblast Differentiation and Mineralized Nodule Formation by Interacting with a Sodium Chloride Co-Transporter in Bone

Thiazide diuretics are used worldwide as a first-choice drug for patients with uncomplicated hypertension. In addition to their antihypertensive effect, thiazides increase bone mineral density and reduce the prevalence of fractures. Traditionally, these effects have been attributed to increased renal calcium reabsorption that occurs secondary to the inhibition of the thiazide-sensitive sodium chloride cotransporter (NCC) in the distal tubule. The aim of the current study was to determine whether thiazides exert a direct bone-forming effect independent of their renal action. We found that the osteoblasts of human and rat bone also express NCC, suggesting that these bone-forming cells may be an additional target for thiazides. In vitro, NCC protein was virtually absent in proliferating human and fetal rat osteoblasts, whereas its expression dramatically increased during differentiation. Thiazides did not affect osteoblast proliferation, but directly stimulated the production of the osteoblast differentiation markers runt-related transcription factor 2 (runx2) and osteopontin. Using overexpression/knockdown studies in fetal rat calvarial cells, we show that thiazides increase the formation of mineralized nodules, but loop diuretics do not. Overall, our study demonstrates that thiazides directly stimulate osteoblast differentiation and bone mineral formation independent of their effects in the kidney. Therefore, in addition to their use as antihypertensive drugs, our results suggest that thiazides may find a role in the prevention and treatment of osteoporosis.

Protein kinase C-mediated phosphorylation of the calcium-sensing receptor is stimulated by receptor activation and attenuated by calyculin-sensitive phosphatase activity.

The agonist sensitivity of the calcium-sensing receptor (CaR) can be altered by protein kinase C (PKC), with CaR residue Thr888 contributing significantly to this effect. To determine whether CaRT888 is a substrate for PKC and whether receptor activation modulates such phosphorylation, a phospho-specific antibody against this residue was raised (CaRpT888). In HEK-293 cells stably expressing CaR (CaR-HEK), but not in cells expressing the mutant receptor CaRT888A, phorbol ester (PMA) treatment increased CaRpT888 immunoreactivity as observed by immunoblotting and immunofluorescence. Raising extracellular Ca2+ concentration from 0.5 to 2.5 mm increased CaRT888 phosphorylation, an effect that was potentiated stereoselectively by the calcimimetic NPS R-467. These responses were mimicked by 5 mm extracellular Ca2+ and abolished by the calcilytic NPS-89636 and also by PKC inhibition or chronic PMA pretreatment. Whereas CaRT888A did exhibit increased apparent agonist sensitivity, by converting intracellular Ca2+ (Ca2+i) oscillations to sustained plateau responses in some cells, we still observed Ca2+i oscillations in a significant number of cells. This suggests that CaRT888 contributes significantly to CaR regulation but is not the exclusive determinant of CaR-induced Ca2+i oscillations. Finally, dephosphorylation of CaRT888 was blocked by the protein phosphatase 1/2A inhibitor calyculin, a treatment that also inhibited Ca2+i oscillations. In addition, calyculin/PMA cotreatment increased CaRT888 phosphorylation in bovine parathyroid cells. Therefore, CaRT888 is a substrate for receptor-induced, PKC-mediated feedback phosphorylation and can be dephosphorylated by a calyculin-sensitive phosphatase.