Orexins, novel excitatory neuropeptides from the lateral hypothalamus, have been strongly implicated in the regulation of sleep and wakefulness. In this study, we explored the effects and mechanisms of orexin A on intracellular free Ca2+ concentration ([Ca2+]i) of freshly dissociated neurons from layers V and VI in prefrontal cortex (PFC). Changes in [Ca2+]i were measured with fluo-4/AM using confocal laser scanning microscopy. The results revealed that application of orexin A (0.1-1 microM) induced increase of [Ca2+]i in a dose-dependent manner. This elevation of [Ca2+]i was completely blocked by pretreatment with selective orexin receptor 1 antagonist SB 334867. While depletion of intracellular Ca2+ stores by the endoplasmic reticulum inhibitor thapsigargin (2 pM), [Ca2+]i in PFC neurons showed no increase in response to orexin A. Under extracellular Ca2+-free condition, orexin A failed to induce any changes of Ca2+ fluorescence intensity in these acutely dissociated cells. Our data further demonstrated that the orexin A-induced increase of [Ca2+]i was completely abolished by the inhibition of intracellular protein kinase C or phospholipase C activities using specific inhibitors, BIS II (1 microM) and D609 (10 microM), respectively. Selective blockade of L-type Ca2+ channels by nifedipine (5 microM) significantly suppressed the elevation of [Ca2+]i induced by orexin A. Therefore, these findings suggest that exposure to orexin A could induce increase of [Ca2+]i in neurons from deep layers of PFC, which depends on extracellular Ca2+ influx via L-type Ca2+ channels through activation of intracellular PLC-PKC signaling pathway by binding orexin receptor 1.

Background The pathogenesis of immune thrombocytopenia (ITP) has not been fully clarified. Anti-αvβ3 integrin autoantibody is detected in chronic ITP patients, but its contribution to ITP is still unclear. Objectives To clarify the potential role of anti-αvβ3 integrin autoantibody in chronic ITP and the related mechanism. Methods Relationship between levels of anti-αvβ3 autoantibody and platelets in chronic ITP patients was evaluated. The influence of anti-αvβ3 antibody on megakaryocyte (MK) survival, differentiation, migration and adhesion was assessed, and the associated signal pathways were investigated. Platelet recovery and MKs' distribution were observed in an ITP mouse model pretreated with different antibodies. Result In this study, we showed that the anti-αvβ3 autoantibody usually coexists with anti-αIIbβ3 autoantibody in chronic ITP patients, and patients with both autoantibodies have lower platelets. In in vitro studies, we showed that the anti-αvβ3 antibody had no significant effect on the survival and proliferation of MKs, whereas it decreased formations of proplatelet significantly. Anti-αvβ3 antibody impeded stromal cell derived facor-1 alpha (SDF-1α)- mediated migration and inhibited the phosphorylation of protein kinase B. Anti-αvβ3 antibody significantly inhibited MKs' adhesion to endothelial cells and Fibrogen. The phosphorylation of focal adhesion kinase and proto-oncogene tyrosine-protein kinase Src induced by adhesion was inhibited when MKs were pretreated with anti-αvβ3 antibody. In in vivo studies, we showed that injection with anti-α antibody delayed platelet recovery in a mouse model of ITP. Conclusions These findings demonstrate that the autoantibody against integrin α β may aggravate thrombocytopenia in ITP patients by impeding MK migration and adhesion to the vascular niche, which provides new insights into the pathogenesis of ITP.

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