P70S6 Kinase Phosphorylation： A New Site to Assess Pharmacodynamy of Sirolimus

Background：The phosphorylation ofp70S6 kinase （p70S6K） represents an important target for sensitive detection on pharmacodynamic effects of sirolimus,but the methods of assessing p70S6K phosphorylation are still unclear.The aim of this study was to investigate p70S6K phosphorylation located down-stream of the mammalian target ofrapamycin （mTOR） pathway in peripheral blood mononuclear cells （PBMCs） of liver transplant patients through different methods.Methods：Seventy-five liver transplant recipients from Beijing Chaoyang Hospital of the Capital Medical University were analyzed in this study.Patients were divided into three groups,patient treated with sirolimus （n =22）,patient treated with tacrolimus （n =30）,patient treated with cyclosporine （n =23）.The p70S6K phosphorylation of PBMCs in patients and healthy control （HC,n =12） were analyzed by phospho-flow cytometry and Western blotting.A correlation analysis of data from phospho-flow cytometry and Western blotting was performed.Intra-assay variability of p70S6K phosphorylation in HC and different patients were measured.Results：Intra-assay variability ofp70S6K phosphorylation in phospho-flow cytometry was from 4.1% to 8.4% and in Western blotting was from 8.2% to 18%.The p70S6K phosphorylation in patients receiving a sirolimus （19.5 ± 7.7） was significantly lower than in HC （50.1 ± 11.3,P 〈 0.001）,tacrolimus （37.7 ± 15.7,P 〈 0.001） or cyclosporine treated patients （41.7 ± 11.7,P 〈 0.001）.The p70S6K phosphorylation in HC （50.1± 11.3） was significantly higher than in tacrolimus （37.7 ± 15.7,P 〈 0.01） or cyclosporine-treated patients （41.7 ± 11.7,P 〈 0.01）.There was correlation between data from phospho-flow cytometry and data from Westem blotting （r =0.88,P 〈 0.001）.Conclusions：The degree of mTOR inhibition by assessing p70S6K phosphorylation was established by phospho-flow cytometry and Westem blotting.Assessment of p70S6K phosphorylation may play an adjunct role to on pharmacodynamically guide and individualize sirolimus based on immunosuppression.

Effects of electroacupuncture on the expression of p70 ribosomal protein S6 kinase and ribosomal protein S6 in the hippocampus of rats with vascular dementia

This study investigated the mechanism underlying electroacupuncture therapy for vascular dementia through electroacupuncture at the acupoints of Baihui （DU20）, Dazhui （DU14）, and bilateral Shenshu （BL23） in a rat model of vascular dementia produced by bilateral middle cerebral artery occlusion. Morris water maze test showed that electroacupuncture improved the learning ability of vascular dementia rats. Western blot assay revealed that the expression of p70 ribosomal protein S6 kinase and ribosomal protein S6 in vascular dementia rats was significantly increased after electroacupuncture, compared with the model group that was not treated with acupuncture. The average escape latency was also shortened after electroacupuncture, and escape strategies in the spatial probe test improved from edge and random searches, to linear and trending swim pathways. The experimental findings indicate that electroacupuncture improves learning and memory ability by up-regulating expression of p70 ribosomal protein S6 kinase and ribosomal protein S6 in the hippocampus of vascular dementia rats.

Angiotensin IV upregulates the activity of protein phosphatase 1α in Neura-2A cells

The peptide angiotensin IV(Ang IV)is a derivative of angiotensin II.While insulin regulated amino peptidase(IRAP)has been proposed as a potential receptor for Ang IV,the signalling pathways of Ang IV through IRAP remain elusive.We applied high-resolution mass spectrometry to perform a systemic quantitative phosphoproteome of Neura-2A(N2A)cells treated with and without Ang IV us-ing sta ble-isotope labeling by amino acids in cell culture(SILAC),and identifi ed a reduction in the phosphorylation of a major Ser/Thr protein phosphorylase 1(PP1)upon Ang IV treatment.In addition,spinophilin(spn),a PP1 reg-ulatory protein that plays important functions in the neural system,was expressed at higher levels.Immunoblotting revealed decreased phosphorylation of p70S6 kinase(p70S6K)and the major cell cycle modulator retinoblas-toma protein(pRB).These changes are consistent with an observed decrease in cell proliferation.Taken together,our study suggests that Ang IV functions via regulating the activity of PP1.

Activation of mammalian target of rapamycin contributes to pain nociception induced in rats by BmK I, a sodium channel-specific modulator

The mammalian target of rapamycin （mTOR） pathway is essential for maintenance of the sensitivity of certain adult sensory neurons. Here, we investigated whether the mTOR cascade is involved in scorpion envenomation-induced pain hypersensitivity in rats. The results showed that intraplantar injection of a neurotoxin from Buthus martensii Karsch, BmK I （10 pg）, induced the activation of mTOR, as well as its downstream molecules p70 ribosomal S6 protein kinase （p70 S6K） and eukaryotic initiation factor 4E-binding protein 1 （4E-BP1）, in lumbar 5-6 dorsal root ganglia neurons on both sides in rats. The activation peaked at 2 h and recovered 1 day after injection. Compared with the control group, the ratios of p-mTOR/p-p70 S6K/p-4E- BP1 in three types of neurons changed significantly. The cell typology of p-mTOR/p-p70 S6K/p-4E-BP1 immuno-reactive neurons also changed. Intrathecal administration of deforolimus, a specific inhibitor of mTOR, attenuated BmK I-induced pain responses （spontaneous flinching, paroxysmal pain-like behavior, and mechanical hypersensitivity）. Together, these results imply that the mTOR signaling pathway is mobilized by and contributes to experimental scorpion sting-induced pain.