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Objective To study the effects of angiotensin Ⅱ (AngⅡ) on insulin signal transduction pathway in skeletal myoblast of L6 rats,and further to explore the possible mechanism of AngⅡ on glucose utilization.Methods Myoblast cells of L6 rats were cultured and induced to differentiate.They were divided into 4 groups according to different treatment by AngⅡ or JAK2-PKA inhibitor H89:normal control group ( NC group),insulin group,insulin + AngⅡ group and insulin + AngⅡ + H89 group.Expression of IRS1 and GLUT4 mRNA was detected by RT-PCR.Expression of IRS1,Ptyr-IRS1 and GLUT4 (total and membrane protein) were detected by Western blot.Results The difference of GLUT4 mRNA expression in the 4 groups detected by RT-PCR had no statistical significance(P ＞ 0.05).The difference of IRS1 mRNA expression among the latter 3 groups had no statistical significance(P ＞ 0.05),however,IRS1 expression in the latter 3 groups was higher than that in NC group(P ＜ 0.05).Western blot results showed expression of IRS1,Ptyr-IRS1 and GLUT4 (membrane protein)was higher in the latter 3 groups than in NC group(P ＜0.05).The difference of IRS1 expression among the latter 3 groups(P ＞ 0.05 ) and GLUT4 (total protein) expression among the 4 groups had no statistical significance (P ＞ 0.05).The expression of of ptyr-IRS1 and GLUT4 membrane protein in Ins + AngⅡ + H89 group was much higher than that in Ins + AngⅡ group,and lower than that in insulin group(P ＜0.05).Conclusion AngⅡ inhibits IRS1's tyrosine phosphorylation and GLUT4's transfer from cytoplasm to plasma membrane in skeletal muscle cells through JAK2-PKA signaling pathway,and therefore induces insulin resistance.
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Aim To assess the effects of N -[2-p-bromo-cinnamylamino-ethyl]-5-isoquinoline-sulfonamide (H-89), a potentially selective inhibitor of Protein Kinase A (PKA), on cardiac membrane ion channels and transporters, which will further fulfill our understanding of pharmacology of PKA inhibitors.Methods Whole-cell patch clamp was used to investigate the effects of H-89 on cardiac L-type Ca~(2+) current (I_(Ca-L)), Na~+ current (I_(Na)), inward rectifier K~+ current (I_(K1)), transient outward K~+ current (I_(to)) and Na~+-Ca~2+ exchanger current (I_(Na/Ca)) in enzymatic dissociated SD rat ventricular myocytes.Results H-89 at 1～10 μmol·L~(-1) could inhibit I_(Ca-L) , I_(Na) , and Ito in a concentration-relative manner (P <0.05). At low concentra-tion (5 μmol·L~(-1)), H-89 completely inhibited I_(K1) (P <0.05) just as the action of 0.5 mmol·L~(-1) BaCl_2.Further, H-89 at 1～10 μmol·L~(-1) had no significant effect on I_(Na/Ca) (P >0.05).Conclusion The direct or PKA-mediated indirect action maybe involved in the effects of H-89 on ion currents and transporter.
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Objective To investigate the signal regulatory mechanism of expression of corticotropin releasing hormone (CRH) stimulated neurons of hypothalamic slices in rats in vitro . Methods Model of hypothalamic slices of rats was established. After CRH stimulatation of corticotropin releasing hormone type 1 receptor (CRH1R) of hypothalamic slices in rats in vitro , the changes of activity of protein kinase A (PKA) signal passway were observed by immunocytochemical method and Western blotting. The relationship between the changes and CRH mRNA expression was also observed. Results CRH could cause the remarkable increase in phosphorylated PKA, phosphorylated CREB, and CRH content in hypothalamic slices in rats. However, CPl54526 or H89 could have significant inhibitory effect on the synthesis of P PKA, P CREB, and CRH. Conclusion PKA signal passway can regulate the ultrashort positive feedback of CRH secretion in the rat hypothalamus in stress due to severe trauma.
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Objective The present study was aimed at investigating the regeneration promoting mechanism of forskolin and IBMX on injured retinal ganglion cells(RGCs). Methods Fluorescent retrograde labeling and autologus sciatic nerve transplanting techniques were used to observe the effects of H 89 or/and wortmannin on the regeneration promoting effects of forskolin and IBMX on injured retinal ganglion cells by intravitreal injection. Results Both H 89 and wortmannin could partly block the promoting effects of forskolin and IBMX on regeneration of injured RGCs, the effects of wortmannin might be more powerful, if united, they could inhibit the effects of forskolin and IBMX completely.Conclusion The regeneration promoting effects of forskolin and IBMX on injured RGCs might be realized by PKA CREB and PI3 K Akt accesses.