| In Vitro | In vitro activity: Go 6983 (300 μM) suppresses PKCμ auto-phosphorylation by 20% reduction in NIH3T3 transfected with PKCμ. In hearts reperfused with PMNs and Go 6983 (100 nM), left ventricular developed pressure (LVDP) and the rate of LVDP recoveres to 89% and 74% of baseline values, respectively, significantly higher than PMNs alone. Go 6983 (100 nM) significantly reduces PMNs adherence to the endothelium and infiltration into the myocardium compared with Ischemia followed by reperfusion (I/R)+ PMN hearts, and significantly inhibits superoxide release from PMNs by 90%. Go 6983 attenuates post-I/R cardiac contractile dysfunction in the presence of PMNs, which may be related in part to decreased superoxide production. Go 6983 significantly inhibits antigen-induced superoxide release from leukocytes of patients previously sensitized to tree pollen. Go 6983 inhibited intracellular Ca(2+) accumulation in human vascular tissue, suggesting a mechanism for its vasodilator properties. Go-6983 (1 μM) combined with Ro-31-8425 (390 nM) slightly inhibits Angiotensin II–induced PLD2 activity in PGSMCs. Go 6983 is isoform-specific PKC inhibitor that target the ATP binding site. Go 6983 inhibits ΔPfPKB activity with an IC50 of 1 μM. In Go 6983 (5 μM)-treated cells, the number of rings in the following cycle is markedly less compared with the control cultures. Go 6983 (5 μM) treatment results in an almost 60% decrease in formation of new rings in P. falciparum cultures.
Kinase Assay: Phosphorylation reactions were carried out in a total volume of 100 μL, containing buffer C (50 mM Tris-HC1, pH 7.5, 10 mM β-mercaptoethanol), 4 mM MgCl2, 10 μg PS, 100 nM TPA, 5 μL of a Sf158 cell extract as a source of recombinant PKCμ or of Sf9 cell extracts as a source of other recombinant PKC isoenzymes, 10 μg of syntide 2 as substrate, and 35 μM ATP containing 1 μCi [γ-32P]ATP. In some experiments PS and TPA were omitted or various inhibitors at concentrations indicated in the text were added. After incubation for 10 mins at 30 °C, the reaction was terminated by transferring 50 μL of the assay mixture onto a 20-mm square piece of phosphocellulose paper, which was washed 3 times in deionized water and twice in acetone. The radioactivity on each paper was determined by liquid scintillation counting.
Cell Assay: In ARCaPE cells, Go 6983 at the dose of 200 nM significantly inhibited the up-regulation of PKCs(PKCα, PKCβ and PKCγ) stimulated by the PMA treatment. In addition, Go 6983 showed complete inhibition at the dose of 1000 NM. |
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