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Bisindolylmaleimide I (bisindolylmaleimide + i)
Selected AbstractsHigh glucose inhibits fructose uptake in renal proximal tubule cells: Involvement of cAMP, PLC/PKC, p44/42 MAPK, and cPLA2JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2004Su Hyung Park The precise signal that regulates fructose transport in renal proximal tubule cells (PTCs) under high glucose conditions is not yet known although fructose has been recommended as a substitute for glucose in the diets of diabetic people. Thus, we investigated that effect of high glucose on fructose uptake and its signaling pathways in primary cultured rabbit renal PTCs. Glucose inhibited the fructose uptake in a time- and dose-dependent manner. A maximal inhibitory effect of glucose on fructose uptake was observed at 25 mM glucose after 48 h, while 25 mM mannitol and l -glucose did not affect fructose uptake. Indeed, 25 mM glucose for 48 h decreased GLUT5 protein level. Thus, the treatment of 25 mM glucose for 48 h was used for this study. Glucose-induced (25 mM) inhibition of fructose uptake was blocked by pertussis toxin (PTX), SQ-22536 (an adenylate cyclase inhibitor), and myristoylated amide 14,22 (a protein kinase A inhibitor). Indeed, 25 mM glucose increased the intracellular cAMP content. Furthermore, 25 mM glucose-induced inhibition of fructose uptake was prevented by neomycin or U-73122 (phospholipase C inhibitors) and staurosporine or bisindolylmaleimide I (protein kinase C inhibitors). In fact, 25 mM glucose increased the total PKC activity and translocation of PKC from the cytosolic to membrane fraction. In addition, PD 98059 (a p44/42 mitogen-activated protein kinase (MAPK) inhibitor) but not SB 203580 (a p38 MAPK inhibitor) and mepacrine or AACOCF3 (phospholipase A2 inhibitors) blocked 25 mM glucose-induced inhibition of fructose uptake. Results of Western blotting using the p44/42 MAPK and GLUT5 antibodies were consistent with the results of uptake experiments. In conclusion, high glucose inhibits the fructose uptake through cAMP, PLC/PKC, p44/42 MAPK, and cytosolic phospholipase A2 (cPLA2) pathways in the PTCs. © 2004 Wiley-Liss, Inc. [source] A cyclic adenosine 3,,5,-monophosphate-dependent protein kinase C activation is involved in the hyperactivation of boar spermatozoa,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 9 2006Hiroshi Harayama Abstract An intracellular cAMP-PKA signaling plays a pivotal role in the expression of fertilizing ability in mammalian spermatozoa. The aim of this study is to disclose biological function of serine/threonine protein kinases that are activated by the action of the cAMP-PKA signaling in boar spermatozoa. Ejaculated spermatozoa were incubated with cBiMPS (a cell-permeable cAMP analog) at 38.5°C up to 180 min, and then they were used for biochemical analyses of PKCs by Western blotting and indirect immunofluorescence and for assessment of flagellar movement. The incubation of spermatozoa with cBiMPS gradually activated PKCs in the connecting piece. The activation of sperm PKCs was accompanied with changes of their electrophoretic mobility by the PKA-mediated serine/threonine phosphorylation. In coincidence with the PKC activation, the cBiMPS-incubated spermatozoa were capable of exhibiting hyperactivation of flagellar movement. Moreover, the cBiMPS-induced hyperactivation was dramatically suppressed by the addition of either of specific PKC inhibitors (Ro-32-0432 and bisindolylmaleimide I) to the sperm suspensions. On the other hand, experiments using a calcium-deficient medium showed that the cBiMPS-induced hyperactivation of flagellar movement and activation of PKCs required the extracellular calcium. Based on the obtained data, we have concluded that a cAMP-PKA signaling can induce activation of calcium-sensitive PKCs that is leading to the hyperactivation of flagellar movement in boar spermatozoa. Moreover, the cAMP may have a unique role as the up-regulator of PKCs during the expression of fertilizing ability in boar spermatozoa. Mol. Reprod. Dev. 1169,1178, 2006. © 2006 Wiley-Liss, Inc. [source] Signal pathways regulating hyaluronan secretion into static and cycled synovial joints of rabbitsTHE JOURNAL OF PHYSIOLOGY, Issue 17 2009K. R. Ingram Joint lubrication, synovial fluid conservation and many pathophysiological processes depend on hyaluronan (HA). Intra-articular HA injection and exercise, which stimulates articular HA production, ameliorate osteoarthritis. We therefore investigated the pathways regulating movement-stimulated articular HA secretion rate () in vivo. Endogenous HA was removed from the knee joint cavity of anaesthetised rabbits by washout. Joints were then cycled passively or remained static for 5 h, with/without intra-articular agonist/inhibitor, after which newly secreted HA was harvested for analysis. Movement almost doubled . Similar or larger increases were elicited in static joints by the intra-articular Ca2+ ionophore ionomycin, prostaglandin E2, cAMP-raising agents, serine/threonine phosphatase inhibitor and activation of protein kinase C (PKC). PKC-stimulated secretion was inhibited by the PKC inhibitor bisindolylmaleimide I and inhibitors of the downstream kinases MEK-ERK (U0126, PD98059). These agents inhibited movement-stimulated secretion of HA (MSHA) only when the parallel p38 kinase path was simultaneously inhibited by SB203580 (ineffective alone). The phospholipase C inhibitor U73122 almost fully blocked MSHA (P= 0.001, n= 10), without affecting static . The ENaC channel blocker amiloride inhibited MSHA, whereas other inhibitors of stretch-activated channels (Gd3+, ruthenium red, SKF96365) did not. It is proposed that MSHA may be mediated by PLC activation, leading to activation of parallel PKC,MEK,ERK and p38 kinase pathways. [source] Tyrosine kinase and phosphatase regulation of slow delayed-rectifier K+ current in guinea-pig ventricular myocytesTHE JOURNAL OF PHYSIOLOGY, Issue 2 2006Sergey Missan The objective of this study was to investigate the involvement of tyrosine phosphorylation in the regulation of the cardiac slowly activating delayed-rectifier K+ current (IKs) that is important for action potential repolarization. Constitutive IKs recorded from guinea-pig ventricular myocytes was suppressed by broad-spectrum tyrosine kinase (TK) inhibitors tyrphostin A23 (IC50, 4.1 ± 0.6 ,m), tyrphostin A25 (IC50, 12.1 ± 2.1 ,m) and genistein (IC50, 64 ± 4 ,m), but was relatively insensitive to the inactive analogues tyrphostin A1, tyrphostin A63, daidzein and genistin. IKs was unaffected by AG1478 (10 ,m), an inhibitor of epidermal growth factor receptor TK, and was strongly suppressed by the Src TK inhibitor PP2 (10 ,m) but not by the inactive analogue PP3 (10 ,m). The results of experiments with forskolin, H89 and bisindolylmaleimide I indicate that the suppression of IKs by TK inhibitors was not mediated via inhibition of (IKs -stimulatory) protein kinases A and C. To evaluate whether the suppression was related to lowered tyrosine phosphorylation, myocytes were pretreated with TK inhibitors and then exposed to the phosphotyrosyl phosphatase inhibitor orthovanadate (1 mm). Orthovanadate almost completely reversed the suppression of IKs induced by broad-spectrum TK inhibitors at concentrations around their IC50 values. We conclude that basal IKs is strongly dependent on tyrosine phosphorylation of Ks channel (or channel-regulatory) protein. [source] Nitric oxide induces acrosome reaction in cryopreserved bovine spermatozoaANDROLOGIA, Issue 5 2005P. C. Rodriguez Summary The aim of this work was to study the effect of nitric oxide on acrosome reaction (AR) and the participation of protein kinases and reactive oxygen species in the AR of cryopreserved bovine spermatozoa. Spermatozoa were capacitated in Tyrode's albumin lactate pyruvate medium with heparin (10 IU ml,1) and then incubated with different concentrations of sodium nitroprusside (SNP) (1,200 ,mol l,1). Methylene blue and haemoglobin were used to confirm the role of nitric oxide as an inducer of the AR. The participation of protein kinase A (PKA) , protein kinase C (PKC) and protein tyrosine kinase was evaluated using specific inhibitors of these enzymes (H-89, 50 ,mol l,1; bisindolylmaleimide I, 0.1 ,mol l,1 and genistein, 3 ,mol l,1). The role of hydrogen peroxide or superoxide anion was evaluated by incubation with catalase or superoxide dismutase respectively. AR percentages were determined by the fluorescence technique with chlortetracycline. The highest levels of AR were obtained in capacitated spermatozoa treated with 5,200 ,mol l,1 SNP (24.8 ± 1.8%). The presence of PKA, PKC and protein tyrosine kinase inhibitors likewise decreased AR percentages. The addition of superoxide dismutase had no effect on the AR level but catalase completely blocked it. These results indicate that nitric oxide induces AR in capacitated spermatozoa involving hydrogen peroxide and the participation of PKA, PKC and protein tyrosine kinase as part of the signal transduction mechanism which lead to the AR in cryopreserved bovine spermatozoa. [source] In vivo evidence for a role of protein kinase C in peripheral nociceptive processingBRITISH JOURNAL OF PHARMACOLOGY, Issue 1 2002Adriano L S Souza The present study was designed to characterize the nociceptive response induced by protein kinase C (PKC) peripheral activation and to investigate if this biochemical event is important for the nociceptive response induced by formaldehyde, and bradykinin (BK). Intraplantar injection of phorbol-12,13-didecanoate (PDD; 0.01, 0.1 or 1 ,g), a PKC activator, but not of 4,-PDD (inactive analogue), dose-dependently induced thermal hyperalgesia in rats. This response was not observed at the contralateral hindpaw. Intraplantar injection of PDD (0.01, 0.1 or 1 ,g) also induced mechanical allodynia. In mice, injection of PDD (0.1 or 1 ,g) into the dorsum of the hindpaw induced a spontaneous licking behaviour. Intraplantar co-injection of chelerythrine (10 or 50 ,g), a PKC inhibitor, attenuated the thermal hyperalgesia induced by PDD (0.1 ,g) in rats. The second phase of the nociceptive response induced by the injection of formaldehyde (0.92%, 20 ,l) into the dorsum of mice hindpaws was inhibited by ipsi-, but not contralateral, pre-treatment with chelerythrine (1 ,g). Intraplantar injection of BK (10 ,g) induced mechanical allodynia in rats. Ipsi- but not contralateral injection of bisindolylmaleimide I (10 ,g), a PKC inhibitor, inhibited BK-induced mechanical allodynia. In conclusion, this study demonstrates that PKC activation at peripheral tissues leads to the development of spontaneous nociceptive response, thermal hyperalgesia and mechanical allodynia. Most importantly, it also gives in vivo evidence that peripheral PKC activation is essential for the full establishment of the nociceptive response induced by two different inflammatory stimuli. British Journal of Pharmacology (2002) 135, 239,247; doi:10.1038/sj.bjp.0704434 [source] | |||||||||||||