h-89 and herbimycin

The aim of the present study was to investigate the acute effect and mechanism of tumor necrosis factor (TNF) on basolateral 50 pS K channels in the thick ascending limb (TAL) of the rat kidney. The TAL tubules were isolated from the rat kidney, and the activity of the 50 pS K channels was recorded using the patch‑clamp technique. The results indicated that the application of TNF (10 nM) significantly activated the 50 pS K channels and the TNF effect was concentration‑dependent. Inhibition of protein kinase A, phospholipase A2 and protein tyrosine kinase using pathway inhibitors (H89, AACOCF3 and Herbimycin A, respectively) did not abolish the stimulatory effect of TNF, indicating that none of these pathways mediated the TNF effect. By contrast, the phenylarsine oxide inhibitor against protein tyrosine phosphatase (PTP) decreased the activity of the 50 pS K channels and blocked the stimulatory effect of TNF on these channels. Furthermore, western blot analysis demonstrated that the application of TNF (10 nM) in the TAL increased the phosphorylation of PTP, an indication of PTP activity stimulation. Thus, it was concluded that the acute application of TNF may stimulate the basolateral 50 pS K channel in the TAL and the stimulatory effect of TNF may be mediated by the PTP‑dependent pathway.

Topics: Animals; Arachidonic Acids; Arsenicals; Cyclic AMP-Dependent Protein Kinases; Isoquinolines; Kidney; Kidney Tubules; Loop of Henle; Male; Patch-Clamp Techniques; Phospholipase A2 Inhibitors; Phospholipases A2; Potassium Channels; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; Rifabutin; Sulfonamides; Tumor Necrosis Factor-alpha

The proteasome is a multicatalytic cellular complex present in human sperm that plays a significant role during several steps of mammalian fertilization. Here, we present evidence that the proteasome is involved in human sperm capacitation. Aliquots of highly motile sperm were incubated with proteasome inhibitors MG132 or epoxomicin. The percentage of capacitated sperm, the chymotrypsin-like activity of the proteasome, cAMP content, and the pattern of protein phosphorylation were assayed by using the chlortetracycline hydrochloride assay, a fluorogenic substrate, the cAMP enzyme immunoassay kit, and Western blot analysis, respectively. Our results indicate that treatment of sperm with proteasome inhibitors blocks the capacitation process, does not alter cAMP concentration, and changes the pattern of protein phosphorylation. To elucidate how proteasome activity is regulated during capacitation, sperm were incubated with: 1) tyrosine kinase (TK) inhibitors (genistein or herbimycin A); 2) protein kinase (PK) A inhibitors or activators (H89 and Rp-cAMPS, and 8-Br-cAMP, respectively); or 3) PKC inhibitors (tamoxifen or staurosporin) at different capacitation times. The chymotrypsin-like activity and degree of phosphorylation of the proteasome were then assayed. The results indicate that sperm treatment with TK and PKA inhibitors significantly decreases the chymotrypsin-like activity of the proteasome during capacitation. Immunoprecipitation and Western blot results suggest that the proteasome is phosphorylated during capacitation in a TK- and PKA-dependent pathway. In conclusion, we suggest that the sperm proteasome participates in the capacitation process, and that its activity is modulated by PKs.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Benzoquinones; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Enzyme Inhibitors; Genistein; Humans; In Vitro Techniques; Isoquinolines; Lactams, Macrocyclic; Leupeptins; Male; Oligopeptides; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein-Tyrosine Kinases; Rifabutin; Sperm Capacitation; Spermatozoa; Sulfonamides; Thionucleotides

Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte-like cell line (U937), a human NK-like cell line (KHYG-1), and a human umbilical vein endothelial cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 cells and by both Rottlerin and Go6976 in the HUV-EC-C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the cell surface of all three cell-lines and induced interleukin-8 (IL-8) production by the U937 cells and interferon-gamma (IFN-gamma) production by the KHYG-1 cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective cell-lines. Whereas recombinant tumor necrosis factor-alpha (rTNF-alpha) slightly up-regulated CD93 expression on the U937 cells, recombinant interleukin-1beta (rIL-1beta), recombinant interleukin-2 (rIL-2), recombinant interferon-gamma (rIFN-gamma) and lipopolysaccharide (LPS) had no effect. Taken together, these findings indicate that the regulation of CD93 expression on these cells involves the PKC isoenzymes.

Topics: Acetophenones; Benzopyrans; Benzoquinones; Carbazoles; Down-Regulation; Endothelial Cells; Flow Cytometry; Humans; Immunoenzyme Techniques; Indoles; Interferon-gamma; Interleukin-8; Isoenzymes; Isoquinolines; Lactams, Macrocyclic; Membrane Glycoproteins; Monocytes; Protein Kinase C; Protein Kinase Inhibitors; Quinones; Receptors, Complement; Rifabutin; Sulfonamides; Tetradecanoylphorbol Acetate; U937 Cells; Up-Regulation

Src tyrosine kinase belongs to a non-receptor tyrosine kinase family and has been shown to be involved in G protein-coupled receptor desensitization and internalization. Stimulation of ovarian thecal cells with lutein-izing hormone (LH) activates adenylyl cyclase via a G protein-coupled LH receptor leading to an increase in cAMP. Subsequently, cAMP activates protein kinase A (PKA) that increases steroidogenesis. In order to evaluate the role of Src in thecal cell steroidogenesis, a pharmacological approach was utilized by treating a population of mouse ovarian theca-enriched cells (TEC) in vitro with two Src inhibitors, geldanamycin (GA) and herbimycin A (HA). Treatment of TEC with either GA or HA increased basal androstenedione secretion without alteration of cAMP. In the presence of forskolin, GA and HA treatment further increased androstenedione secretion. RT-PCR analysis of RNA from cells treated with GA for 8, 24, and 48 h revealed that GA increased cytochrome P450 17alpha-hydroxylase/lyase (CYP17) mRNA at 48 h. CYP17 promoter activity also increased after treatment of cells with GA and after co-transfection with a Src dominant negative plasmid. Inhibition of PKA using H89 blocked the effect GA and HA on androstenedione secretion. These results indicate that the pharmacological inhibitors of Src, GA and HA, tested in vitro increased thecal CYP17 promoter activity, CYP17 mRNA, and androstenedione secretion. In addition, GA and HA induced thecal androstenedione secretion may be cAMP independent but possibly requires PKA.

Topics: 3-Hydroxysteroid Dehydrogenases; Androstenedione; Animals; Benzoquinones; Colforsin; Cyclic AMP; Female; Isoquinolines; Lactams, Macrocyclic; Mice; Mice, Inbred C57BL; Ovary; Progesterone; Promoter Regions, Genetic; Protein Kinase Inhibitors; Quinones; Reverse Transcriptase Polymerase Chain Reaction; Rifabutin; RNA, Messenger; src-Family Kinases; Steroid 17-alpha-Hydroxylase; Sulfonamides; Theca Cells

Platelet-activating factor (PAF) is a potent stimulator of human eosinophils involved in the pathogenesis of allergic diseases. However, intracellular signaling mechanisms in eosinophils involving the PAF receptor are incompletely understood.. We sought to determine the roles of protein kinase C (PKC) and cyclic AMP-dependent protein kinase (protein kinase A [PKA]) in signaling pathways of human eosinophils stimulated with PAF.. After pretreatment with a PKC inhibitor, bisindolylmaleimide I, or a PKA inhibitor, H89, we investigated PAF-evoked functions, such as CD11b expression, cellular adhesion, superoxide anion generation, and degranulation in human eosinophils.. Preincubation of eosinophils with bisindolylmaleimide I resulted in enhancement of upregulated CD11b expression and adhesion induced by PAF. H89 pretreatment also enhanced PAF-induced cellular adhesion. Superoxide anion generation and degranulation were suppressed by means of inhibition of either PKC or PKA.. PKC and PKA negatively regulate PAF-induced CD11b upregulation and cellular adhesion but promote eosinophil effector functions, such as superoxide anion generation and degranulation. PKC and PKA modulate PAF-evoked intracellular signaling of the eosinophil function in distinct ways.

Topics: Benzoquinones; Cell Adhesion; Cell Degranulation; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Eosinophils; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Indoles; Isoquinolines; Lactams, Macrocyclic; Macrophage-1 Antigen; Maleimides; Platelet Activating Factor; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Rifabutin; Signal Transduction; Sulfonamides; Superoxides; Up-Regulation; Virulence Factors, Bordetella

Glial fibrillary acidic protein (GFAP) is expressed upon cAMP-mediated induction of differentiation of glial progenitor cells into type II astrocytes. The protein is regulated by hormones, growth factors and cytokines but the signal transduction pathways involved in the regulation of GFAP expression are largely unknown. Specific protein kinase inhibitors were used to study their effect on the expression of GFAP in rat C6 glioma cells. Herbimycin A, a selective protein tyrosine kinase inhibitor, reduced GFAP mRNA and protein expression upon cAMP analog or beta-adrenergic receptor-mediated induction of differentiation. The latter inhibitor attenuated the elevation of cAMP by adenylate cyclase and abolished the activity of phosphatidylinositol 3-kinase (PI 3-K). These data indicate that GFAP expression is regulated by protein tyrosine phosphorylations, modulating the cAMP concentration and PI 3-K activity in C6 glioma cells.

Topics: Adenylyl Cyclases; Adrenergic beta-Agonists; Androstadienes; Animals; Anti-Bacterial Agents; Benzoquinones; Cell Differentiation; Chromones; Cyclic AMP; Enzyme Inhibitors; Flavonoids; Gene Expression; Glial Fibrillary Acidic Protein; Glioma; Indoles; Isoproterenol; Isoquinolines; Lactams, Macrocyclic; Maleimides; Morpholines; Phosphatidylinositol 3-Kinases; Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; Sirolimus; Sulfonamides; Tumor Cells, Cultured; Wortmannin

Gangliosides are sialic acid-containing glycosphingolipids and exhibit various physiologic functions. Gangliosides GD1a and GM3 strongly induced interleukin-10 (IL-10) protein secretion and mRNA expression in T cells from normal human subjects while the other gangliosides were ineffective. IL-10 induction by both gangliosides was completely blocked by protein tyrosine kinase (PTK) inhibitors, herbimycin A, genistein, and tyrphostin AG 1288, but not by other signal transduction inhibitors. These results suggest that GD1a and GM3 may induce IL-10 production in T cells by regulating the PTK-dependent signaling pathway. These gangliosides may thus act as important immunoregulators via IL-10.

Topics: Benzoquinones; Cell Division; Cell Survival; Cells, Cultured; DNA; Dose-Response Relationship, Drug; G(M3) Ganglioside; Gangliosides; Humans; Interleukin-10; Isoquinolines; Lactams, Macrocyclic; Protein Kinase Inhibitors; Protein Kinases; Quinones; Rifabutin; RNA, Messenger; Signal Transduction; Staurosporine; Sulfonamides; T-Lymphocytes; Time Factors; Transcriptional Activation

1-(5-Isoquinolinesulfonyl1)-2-methylpiperazine hydrochloride (H-7), an inhibitor of protein kinases, has been shown to inhibit the thymocyte apoptosis induced by various apoptogenic agents. In the present study, when mouse thymocytes were pretreated with H-7, washed, and cultured for an additional time, apoptosis was induced depending on the preincubation time and the dose of H-7. The protein kinase C activity in the H-7-pretreated and -washed cells was not altered, suggesting that an alteration of a certain PKC isoform is related to both the triggering and the progression of apoptosis.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Apoptosis; Benzoquinones; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Cycloheximide; Dactinomycin; Enzyme Inhibitors; Isoenzymes; Isoquinolines; Lactams, Macrocyclic; Male; Mice; Mice, Inbred BALB C; Nucleic Acid Synthesis Inhibitors; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Protein Synthesis Inhibitors; Quinones; Rifabutin; RNA; Sulfonamides; T-Lymphocytes; Thymus Gland