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Hypertension Research : Official... Jun 1995The modulation of dopamine DA1 receptors of cultured rat renal arterial smooth muscle cells by phorbol ester, glucocorticoid and sodium chloride was studied. The extent...
The modulation of dopamine DA1 receptors of cultured rat renal arterial smooth muscle cells by phorbol ester, glucocorticoid and sodium chloride was studied. The extent of [3H]Sch-23390 binding to phorbol ester-treated cell was increased without any change in the dissociation constant (Kd). At a concentration of 10 nmol/l, the synthetic glucocorticoid dexamethasone increased maximum receptor binding (Bmax) but had no effect on the Kd. 100 mmol/l sodium chloride did not change Bmax, but increased the Kd for DA1 receptor. The production of cAMP in response to DA1 receptor stimulation was enhanced without any change of the adenylate cyclase activity. The glucocorticoid effect on DA1 of arterial smooth muscle cells became apparent after hours of incubation in the presence of the steroid and was significantly inhibited by cycloheximide (10 micrograms/ml) and by the glucocorticoid receptor antagonist RU-38486, indicating that the effect required protein synthesis through glucocorticoid receptors. Treatment of cells with 1 mumol/l dexamethasone for 24 h increased basal and DA1-stimulated adenylate cyclase activity. Basal adenylate cyclase was decreased by sodium chloride in a dose-dependent manner. These results suggest differential control of DA1 receptors on vascular smooth muscle cells by protein kinase C, glucocorticoid or sodium chloride.
Topics: Adenylyl Cyclases; Animals; Benzazepines; Binding, Competitive; Cyclic AMP; Dopamine Antagonists; Glucocorticoids; In Vitro Techniques; Kinetics; Muscle, Smooth, Vascular; Phorbol Esters; Rats; Rats, Wistar; Receptors, Dopamine D1; Sodium Chloride
PubMed: 8529070
DOI: 10.1291/hypres.18.supplementi_s29 -
The Journal of Cell Biology Jan 1986Embryonic rat neurons cultured in defined medium, essentially in the absence of glia, were highly enriched in phorbol ester receptors. The neurons displayed a single...
Embryonic rat neurons cultured in defined medium, essentially in the absence of glia, were highly enriched in phorbol ester receptors. The neurons displayed a single class of phorbol 12,13-dibutyrate binding sites with a maximum binding capacity, after 10 d in culture, of 18.6 pmol/mg protein and an apparent dissociation constant of 7.1 nM. Phorbol ester binding sites were associated with protein kinase C, which represented a major protein kinase activity in primary neuronal cultures. Ca2+-phosphatidylserine-sensitive phosphorylation of endogenous substrates was more marked than that observed in the presence of cyclic AMP or Ca2+ and calmodulin. Phorbol ester receptors and protein kinase C levels were critically dependent on the culture age. Thus, about a 20-fold increase in binding sites occurred during the first week in culture and was accompanied by a corresponding increase in Ca2+-phosphatidylserine-sensitive protein phosphorylation in soluble neuronal extracts. These changes largely paralleled a similar rise in phorbol ester binding during fetal development in vivo. The apparent induction of phorbol ester receptors was specific relative to other cellular proteins and could be inhibited by cycloheximide or Actinomycin D. Phosphorylation of endogenous substrates in intact cultured neurons paralleled the age-dependent increase in protein kinase C. Furthermore, 32P incorporation into several major phosphoproteins was markedly augmented by treating the neuronal cultures with phorbol esters. Such phosphorylation events may provide a clue to the significance of protein kinase C in developing neurons.
Topics: Animals; Caenorhabditis elegans Proteins; Carrier Proteins; Cell Differentiation; Cell Survival; Cells, Cultured; Molecular Weight; Nerve Tissue Proteins; Neuroglia; Neurons; Phorbol Esters; Phosphorylation; Protein Kinase C; Rats; Receptors, Drug; Receptors, Immunologic
PubMed: 3941157
DOI: 10.1083/jcb.102.1.312 -
The Journal of Biological Chemistry Nov 2011The product of the SSeCKS/GRAVIN/AKAP12 gene ("SSeCKS") is a major protein kinase (PK) C substrate that exhibits tumor- and metastasis-suppressing activity likely...
The product of the SSeCKS/GRAVIN/AKAP12 gene ("SSeCKS") is a major protein kinase (PK) C substrate that exhibits tumor- and metastasis-suppressing activity likely through its ability to scaffold multiple signaling mediators such as PKC, PKA, cyclins, calmodulin, and Src. Although SSeCKS and PKCα bind phosphatidylserine, we demonstrate that phosphatidylserine-independent binding of PKC by SSeCKS is facilitated by two homologous SSeCKS motifs, EG(I/V)(T/S)XWXSFK(K/R)(M/L)VTP(K/R)K(K/R)X(K/R)XXXEXXXE(E/D) (amino acids 592-620 and 741-769). SSeCKS binding to PKCα decreased kinase activity and was dependent on the two PKC-binding motifs. SSeCKS scaffolding of PKC was increased in confluent cell cultures, correlating with significantly increased SSeCKS protein levels and decreased PKCα activity, suggesting a role for SSeCKS in suppressing PKC activation during contact inhibition. SSeCKS-null mouse embryo fibroblasts displayed increased relative basal and phorbol ester (phorbol 12-myristate 13-acetate)-induced PKC activity but were defective in phorbol 12-myristate 13-acetate-induced actin cytoskeletal reorganization and cell shape change; these responses could be rescued by the forced expression of full-length SSeCKS but not by an SSeCKS variant deleted of its PKC-binding domains. Finally, the PKC binding sites in SSeCKS were required to restore cell rounding and/or decreased apoptosis in phorbol ester-treated LNCaP, LNCaP-C4-2, and MAT-LyLu prostate cancer cells. Thus, PKC-mediated remodeling of the actin cytoskeleton is likely regulated by the ability of SSeCKS to control PKC signaling and activity through a direct scaffolding function.
Topics: A Kinase Anchor Proteins; Animals; Apoptosis; Binding Sites; Cell Cycle Proteins; Cell Survival; Cytoskeleton; Fibroblasts; HEK293 Cells; Humans; Mice; Mice, Knockout; Phorbol Esters; Protein Isoforms; Protein Kinase C
PubMed: 21903576
DOI: 10.1074/jbc.M111.258830 -
Kidney International Sep 2001Hyperglycemia-induced overexpression of prosclerotic transforming growth factor-beta 1 (TGF-beta 1) has been implicated in the pathogenesis of diabetic nephropathy....
BACKGROUND
Hyperglycemia-induced overexpression of prosclerotic transforming growth factor-beta 1 (TGF-beta 1) has been implicated in the pathogenesis of diabetic nephropathy. Since previous in vivo studies demonstrated a renoprotective effect of low-molecular-weight (LMW) heparin in experimental animals, and recent in vitro data showed an interaction of this drug with the overactivated TGF-beta 1 cascade in high glucose- and phorbol ester-stimulated mesangial cells, we studied the molecular mechanism of these effects on TGF-beta 1 gene expression.
METHODS
Mesangial cells were stimulated with 30 mmol/L glucose or with 0.5 micromol/L phorbol ester [phorbol myristate acetate (PMA)] in the absence or presence of LMW heparin. TGF-beta 1 promoter activity was determined in promoter-reporter luciferase assays. The effect of LMW heparin on the binding of nuclear proteins to a regulatory activator protein-1 (AP-1) site, which mediates the high glucose and PMA responsiveness of the TGF-beta 1 promoter, was studied by electrophoretic mobility shift assays.
RESULTS
The presence of LMW heparin completely prevented TGF-beta 1 gene activation in both high glucose- and PMA-stimulated cells. Preincubation of the cells with LMW heparin and subsequent stimulation of the cells with both stimuli yielded the same result. Furthermore, treatment with LMW heparin prevented the enhanced binding of nuclear proteins to the regulatory AP-1 site, while binding to a Sp1 site was unaffected. Basal promoter activity and basal AP-1 binding also was reduced by LMW heparin. The LMW heparin effect on basal promoter activity was abolished by mutation of the regulatory AP-1 box B and by deletion of this AP-1 binding site.
CONCLUSIONS
LMW heparin prevents high glucose- and PMA-mediated TGF-beta 1 expression by inhibiting the activation of the TGF-beta 1 promoter and by preventing the enhanced binding of nuclear proteins to the regulatory AP-1 site.
Topics: Animals; Binding Sites; Cells, Cultured; Dose-Response Relationship, Drug; Drug Interactions; Gene Expression Regulation; Glomerular Mesangium; Glucose; Heparin, Low-Molecular-Weight; Phorbol Esters; Promoter Regions, Genetic; Protein Binding; Swine; Transcription Factor AP-1; Transcriptional Activation; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1
PubMed: 11532088
DOI: 10.1046/j.1523-1755.2001.060003935.x -
Proceedings of the National Academy of... Apr 1981Utilizing [3H]phorbol dibutyrate [P(Bu)2], we have developed an assay for high-affinity phorbol ester receptors in intact rat embryo fibroblasts. At 37 degrees C,...
Utilizing [3H]phorbol dibutyrate [P(Bu)2], we have developed an assay for high-affinity phorbol ester receptors in intact rat embryo fibroblasts. At 37 degrees C, binding of [3H]P(Bu)2 reached a maximum within 10 min and was rapidly reversible. The tumor promoters 12-O-tetradecanoyl-phorbol 13-acetate, teleocidin B, and mezerein were potent inhibitors of [3H]P(Bu)2 binding. Phorbol and 4-alpha-phorbol didecanoate, which lack tumor-promoting activity, did not inhibit [3H]P(Bu)2 binding. Epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, arginine and lysine vasopressin, luteinizing-hormone releasing hormone, and diazepam did not inhibit [3H]P(Bu)2 binding. A Scatchard analysis was compatible with two classes of binding sites, one with Kd = 8 nM and about 1--2 x 10(5) sites per cell and the other with Kd = 710 nM and about 3 x 10(6) sites per cell. Sera from various species, human amniotic fluid, and certain tissue extracts inhibited specific binding of [3H]P(Bu)2. Fractionation of human serum led to 135-fold purification of an inhibitory factor with a molecular weight in the range 40,000 to 80,000.
Topics: Animals; Binding, Competitive; Blood; Caenorhabditis elegans Proteins; Carrier Proteins; Cells, Cultured; Cocarcinogenesis; Kinetics; Phorbol Esters; Phorbols; Protein Kinase C; Rats; Receptors, Drug; Structure-Activity Relationship
PubMed: 6941290
DOI: 10.1073/pnas.78.4.2315 -
Scientific Reports Apr 2019Despite our extensive knowledge on the biology of protein kinase C (PKC) and its involvement in disease, limited success has been attained in the generation of PKC...
Despite our extensive knowledge on the biology of protein kinase C (PKC) and its involvement in disease, limited success has been attained in the generation of PKC isozyme-specific modulators acting via the C1 domain, the binding site for the lipid second messenger diacylglycerol (DAG) and the phorbol ester tumor promoters. Synthetic efforts had recently led to the identification of AJH-836, a DAG-lactone with preferential affinity for novel isozymes (nPKCs) relative to classical PKCs (cPKCs). Here, we compared the ability of AJH-836 and a prototypical phorbol ester (phorbol 12-myristate 13-acetate, PMA) to induce changes in gene expression in a lung cancer model. Gene profiling analysis using RNA-Seq revealed that PMA caused major changes in gene expression, whereas AJH-836 only induced a small subset of genes, thus providing a strong indication for a major involvement of cPKCs in their control of gene expression. MMP1, MMP9, and MMP10 were among the genes most prominently induced by PMA, an effect impaired by RNAi silencing of PKCα, but not PKCδ or PKCε. Comprehensive gene signature analysis and bioinformatics efforts, including functional enrichment and transcription factor binding site analyses of dysregulated genes, identified major differences in pathway activation and transcriptional networks between PMA and DAG-lactones. In addition to providing solid evidence for the differential involvement of individual PKC isozymes in the control of gene expression, our studies emphasize the importance of generating targeted C1 domain ligands capable of differentially regulating PKC isozyme-specific function in cellular models.
Topics: A549 Cells; Diglycerides; Drug Discovery; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; Isoenzymes; Lactones; Ligands; Lung Neoplasms; Phorbol Esters; Protein Kinase C; Transcriptome
PubMed: 30988374
DOI: 10.1038/s41598-019-42581-4 -
Blood Aug 1982Phorbol esters are potent stimulants of the respiratory burst of the human neutrophil as assessed by superoxide (O2-) generation in whole cells and by NADPH-oxidase...
Phorbol esters are potent stimulants of the respiratory burst of the human neutrophil as assessed by superoxide (O2-) generation in whole cells and by NADPH-oxidase activity in a broken-cell 27,000-g particulate fraction. Phorbol 12-myristate, 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) stimulate production of O2- by human neutrophils with ED50 concentrations of 3.9 +/- 2.1 and 41.7 +/- 7.1 nM, respectively. The relation of biologic activity to receptor occupancy was assessed with binding studies of PMA and PDBu. Phorbol ester binding revealed a single high affinity phorbol ester receptor present at 7.6 x 10(5) sites/cell. The binding affinities for PMA and PDBu, 4.9 nM and 38.4 nM, respectively, agreed quantitatively with that of biologic potencies. Because of the high concentration of phorbol ester receptors (up to 125 nM) and the large amount of nonspecific binding at high cell density, apparent discrepancies between ED50's for NADPH-oxidase and whole cell O2- generation were noted. With the use of low cell concentrations, quantitative agreement between intact cell production of O2-, NADPH-oxidase activity, and receptor binding was found. These results further support the identity of the NADPH-oxidase as the enzymatic source of respiratory burst O2- production in human neutrophils.
Topics: Caenorhabditis elegans Proteins; Carrier Proteins; Humans; NADH, NADPH Oxidoreductases; Neuraminidase; Neutrophils; Peroxides; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; Receptors, Drug
PubMed: 6953983
DOI: No ID Found -
Japanese Journal of Pharmacology Nov 1995Effects of phorbol ester, 12-deoxyphorbol 13-isobutyrate (DPB), on muscle tension and cytosolic Ca2+ ([Ca2+]i) level was investigated in rat anococcygeus muscle in...
Effects of phorbol ester, 12-deoxyphorbol 13-isobutyrate (DPB), on muscle tension and cytosolic Ca2+ ([Ca2+]i) level was investigated in rat anococcygeus muscle in comparison with other smooth muscles. 1) DPB (10(-6) M) induced a large contraction and an elevation of [Ca2+]i level in rat aorta and small and rhythmic changes in tension and [Ca2+]i level in guinea pig ileum. However, DPB did not change either of the parameters in rat anococcygeus muscle. 2) DPB caused tension development without changing the [Ca2+]i level elevated by high K+, ionomycin or beta-escin in the anococcygeus muscle. 3) In the beta-escin permeabilized muscles of guinea pig ileum and urinary bladder, rabbit mesenteric artery and rat anococcygeus muscle, DPB enhanced the Ca(2+)-developed tension. Moreover, the enhancement was inhibited by H-7 (3 x 10(-5) M). 4) DPB did not cause muscle tension to develop in the muscle of rat aorta, guinea pig ileum and rat anococcygeus muscle, pretreated with phorbol 12-myristate 13-acetate for 24 hr. In conclusion, DPB showed different contractile effects on the aorta, ileum and anococcygeus muscle, respectively. The initiation of muscle tension by DPB probably requires [Ca2+]i and the DPB-induced enhancement may be due to a Ca2+ sensitization of contractile elements in the anococcygeus muscle. Therefore, the difference between the DPB-induced response of the anococcygeus muscle and those of the other muscles seems to be due to a different Ca2+ movement caused by DPB. Moreover, it is suggested that DPB develops muscle tension by increasing [Ca2+]i and enhances it through the mediation of protein kinase C in the anococcygeus muscle as well as the other smooth muscles.
Topics: Animals; Aorta; Calcium; Female; Guinea Pigs; Ileum; Male; Muscle, Smooth; Phorbol Esters; Rats; Rats, Wistar; Trachea; Urinary Bladder
PubMed: 8699627
DOI: 10.1254/jjp.69.195 -
International Journal of Molecular... Mar 2016The Aquilaria malaccensis (Thymelaeaceae) tree is a source of precious fragrant resin, called agarwood, which is widely used in traditional medicines in East Asia...
The Aquilaria malaccensis (Thymelaeaceae) tree is a source of precious fragrant resin, called agarwood, which is widely used in traditional medicines in East Asia against diseases such as asthma. In our continuous search for active natural products, A. malaccensis seeds ethanolic extract demonstrated antiallergic effect with an IC50 value less than 1 µg/mL. Therefore, the present research aimed to purify and identify the antiallergic principle of A. malaccensis through a bioactivity-guided fractionation approach. We found that phorbol ester-rich fraction was responsible for the antiallergic activity of A. malaccensis seeds. One new active phorbol ester, 12-O-(2Z,4E,6E)-tetradeca-2,4,6-trienoylphorbol-13-acetate, aquimavitalin (1) was isolated. The structure of 1 was assigned by means of 1D and 2D NMR data and high-resolution mass spectrometry (HR-MS). Aquimavitalin (1) showed strong inhibitory activity in A23187- and antigen-induced degranulation assay with IC50 values of 1.7 and 11 nM, respectively, with a therapeutic index up to 71,000. The antiallergic activities of A. malaccensis seeds and aquimavitalin (1) have never been revealed before. The results indicated that A. malaccensis seeds and the pure compound have the potential for use in the treatment of allergy.
Topics: Animals; Anti-Allergic Agents; Cell Line, Tumor; Phorbol Esters; Plant Extracts; Rats; Seeds; Thymelaeaceae
PubMed: 27007372
DOI: 10.3390/ijms17030398 -
The Journal of Biological Chemistry Sep 1986The effects of long-chain (sphingoid) bases on the phorbol ester-dependent differentiation of HL-60 cells were investigated since these molecules are potent inhibitors...
The effects of long-chain (sphingoid) bases on the phorbol ester-dependent differentiation of HL-60 cells were investigated since these molecules are potent inhibitors of protein kinase C (Hannun, Y. A., Loomis, C. R., Merrill, A. H., Jr., and Bell, R. M. (1986) J. Biol. Chem. 261, 12604-12609). After 24 h, low concentrations of sphinganine (1-5 microM blocked both cell adherence and the inhibition of growth in response to phorbol 12-myristate 13-acetate, as measured by cell number and acid phosphatase activity. Sphinganine and sphingosine decreased adherence by 50% at 1-3 microM; other long-chain bases were effective in parallel to their inhibition of protein kinase C. Sphinganine decreased the binding of [3H]phorbol dibutyrate by the phorbol receptor of HL-60 cells, protein kinase C, and inhibited the response of HL-60 cells to dioctanoylglycerol, a cell permeable activator of this enzyme. Long-chain base uptake by HL-60 cells was demonstrated with [3-3H]sphinganine and within 1-3 days much had been converted to ceramides. By day 3, most of the cells had recovered the ability to adhere and exhibited macrophage characteristics, whereas cells in suspension did not differentiate. The level of free sphinganine in HL-60 cells was determined to be 12.3 +/- 1.2 pmol/10(6) cells. These results establish that sphingoid bases inhibit protein kinase C in HL-60 cells and may function physiologically as negative effectors of this enzyme.
Topics: Acid Phosphatase; Binding, Competitive; Cell Adhesion; Cell Differentiation; Cell Line; Humans; Kinetics; Leukemia, Myeloid, Acute; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; Sphingosine; Structure-Activity Relationship; Tetradecanoylphorbol Acetate
PubMed: 3462189
DOI: No ID Found