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The Journal of Cell Biology Jul 1984The human, leukemic cell line, HL-60, undergoes differentiation in response to tumor-promoting phorbol esters. Recent studies have implicated stimulation of a Na+/H+...
The human, leukemic cell line, HL-60, undergoes differentiation in response to tumor-promoting phorbol esters. Recent studies have implicated stimulation of a Na+/H+ antiporter as an initial event in cellular differentiation and/or proliferation. The effects of phorbol esters on Na+-dependent H+ efflux from HL-60 cells were studied by pH-stat titration. Tumor-promoting phorbol diesters, but not the inactive parent alcohol, stimulated Na+-dependent H+ efflux in a rapid (within 1 min at 37 degrees C) and reversible manner. Stimulation was dependent on the concentration of extracellular sodium; lithium could substitute for sodium, but choline could not. Stimulation was dependent on the activity of extracellular protons and was inhibited completely by amiloride. The concentrations of phorbol diesters at which we observed half-maximal stimulation of Na+-dependent H+ efflux are very similar to the Kd reported in the literature for binding of these phorbol diesters to the phorbol ester receptor and the Km for phorbol diester activation of protein kinase C. Overall characterization of basal and phorbol ester-stimulated H+ efflux indicate that stimulation of a Na+/H+ antiporter constitutes a primary event in phorbol ester interaction with HL-60 cells.
Topics: Amiloride; Carrier Proteins; Cell Line; Humans; Kinetics; Leukemia, Myeloid, Acute; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Pyrazines; Sodium-Hydrogen Exchangers; Tetradecanoylphorbol Acetate
PubMed: 6330130
DOI: 10.1083/jcb.99.1.340 -
Journal of Bioscience and Bioengineering Mar 2014A novel enzymatic degradation of phorbol esters (PE) in the jatropha seed cake was developed using lipase. Cihera rice bran lipase had the highest ability to hydrolyze...
A novel enzymatic degradation of phorbol esters (PE) in the jatropha seed cake was developed using lipase. Cihera rice bran lipase had the highest ability to hydrolyze PE, and reduced PE to a safe level after 8 h of incubation. Enzymatic degradation may be a promising method for PE degradation.
Topics: Biodegradation, Environmental; Chromatography, Thin Layer; Jatropha; Lipase; Oryza; Phorbol Esters; Seeds
PubMed: 24099956
DOI: 10.1016/j.jbiosc.2013.08.006 -
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 -
The American Journal of Physiology Dec 1987Intracellular calcium concentration ([Ca2+]i) was measured with aequorin in ferret and rat aortic strips contracted with phorbol esters. In ferret aorta, 12-deoxyphorbol...
Intracellular calcium concentration ([Ca2+]i) was measured with aequorin in ferret and rat aortic strips contracted with phorbol esters. In ferret aorta, 12-deoxyphorbol 13-isobutyrate 20-acetate (DPBA, 1 microM) induced contractions without significantly increasing [Ca2+]i, whereas 21 mM K+ induced smaller contractions with a significant rise in [Ca2+]i. Ca2+-free 2.5 mM ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA)-physiological saline solution (PSS) had no effect on DPBA-induced tension, whereas it abolished contractions induced by 66 mM K+. The alpha 1-adrenergic agonist phenylephrine (10(-5) M) induced less than 10% of the tension with no initial [Ca2+]i spike under Ca-free conditions. In rat aorta, both phorbol 12-myristate 13-acetate (PMA, 2 microM) and DPBA (1 microM) induced contractions without increasing [Ca2+]i; Ca2+-free EGTA-PSS or the addition of the calcium channel blocker gallopamil (D600, 1 microM), however, abolished greater than 50% of the tension induced by either phorbol ester with a decrease in [Ca2+]i. These results are consistent with the idea that 1) resting [Ca2+]i is both sufficient and required to support phorbol ester-induced contractions in two vascular smooth muscles, suggesting an increased sensitivity of the contractile apparatus for Ca2+, and 2) there are differences in the mechanisms by which phorbol esters and alpha 1-agonists may activate vascular smooth muscle.
Topics: Animals; Aorta; Calcium; Ferrets; Isometric Contraction; Male; Muscle Contraction; Muscle, Smooth, Vascular; Phenylephrine; Phorbol Esters; Protein Kinase C; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate
PubMed: 3425738
DOI: 10.1152/ajpheart.1987.253.6.H1365 -
Cancer Research Dec 1983Sustained exposure of GH4C1 rat pituitary cells to phorbol esters or the hypothalamic tripeptide thyrotropin-releasing hormone decreases the number of phorbol ester...
Sustained exposure of GH4C1 rat pituitary cells to phorbol esters or the hypothalamic tripeptide thyrotropin-releasing hormone decreases the number of phorbol ester binding sites without changing affinity (down modulation; Jaken, S., Tashjian, A. H., Jr., and Blumberg, P. M. Cancer Res., 41: 2175-2181, 1981). In untreated control cultures, the concentration of receptors in lysates was not significantly different from the concentration in intact cells. In contrast, in down-modulated cultures, the concentration of receptors in lysates was greater than the concentration in the corresponding intact cells and instead was equal to the concentration in lysates of control cultures. Therefore, the processes of both homologous (phorbol ester-induced) and heterologous (thyrotropin-releasing hormone-induced) phorbol ester receptor down modulation involves the generation of a cryptic receptor state that is revealed upon cell lysis. Furthermore, comparison of receptor properties in cells and lysates revealed that the affinity of the receptor for phorbol dibutyrate was decreased from a Kd of 11.1 +/- 0.6 nM (S.D.) in intact cells to 40 +/- 10 nM in cell lysates. Binding of the receptor for the structurally related diterpene, mezerein, fit a one-component model in intact cells, but was better fit to a two-component model in cell lysates. That is, the apparently homogeneous phorbol 12,13-dibutyrate receptor population in cell lysates appeared heterologous with respect to mezerein binding. This is not due to a preferential recovery of a subset of receptors in the lysates, because no substantial loss of receptor number was associated with cell lysis. Instead, these results suggest that the affinity and ligand specificity of the phorbol ester receptor may depend on its cellular environment.
Topics: Animals; Caenorhabditis elegans Proteins; Carrier Proteins; Cell Line; Cell Nucleus; Kinetics; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Pituitary Neoplasms; Protein Kinase C; Rats; Receptors, Cell Surface; Receptors, Drug; Thyrotropin-Releasing Hormone
PubMed: 6315217
DOI: No ID Found -
Journal of Biomolecular Structure &... Jul 2016Protein kinase C (PKC) isozymes are important regulatory enzymes that have been implicated in many diseases, including cancer, Alzheimer's disease, and in the...
Protein kinase C (PKC) isozymes are important regulatory enzymes that have been implicated in many diseases, including cancer, Alzheimer's disease, and in the eradication of HIV/AIDS. Given their potential clinical ramifications, PKC modulators, e.g. phorbol esters and bryostatin, are also of great interest in the drug development. However, structural details on the binding between PKC and its modulators, especially bryostatin - the highly potent and non-tumor promoting activator for PKCs, are still lacking. Here, we report the first comparative molecular dynamics study aimed at gaining structural insight into the mechanisms by which the PKC delta cys2 activator domain is used in its binding to phorbol ester and bryostatin-1. As anticipated in the phorbol ester binding, hydrogen bonds are formed through the backbone atoms of Thr242, Leu251, and Gly253 of PKC. However, the opposition of H-bond formation between Thr242 and Gly253 may cause the phorbol ester complex to become less stable when compared with the bryostatin binding. For the PKC delta-bryostatin complex, hydrogen bonds are formed between the Gly253 backbone carbonyl and the C30 carbomethoxy substituent of the ligand. Additionally, the indole Nε1 of the highly homologous Trp252 also forms an H-bond to the C20 ester group on bryostatin. Backbone fluctuations also suggest that this latter H-bond formation may abrogate the transient interaction between Trp252 and His269, thus dampening the fluctuations observed on the nearby Zn(2+)-coordinating residues. This new dynamic fluctuation dampening model can potentially benefit future design of new PKC modulators.
Topics: Binding Sites; Bryostatins; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Ligands; Models, Molecular; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Phorbol Esters; Protein Binding; Protein Interaction Domains and Motifs; Protein Kinase C
PubMed: 26292580
DOI: 10.1080/07391102.2015.1084479 -
Cancer Research Jun 1981Phorbol ester binding to intact GH4C1 cells, a continuous strain of rat pituitary cells, was measured using [3H]phorbol 12,13-dibutyrate. The binding was saturable;...
Phorbol ester binding to intact GH4C1 cells, a continuous strain of rat pituitary cells, was measured using [3H]phorbol 12,13-dibutyrate. The binding was saturable; Scatchard analysis indicated one class of high-affinity binding sites (Kd, 11 nM) with 6.4 pmol [3H]phorbol 12,13-dibutyrate bound per mg cell protein at saturation. The relative binding affinities for other phorbol esters and analogs were similar to those demonstrated for binding to homogenates of chick embryo fibroblasts and mouse skin, as well as for tumor promotion in vivo. A close correlation was shown to exist between the binding affinities of these derivatives and their potencies for inducing biological responses in GH4C1 cells, such as decreases in binding of epidermal growth factor and thyrotropin-releasing hormone. A distinctive new finding is the down modulation of phorbol ester binding sites on GH4C1 cells by both homologous and heterologous ligands. Prolonged exposure to phorbol esters or thyrotropin-releasing hormone produced a loss of available [3H]phorbol 12,13-dibutyrate binding sites with a maximal decrease to about 20% of control after 24 hr of treatment. Scatchard analysis indicated that the decrease in binding was due to a loss of receptors with no change in affinity. The biological significance of phorbol ester receptor down modulation is not yet known; however, it may represent a mechanism for attenuating cellular responsiveness to phorbol esters in their continued presence.
Topics: Animals; Binding Sites; Caenorhabditis elegans Proteins; Carrier Proteins; Dose-Response Relationship, Drug; Kinetics; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Pituitary Gland; Protein Kinase C; Rats; Receptors, Drug; Thyrotropin-Releasing Hormone; Time Factors
PubMed: 6786733
DOI: No ID Found -
Cardiovascular Research Mar 1993The aim was to examine the cardiac effects of phorbol esters over a wide concentration range and to determine if the effects are related to Ca2+ availability.
OBJECTIVE
The aim was to examine the cardiac effects of phorbol esters over a wide concentration range and to determine if the effects are related to Ca2+ availability.
METHODS
Studies were carried out using isolated rat hearts exposed for 60 min either to phorbol 12-myristate 13-acetate (PMA, 10(-11) to 10(-6) M) or phorbol 12,13-dibutyrate (PDBu, 10(-12) to 10(-7) M) in the presence of either 1.25 or 2.50 mM CaCl2. Experiments were also done to assess the effect of BAY K8644, a Ca2+ agonist, on phorbol ester effects. After treatment, hearts were freeze clamped for later analysis of energy products.
RESULTS
At the lowest concentrations studied, both PMA and PDBu produced positive inotropic effects, whereas higher concentrations resulted in a loss of contractile force in hearts perfused with 1.25 mM CaCl2. Doubling the CaCl2 concentration or the presence of BAY K8644 had little effect on the negative inotropic influence of either phorbol ester but reversed the positive inotropic effect to a negative inotropic one. Neither treatment had any effect on the coronary constricting effects of phorbol esters. Increases in resting tension and reductions in high energy phosphate content were evident only with the highest phorbol ester concentrations and were unaffected either by changes in CaCl2 concentrations or the presence of BAY K8644.
CONCLUSIONS
At picomolar and nanomolar concentrations phorbol esters produce positive inotropic actions which are probably mediated by enhanced Ca2+ influx. Although higher concentrations produce negative inotropic effects, these are not influenced by [Ca2+]o nor are they related to disturbances in energy metabolism except at the highest concentrations. We conclude that phorbol esters produce complex concentration dependent cardiac effects which are not mediated by a single mechanism of action.
Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcium Chloride; Dose-Response Relationship, Drug; Drug Interactions; Energy Metabolism; Male; Myocardial Contraction; Myocardium; Phorbol 12,13-Dibutyrate; Rats; Rats, Sprague-Dawley; Tetradecanoylphorbol Acetate; Time Factors
PubMed: 7683974
DOI: 10.1093/cvr/27.3.390 -
IARC Scientific Publications 1984Specific phorbol ester receptors are found in the particulate fraction of cells. In addition, cytosol contains a phorbol ester apo-receptor, which requires phospholipids...
Specific phorbol ester receptors are found in the particulate fraction of cells. In addition, cytosol contains a phorbol ester apo-receptor, which requires phospholipids for reconstitution. The apo-receptor corresponds to protein kinase C, and the quantitatively major membrane receptor appears to be a protein kinase C-phospholipid complex. The ability to reconstitute the phorbol ester apo-receptor into different lipid domains now makes it possible to begin elucidation of the role of the lipid domain in phorbol ester action. Studies reviewed here indicate that diglycerides competitively inhibit phorbol ester binding, consistent with their being the postulated endogenous phorbol ester analogues. Highly lipophilic phorbol esters inhibit effectively only if they are incorporated into the lipid phase, indicating that it is the membrane-dissolved form of the ligand which is recognized. The binding affinity of 3H-phorbol 12,13-dibutyrate for holo-receptor depends markedly (greater than 20-fold range) on the phospholipid environment, and heterogeneous phorbol ester binding (i.e., curved Scatchard plots) can be generated by use of heterogeneous lipid environments in the reconstitution. The possible existence of other phorbol ester receptors in addition to protein kinase C-phospholipid complexes remains to be resolved.
Topics: Animals; Apoproteins; Caenorhabditis elegans Proteins; Carrier Proteins; Cell Membrane; Cytosol; Diglycerides; Kinetics; Membrane Lipids; Phorbol Esters; Phorbols; Phospholipids; Protein Kinase C; Protein Kinases; Receptors, Drug; Receptors, Immunologic
PubMed: 6242146
DOI: No ID Found -
Biochimica Et Biophysica Acta Apr 1988Both phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (10(-8)-10(-6) M) induced concentration-dependent increases in prostaglandin E2 (PGE2) production...
Both phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (10(-8)-10(-6) M) induced concentration-dependent increases in prostaglandin E2 (PGE2) production by human amnion cells, with maximum stimulations of 10.8-fold and 5.9-fold, respectively. 4 alpha-Phorbol 12,13-didecanoate, an inactive phorbol ester analogue, had little or no effect on PGE2 production by amnion cells. PMA and phorbol 12,13-dibutyrate (10(-7) M) induced a maximal increase in the rate of PGE2 biosynthesis within 15 min of treatment. These results suggest that there is an active protein kinase C present in amnion cells that is linked to arachidonic acid release and/or metabolism.
Topics: Amnion; Dinoprostone; Humans; Phorbol 12,13-Dibutyrate; Phorbol Esters; Prostaglandins E; Tetradecanoylphorbol Acetate
PubMed: 3162688
DOI: 10.1016/0005-2760(88)90214-7