Evidence that phorbol ester interferes with stimulated Ca2+ redistribution by activating Ca2+ efflux in neutrophil leucocytes.
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ABSTRACT:
SUBMITTER: Rickard JE
PROVIDER: S-EPMC1152794 | biostudies-other | 1985 Nov
REPOSITORIES: biostudies-other
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Project description:Endothelin-1 (ET-1) and activation of protein kinase C (PKC) have been implicated in alterations of myocyte function in cardiac hypertrophy and heart failure. Changes in cellular Ca2+ handling and electrophysiological properties also occur in these states and may contribute to mechanical dysfunction and arrhythmias. While ET-1 or PKC stimulation induces cellular hypertrophy in cultured neonatal rat ventricular myocytes (NRVMs), a system widely used in studies of hypertrophic signaling, there is little data about electrophysiological changes. Here we studied the effects of ET-1 (100 nM) or the PKC activator phorbol 12-myristate 13-acetate (PMA, 1 ?M) on ionic currents in NRVMs. The acute effects of PMA or ET-1 (?30 min) were small or insignificant. However, PMA or ET-1 exposure for 48-72 h increased cell capacitance by 100 or 25%, respectively, indicating cellular hypertrophy. ET-1 also slightly increased Ca2+ current density (T and L type). Na+/Ca2+ exchange current was increased by chronic pretreatment with either PMA or ET-1. In contrast, transient outward and delayed rectifier K+ currents were strongly downregulated by PMA or ET-1 pretreatment. Inward rectifier K+ current tended toward a decrease at larger negative potential, but time-independent outward K+ current was unaltered by either treatment. The enhanced inward and reduced outward currents also result in action potential prolongation after PMA or ET-1 pretreatment. We conclude that chronic PMA or ET-1 exposure in cultured NRVMs causes altered functional expression of cardiac ion currents, which mimic electrophysiological changes seen in whole animal and human hypertrophy and heart failure.
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Project description:The signalling lipid phosphatidic acid (PA) is generated by the hydrolysis of phosphatidylcholine (PC), which is catalysed by phospholipase D (PLD) enzymes. Neutrophils, important cells of the innate immune system, maintain the body's defence against infection. Previous studies have implicated PLD-generated PA in neutrophil function; these have relied heavily on the use of primary alcohols to act as inhibitors of PA production. The recent development of isoform-selective small molecule inhibitors and the generation of a knockout mouse model provide us with accurate tools to study the role of PLDs in neutrophil responses. We show that PLD1 is a regulator of phorbol-ester-, chemoattractant, adhesion-dependent and Fc?-receptor-stimulated production of reactive oxygen species (ROS) in neutrophils. Significantly we found that this role of PLD is isoform specific: the absence of PLD2 does not negatively affect these processes. Contrary to expectation, other functions required for an efficient immune response operate effectively in Pld2-deficient neutrophils or when both isoforms are inhibited pharmacologically. We conclude that although PLD1 does have important regulatory roles in neutrophils, the field has been confused by the use of primary alcohols; now that gold standard Pld-knockout mouse models are available, previous work might need to be reassessed.
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Project description: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.
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Project description:Down-regulation of the KAI1 (CD82) metastasis suppressor is common in advanced human cancer, but underlying mechanism(s) regulating KAI1 expression are only now being elucidated. Recent data provide evidence that low levels of KAI1 mRNA in LNCaP cells are caused by binding of beta-catenin/Reptin complexes to a specific motif in the proximal promoter, which prevents binding of Tip60/Pontin activator complexes to the same motif, thus inhibiting transcription. Here, we explored a pathway by which phorbol 12-myristate 13-acetate (PMA) up-regulates KAI1 transcription in LNCaP prostate cancer cells. Pretreatment with specific inhibitors showed that induction of KAI1 by PMA uses classic isoforms of protein kinase C (cPKC), is independent of Ras and Raf, and requires activation of MEK1/2 and ERK1/2, but does not involve p38MAPK. Induction of KAI1 transcription by PMA was associated with enhanced overall acetylation of histones H3 and H4, but only acetylation of H3 was blocked by a PKC inhibitor. Chromatin immunoprecipitation showed that PMA induces recruitment of Tip60/Pontin activator complexes to NFkappaB-p50 motifs in the proximal promoter, and this was blocked by a PKC inhibitor. These changes were not associated with differences in overall levels of Tip60, Pontin, beta-catenin, or Reptin protein expression but with PMA-induced nuclear translocation of Tip60.
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Project description:White rot fungi are well known for their ability to degrade a wide range of xenobiotics due to their enzymatic systems. Therefore, the present investigation was aimed at screening ten different white rot fungi for degradation of phorbol esters from Jatropha seedcake (JSC). The JSC was fermented with pure cultures of white rot fungi for 20 days under solid state condition. All the white rot fungi tested exhibited degradation of phorbol ester during fermentation of JSC without adversely influencing the nutritional properties of the seedcake. Ganoderma lucidum and Trametes zonata were found to degrade phorbol ester in JSC to undetectable levels. This study demonstrates the potential of white rot fungi for degradation of phorbol esters, a major anti-nutritional factor, in JSC preventing its utilization as cattle feed.
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Project description:The membrane-bound transglutaminase of cultured keratinocytes became radioactively labelled upon addition of [32P]Pi to the medium. Transglutaminase phosphorylation was also demonstrable using particulate material isolated from cell homogenates. Compatible with mediation of the labelling by protein kinase C, the degree of phosphorylation in intact cells was stimulated approx. 5-fold in 4 h on treatment with the tumour-promoting phorbol ester phorbol 12-myristate 13-acetate, but not by phorbol. The extent of labelling was virtually unaffected by cycloheximide inhibition of protein synthesis, indicating that it arose primarily through turnover of phosphate in the membrane-bound enzyme. Phosphoamino acid analysis detected labelling only of serine residues. Most of the label was removed by trypsin release of the enzyme from the particulate fraction of cell homogenates, which deletes a membrane anchorage region of approximately 10 kDa. Upon trypsin treatment of the enzyme after immunoprecipitation, the phosphate label was recovered in soluble peptide material with a size of several thousand Da or less. Indicative of fragmentation of the membrane anchorage region, this material was separable by h.p.l.c. into two equally labelled peptides. Moreover, when the enzyme was labelled with [3H]palmitate or [3H]myristate, the fatty-acid-labelled peptide material required non-ionic detergent for solubilization and was separable from the phosphate-labelled material by gel filtration. Phorbol ester treatment of cultured keratinocytes in high- or low- Ca2(+)-containing medium was not accompanied by an appreciable protein-synthesis-independent change in transglutaminase activity. Independent of possible alteration of the intrinsic catalytic activity of the enzyme, phosphorylation may well modulate its interaction with substrate proteins, a potential site for physiological regulation.
| S-EPMC1149509 | biostudies-other
Project description:Phorbol 12-myristate 13-acetate (PMA)-stimulated phosphorylation of the human insulin receptor (IR) was characterized and compared in two cell types of different lineage: normal rat kidney epithelial (NRK) cells and Chinese hamster ovary (CHO) fibroblasts. PMA stimulation increased IR beta-subunit phosphorylation to 252 +/- 43 and 25- +/- 47% (+/- S.D.) of the unstimulated control in NRK and CHO cells respectively. Tryptic phosphopeptide analysis by Tricine/SDS/PAGE revealed significant differences in the PMA-stimulated phosphorylation of the IR in these two cell types. This phosphorylation of the IR was predominantly located in two tryptic phosphopeptides, and these phosphopeptides were absent in an IR mutant truncated by 43 C-terminal amino acids. The major PMA-stimulated tryptic phosphopeptide from in vivo-labelled CHO/IR was immunoprecipitated with an antibody against residues Ser1315 to Lys1329, and this precipitation was blocked with excess unlabelled peptide containing this sequence. Radiosequencing by manual Edman degradation revealed that this tryptic phosphopeptide was phosphorylated at Ser1315. This PMA-stimulated phosphorylation did not inhibit autophosphorylation of the IR in vivo. These results demonstrate that PMA-stimulated phosphorylation of the IR can exhibit significant differences when expressed in different cell types, and that Ser1315 is a major PMA-stimulated phosphorylation site on the human IR.
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Project description:The antagonization of phorbol 12-myristate 13-acetate (PMA)-stimulated phosphatidylcholine (PtdCho) biosynthesis by the phospholipid analogue hexadecylphosphocholine (HePC) in MDCK cells was investigated and compared with the corresponding influence in HeLa cells. In both cell lines, PMA-stimulated PtdCho biosynthesis was antagonized by 50 microM HePC. However, subsequent experiments provided evidence that PMA enhances PtdCho biosynthesis by at least two mechanisms: (i) by stimulation of choline uptake and (ii) by translocation of CTP:choline phosphate cytidylyltransferase to membranes. In MDCK cells, 5 nM PMA caused a 4-fold increase in [methyl-3H]choline incorporation into PtdCho, which was paralleled by an approx. 2-fold stimulation of choline uptake. These data indicate that choline uptake might play an important role in the regulation of PtdCho biosynthesis in this cell line, especially since we could not detect any significant increase in membrane-bound cytidyltransferase activity in PMA-treated MDCK cells. In contrast, enhanced PtdCho biosynthesis in HeLa cells is achieved by a 2-fold increase in particulate cytidylyltransferase activity after PMA stimulation. Translocation of cytidylyltransferase from the cytosol to membranes is therefore important in HeLa cells. Nevertheless, in both cell lines, the main target of HePC seems to be the translocation process. In MDCK cells, addition of 50 microM HePC decreases membrane-bound cytidylyltransferase activity by about 45%, compared with control cells and PMA-treated cells. In HeLa cells, PMA-induced translocation of cytidylyltransferase to membranes is totally abolished by HePC.
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Project description:BackgroundResveratrol (1) is a naturally occurring polyphenol that has been implicated in neuroprotection. One of resveratrol's several biological targets is Ca2+-sensitive protein kinase C alpha (PKCα). Resveratrol inhibits PKCα by binding to its activator-binding C1 domain. Munc13-1 is a C1 domain-containing Ca2+-sensitive SNARE complex protein essential for vesicle priming and neurotransmitter release.MethodsTo test if resveratrol could also bind and inhibit Munc13-1, we studied the interaction of resveratrol and its derivatives, (E)-1,3-dimethoxy-5-(4-methoxystyryl)benzene, (E)-5,5'-(ethene-1,2-diyl)bis(benzene-1,2,3-triol), (E)-1,2-bis(3,4,5-trimethoxyphenyl)ethane, and (E)-5-(4-(hexadecyloxy)-3,5-dihydroxystyryl)benzene-1,2,3-triol with Munc13-1 by studying its membrane translocation from cytosol to plasma membrane in HT22 cells and primary hippocampal neurons.ResultsResveratrol, but not the derivatives inhibited phorbol ester-induced Munc13-1 translocation from cytosol to membrane in HT22 cells and primary hippocampal neurons, as evidenced by immunoblot analysis and confocal microscopy. Resveratrol did not show any effect on Munc13-1H567K, a mutant which is not sensitive to phorbol ester. Binding studies with Munc13-1 C1 indicated that resveratrol competes with phorbol ester for the binding site. Molecular docking and dynamics studies suggested that hydroxyl groups of resveratrol interact with phorbol-ester binding residues in the binding pocket.Conclusions and significanceThis study characterizes Munc13-1 as a target of resveratrol and highlights the importance of dietary polyphenol in the management of neurodegenerative diseases.
| S-EPMC5623624 | biostudies-literature
Project description:Ser910 of FAK (focal adhesion kinase) was phosphorylated in fibroblasts treated with the phorbol ester PMA and dephosphorylated by PP1d (protein phosphatase 1d), as indicated by shRNA (small-hairpin RNA) gene silencing. Ser910 of FAK was reported previously to be an ERK (extracellular-signal-regulated kinase) 1/2 target in cells treated with phorbol esters. In contrast, various approaches, including the use of the MEK (mitogen-activated protein kinase/ERK kinase) inhibitors UO126 and CI-1040 to inhibit ERK1/2 pointed to the involvement of ERK5. This hypothesis was confirmed by: (i) shRNA ERK5 gene silencing, which resulted in complete pSer910 loss in non-stimulated and PMA-stimulated cells; (ii) direct phosphorylation of recombinant FAK by ERK5; and (iii) ERK5 activation by PMA. PMA stimulation and ERK5 silencing in MDA-MB 231 and MDA-MB 361 breast cancer cells indicated Ser910 targeting by ERK5 also in these cells. Given the proximity of Ser910 to the FAT (focal adhesion targeting) regulatory domain of FAK, cell proliferation and morphology were investigated in FAK-/- cells expressing S910A mutant FAK. The cell growth rate decreased and exposure to PMA induced peculiar morphological changes in cells expressing S910A, with respect to wild-type FAK, suggesting a role for Ser910 in these processes. The present study indicates, for the first time, the phosphorylation of Ser910 of FAK by ERK5 and its dephosphorylation by PP1d, and suggested a role for Ser910 in the control of cell shape and proliferation.
| S-EPMC2049076 | biostudies-other