Phorbol ester inhibition of chicken intestinal brush-border sodium-proton exchange

1991 ◽  
Vol 260 (6) ◽  
pp. C1264-C1272 ◽  
Author(s):  
E. B. Chang ◽  
M. W. Musch ◽  
D. Drabik-Arvans ◽  
M. C. Rao
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Brush Border ◽  
Time Course ◽  
Brush Border Membrane ◽  
Phorbol Esters ◽  
Kinase C ◽  
Na Uptake ◽  
Exchange Activity

Phorbol esters, specific activators of protein kinase C, inhibit amiloride-sensitive Na uptake from the mucosal medium in intact intestinal mucosa as well as in isolated chicken villus enterocytes. In isolated cells, maximal inhibition is observed at 60 s, and influx returns to control values within 15 min. This effect can be measured either as initial 22Na influx rates or by following changes in intracellular pH using the pH-sensitive fluorescent dye 5,6-carboxyfluorescein. The effects of amiloride and phorbol esters were not additive, suggesting inhibition of a common transport system, i.e., Na-H exchange. In brush-border membrane vesicles (BBMV) made from villus enterocytes, amiloride-sensitive Na-H exchange activity was significantly inhibited in phorbol ester-treated cells. The degree of inhibition of 22Na uptake by BBMV had the same time course and dose-effect relationship as phorbol ester-inhibited cellular Na uptake. Similarly, the time course of protein kinase C translocation from cytosol to particulate or brush-border membrane fractions correlated with Na uptake measurements made in whole cells and BBMV. These results suggest that protein kinase C activation in chicken villus enterocytes inhibits brush-border membrane Na-H exchange activity.

scholarly journals Osmotic and phorbol ester-induced activation of Na+/H+ exchange: possible role of protein phosphorylation in lymphocyte volume regulation.

1985 ◽  
Vol 101 (1) ◽  
pp. 269-276 ◽  
Author(s):  
S Grinstein ◽  
S Cohen ◽  
J D Goetz ◽  
A Rothstein
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Time Course ◽  
Phorbol Esters ◽  
Underlying Mechanism ◽  
Atp Depletion ◽  
Sequence Of Events ◽  
Kinase C ◽  
Stimulation Of

The Na+/H+ antiport is stimulated by 12-O-tetradecanoylphorbol-13, acetate (TPA) and other phorbol esters in rat thymic lymphocytes. Mediation by protein kinase C is suggested by three findings: (a) 1-oleoyl-2-acetylglycerol also activated the antiport; (b) trifluoperazine, an inhibitor of protein kinase C, blocked the stimulation of Na+/H+ exchange; and (c) activation of countertransport was accompanied by increased phosphorylation of specific membrane proteins. The Na+/H+ antiport is also activated by osmotic cell shrinking. The time course, extent, and reversibility of the osmotically induced and phorbol ester-induced responses are similar. Moreover, the responses are not additive and they are equally susceptible to inhibition by trifluoperazine, N-ethylmaleimide, and ATP depletion. The extensive analogies between the TPA and osmotically induced effects suggested a common underlying mechanism, possibly activation of a protein kinase. It is conceivable that osmotic shrinkage initiates the following sequence of events: stimulation of protein kinase(s) followed by activation of the Na+/H+ antiport, resulting in cytoplasmic alkalinization. The Na+ taken up through the antiport, together with the HCO3- and Cl- accumulated in the cells as a result of the cytoplasmic alkalinization, would be followed by osmotically obliged water. This series of events could underlie the phenomenon of regulatory volume increase.

Tamoxifen inhibits phorbol ester stimulated osteoclastic bone resorption: An effect mediated by calmodulin

2000 ◽  
Vol 78 (6) ◽  
pp. 715-723 ◽  
Author(s):  
John P Williams ◽  
Margaret A McKenna ◽  
Allyn M Thames III ◽  
Jay M McDonald
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Bone Resorption ◽  
Phorbol Ester ◽  
Phorbol Esters ◽  
Fold Increase ◽  
Dependent Manner ◽  
Osteoclast Activity ◽  
Kinase C ◽  
Protein Kinase Cα

Tamoxifen inhibits bone resorption by disrupting calmodulin-dependent processes. Since tamoxifen inhibits protein kinase C in other cells, we compared the effects of tamoxifen and the phorbol ester, phorbol myristate acetate, on osteoclast activity. Phorbol esters stimulate bone resorption and calmodulin levels four-fold (k0.5 = 0.1–0.3 µM). In contrast, tamoxifen inhibited osteoclast activity ~60% with an IC50 of 1.5 µM, had no apparent effect on protein kinase C activity in whole-cell lysates, and reduced protein kinase Cα recovered by immunoprecipitation 75%. Phorbol esters stimulated resorption in a time-dependent manner that was closely correlated with a similar-fold increase in calmodulin. Protein kinase Cα, β, δ, ε, and ζ were all down-regulated in response to phorbol ester treatment. Tamoxifen and trifluoperazine inhibited PMA-dependent increases in bone resorption and calmodulin by 85 ± 10%. Down-regulation of protein kinase C isoforms by phorbol esters suggests that the observed increases in bone resorption and calmodulin levels are most likely due to a mechanism independent of protein kinase C and dependent on calmodulin. In conclusion, the data suggest that protein kinase C negatively regulates calmodulin expression and support the hypothesis that the effects of both phorbol esters and tamoxifen on osteoclast activity is mediated by calmodulin.Key words: osteoclast, calmodulin, tamoxifen, osteoporosis, protein kinase C.

Regulation of p-aminohippurate transport by protein kinase C in OK kidney epithelial cells

1996 ◽  
Vol 271 (2) ◽  
pp. F469-F475 ◽  
Author(s):  
M. Takano ◽  
J. Nagai ◽  
M. Yasuhara ◽  
K. Inui
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Phorbol Esters ◽  
Basolateral Membrane ◽  
Significant Inhibition ◽  
Regulatory Control ◽  
Ok Cells ◽  
Kinase C ◽  
Apical Side

We studied the effect of phorbol 12-myristate 13-acetate (PMA), a phorbol ester which activates protein kinase C, on p-aminohippurate (PAH) transport in OK cells. PMA (10(-7) M) almost completely inhibited the transcellular transport of PAH across OK cell monolayers from the basal to the apical side, as well as the accumulation of PAH in the cells. The uptake of PAH across the basolateral membrane of OK cells was inhibited by PMA in a time-and dose-dependent fashion. Exposing the cells with other protein kinase C activators such as active phorbol esters and diacylglycerols also resulted in a significant inhibition of basolateral PAH uptake, but the inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, had no effect. The inhibition of basolateral PAH uptake by PMA was blocked by staurosporine, an inhibitor of protein kinase C. Cycloheximide, actinomycin D, colchicine, and cytochalasin D did not affect the inhibitory effect of PMA on basolateral PAH uptake. These results suggested that the PAH transport system in OK cells is under the regulatory control of protein kinase C.

scholarly journals Protein kinase C-dependent and -independent mechanisms regulating the parotid substance P receptor as revealed by differential effects of protein kinase C inhibitors

1988 ◽  
Vol 256 (2) ◽  
pp. 677-680 ◽  
Author(s):  
H Sugiya ◽  
J W Putney
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Substance P ◽  
Time Course ◽  
Phorbol Esters ◽  
Inhibitory Effect ◽  
Protein Kinase C Inhibitors ◽  
Kinase C ◽  
Substance P Receptor ◽  
Rat Parotid

Substance P-induced inositol trisphosphate (InsP3) formation was inhibited by 1 microM-4 beta-phorbol 12,13-dibutyrate (PDBu) in rat parotid acinar cells. The inhibitory effect of PDBu was reversed by the protein kinase C inhibitors H-7 or K252a. Substance P also elicits a persistent desensitization of subsequent substance P-stimulated InsP3 formation. However, this desensitization was not inhibited by H-7. In addition, H-7 had no effect on the time course of substance P-induced InsP3 formation. These results suggest that, although activation of protein kinase C by phorbol esters can inhibit the substance P receptor-linked phospholipase C pathway, this mechanism apparently plays little, if any, role in regulating this system after activation by substance P.

scholarly journals Characterization of the Rac-GAP (Rac-GTPase-activating protein) activity of β2-chimaerin, a ‘non-protein kinase C’ phorbol ester receptor

2003 ◽  
Vol 375 (2) ◽  
pp. 313-321 ◽  
Author(s):  
Maria Jose CALOCA ◽  
HongBin WANG ◽  
Marcelo G. KAZANIETZ
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Phorbol Esters ◽  
Gtpase Activating Protein ◽  
Protein Activity ◽  
Rac Gtpase ◽  
Kinase C ◽  
Phorbol Ester Receptor

The regulation and function of β2-chimaerin, a novel receptor for the phorbol ester tumour promoters and the second messenger DAG (diacylglycerol), is largely unknown. As with PKC (protein kinase C) isoenzymes, phorbol esters bind to β2-chimaerin with high affinity and promote its subcellular distribution. β2-Chimaerin has GAP (GTPase-activating protein) activity for the small GTP-binding protein Rac1, but for not Cdc42 or RhoA. We show that acidic phospholipids enhanced its catalytic activity markedly in vitro, but the phorbol ester PMA had no effect. β2-Chimaerin and other chimaerin isoforms decreased cellular levels of Rac-GTP markedly in COS-1 cells and impaired GTP loading on to Rac upon EGF (epidermal growth factor) receptor stimulation. Deletional and mutagenesis analysis determined that the β2-chimaerin GAP domain is essential for this effect. Interestingly, PMA has a dual effect on Rac-GTP levels in COS-1 cells. PMA increased Rac-GTP levels in the absence of a PKC inhibitor, whereas under conditions in which PKC activity is inhibited, PMA markedly decreased Rac-GTP levels and potentiated the effect of β2-chimaerin. Chimaerin isoforms co-localize at the plasma membrane with active Rac, and these results were substantiated by co-immunoprecipitation assays. In summary, the novel phorbol ester receptor β2-chimaerin regulates the activity of the Rac GTPase through its GAP domain, leading to Rac inactivation. These results strongly emphasize the high complexity of DAG signalling due to the activation of PKC-independent pathways, and cast doubts regarding the selectivity of phorbol esters and DAG analogues as selective PKC activators.

scholarly journals Phosphorylation of p90 and p52 in response to phorbol-esters in Swiss/3T3 cells overexpressing protein kinase C-alpha.

1992 ◽  
Vol 3 (9) ◽  
pp. 1049-1056 ◽  
Author(s):  
H Eldar ◽  
E Livneh
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Cell Lines ◽  
Phorbol Ester ◽  
Phorbol Esters ◽  
Tumor Promotion ◽  
3T3 Cells ◽  
Kinase C ◽  
Pkc Alpha

Cell lines stably overexpressing protein kinase C (PKC)-alpha were previously described by us. These cell lines were generated by the introduction of the full length cDNA coding for PKC-alpha into Swiss/3T3 cells. Here we show that activation of PKC-alpha by phorbol-esters induced in these cells specific phosphorylation of two cellular proteins p90 and p52. Phosphorylation of p80 (MARCKS protein), previously identified as a substrate for PKC, was also enhanced. Phosphorylated p90 and p52 proteins were associated with particulate membrane-enriched fractions and were extractable with the use of nonionic detergents. Time course analysis of phorbol-ester induced phosphorylation of p90 and p52 revealed maximal stimulation of phosphorylation after 15-30 min. Phosphamino acid analysis showed that phosphorylation of p90 and p52 occurred mainly on serine residues. Phosphorylation of p52 was also on threonine residues. Whereas, phorbol ester activation induced phosphorylation of both p90 and p52, the mitogens platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) enhanced phosphorylation of p90, but not p52. Thus, our studies showed the involvement of PKC-alpha in the regulation of p90 and p52 phosphorylation and provided direct evidence for the role of PKC-alpha in cellular signaling by PDGF and FGF. Moreover, the fact that phosphorylation of p52 was specific to phorbol ester activation may suggest its involvement in tumor promotion. Characterization of p90 and p52 will enable us to reveal the phosphorylation cascade activated downstream to PKC-alpha and to determine their role in mitogenic signaling and tumor promotion.

scholarly journals Expression of mammalian protein kinase C in Schizosaccharomyces pombe: isotype-specific induction of growth arrest, vesicle formation, and endocytosis.

1994 ◽  
Vol 5 (8) ◽  
pp. 907-920 ◽  
Author(s):  
N T Goode ◽  
M A Hajibagheri ◽  
G Warren ◽  
P J Parker
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Growth Inhibition ◽  
Schizosaccharomyces Pombe ◽  
Phorbol Ester ◽  
Phorbol Esters ◽  
Vesicle Formation ◽  
Vesicular Traffic ◽  
Kinase C ◽  
Mammalian Protein

Mammalian protein kinase C (PKC) isotypes elicit a number of effects on expression in Schizosaccharomyces pombe. A small decrease in growth rate results from PKC-gamma expression, and treatment of these cells with phorbol esters leads to marked growth inhibition and vesicle formation. PKC-delta and -eta expression causes growth inhibition and vesiculation, and the magnitude of both of these effects is increased by phorbol esters. In contrast, PKC-epsilon expression produces growth inhibition but no vesicle accumulation, and this effect is not responsive to phorbol ester. Finally, PKC-zeta has no observable effect. Thus, isotype-specific biological effects are observed. The accumulation of vesicles correlates with phorbol ester-dependent growth inhibition and occurs only with expression of those isotypes that down-regulate in response to phorbol esters in these cells. Antibodies against mammalian clathrin light chain 1a identified clathrin-coated vesicles and up-regulation of clathrin expression in those cells where vesicles accumulate; the increased vesicular traffic includes an element of endocytosis. Thus expression of specific mammalian PKC isotypes up-regulates endocytosis in S. pombe, providing a likely explanation for PKC-mediated receptor internalization in higher eukaryotes.

scholarly journals Bryostatin 1 activates protein kinase C and induces monocytic differentiation of HL-60 cells

Blood ◽  
1988 ◽  
Vol 72 (1) ◽  
pp. 208-213 ◽  
Author(s):  
RM Stone ◽  
E Sariban ◽  
GR Pettit ◽  
DW Kufe
Keyword(s):  
Gene Expression ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Growth Inhibition ◽  
Phorbol Ester ◽  
Phorbol Esters ◽  
Cell Protein ◽  
Monocytic Differentiation ◽  
Bryostatin 1 ◽  
Kinase C

Phorbol esters induce the human HL-60 promyelocytic cell line to differentiate along a monocytic pathway. This induction of differentiation may involve phorbol ester-induced activation of the phospholipid- and calcium-dependent protein kinase C. Bryostatin 1, a macrocyclic lactone, has been shown to compete with phorbol esters for binding to protein kinase C. We have confirmed that bryostatin 1 translocates activity of protein kinase C from the cytosolic to membrane fractions of HL-60 cells. The present results also demonstrate that bryostatin 1 (10 nmol/L) induces monocytic differentiation of HL- 60 cells as determined by adherence, growth inhibition, appearance of monocyte cell surface antigens, and alpha-naphthyl acetate esterase staining. Furthermore, bryostatin 1 (10 nmol/L) downregulated c-myc expression and induced c-fos, c-fms, and tumor necrosis factor transcripts. These changes in gene expression induced by bryostatin 1 are similar to those associated with phorbol ester-induced monocytic differentiation of HL-60 cells. In contrast, exposure to a higher concentration of bryostatin 1 (100 nmol/L) had less of an effect on growth inhibition of HL-60 cells and changes in gene expression. Moreover, 100 nmol/L bryostatin 1 antagonized the cytostatic effects and adherence induced by phorbol esters. Our results thus suggest that bryostatin 1 activates HL-60 cell protein kinase C and that this effect is associated with induction of monocytic differentiation.

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