scholarly journals Agonist-induced desensitization of cholinergically stimulated phosphoinositide breakdown is independent of endogenously activated protein kinase C in HT-29 human colon carcinoma cells

1990 ◽  
Vol 269 (1) ◽  
pp. 73-78 ◽  
Author(s):  
R Kopp ◽  
P Mayer ◽  
A Pfeiffer
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
G Protein ◽  
Muscarinic Receptors ◽  
Phorbol Esters ◽  
Fold Increase ◽  
Human Colon ◽  
Kinase C ◽  
Ht 29

Activation of M3 muscarinic receptors in HT-29 cells by carbachol rapidly increases polyphosphoinositide breakdown. Pretreatment of these cells with carbachol (0.1 mM) for 5 h completely inhibits the subsequent ability of carbachol to increase [3H]inositol monophosphate ([3H]InsP) accumulation, paralleled by a total loss of muscarinic binding sites. In contrast, protein kinase C (PK-C)-mediated desensitization by incubation with phorbol esters [PMA (phorbol 12-myristate 13-acetate)], leading to a time- and dose-dependent inhibition of cholinergically stimulated InsP release (95% inhibition after 4 h with 0.1 microM-PMA), is accompanied by only a 40% decrease in muscarinic receptor binding, which suggests an additional mechanism of negative-feedback control. Neither carbachol nor PMA pretreatment had any effect on receptor affinity. Incubation with carbachol for 15 min caused a small increase of membrane-associated PK-C activity (15% increase, P less than 0.05) as compared with the potency of phorbol esters (PMA) (3-4-fold increase, P less than 0.01). Long-term incubation (4-24 h) with PMA resulted in a complete down-regulation of cytosolic and particulate PK-C activity. Stimulation of InsP release by NaF (20 mM) was not affected after a pretreatment with phorbol esters or carbachol, demonstrating an intact function of G-protein and phospholipase-C (PL-C) at the effector side. Determination of PL-C activity in a liposomal system with [3H]PtdInsP2 as substrate, showed no change in PL-C activity after carbachol (13 h) and short-term PMA (2.5 h) pretreatment, whereas long-term preincubation with phorbol esters (13 h) caused a small but significant decrease in PL-C activity (19%, P less than 0.05). Our results indicate that agonist-induced desensitization of phosphoinositide turnover occurs predominantly at the receptor level, with a rapid loss of muscarinic receptors. Exogenous activation of PK-C by phorbol esters seems to dissociate the interaction between receptor and G-protein/PL-C, without major effects on total cellular PL-C activity.

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.

Protein kinase C and casein kinase II activities in two human colon carcinoma cell lines, HT-29 and CaCo-2: Possible correlation with differentiation

1990 ◽  
Vol 10 (3) ◽  
pp. 293-299 ◽  
Author(s):  
Ewa Rydell ◽  
Karl-Eric Magnusson ◽  
Anita Sjö ◽  
Krister Axelsson
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Colon Carcinoma ◽  
Carcinoma Cell ◽  
Human Colon ◽  
Human Colon Carcinoma Cell ◽  
Carcinoma Cell Lines ◽  
Kinase C ◽  
Human Colon Carcinoma ◽  
Ht 29

Protein kinase C (PK-C) and casein kinase II (CK-II) activities were studied in two human colon carcinoma cell lines (HT-29 and CaCO-2) undergoing differentiation in vitro resulting, in small-intestine-like cells. CaCo-2 cells, when grown under standard conditions, appear to undergo spontaneous differentiation. In these cells PK-C and CK-II activities were determined on day 5, 10 and 15. No significant differences in activities were seen either in PK-C or CK-II activity. HT-29 cells, when grown in glucose-free medium can be stimulated to undergo differentiation which is completed within 20 days. PK-C and CK-II activities were determined after 5, 10, 15, 20 and 25 days, respectively. PK-C activity rose from 7.9±3.5 pmole32P/mg protein/min at day 5 to 37.5±14.8 pmole32P/mg protein/min at day 20. After 25 days the activity was reduced to 20.0±7.8 pmole32P/mg protein/min. CK-II activity did not change significantly during day 5 to 20, but on day 25 there was a significant decrease in CK-II activity from 94.9±6.4 pmole32P/mg protein/min (day 20) to 62.6±3.9 pmole32P/mg protein/min (day 25) p=0.003. The results in this study indicate a role for PK-C and CK-II in cell growth and differentiation.

scholarly journals Protein kinase C is involved in desensitization of muscarinic receptors induced by phorbol esters but not by receptor agonists

1990 ◽  
Vol 267 (1) ◽  
pp. 23-29 ◽  
Author(s):  
W S Lai ◽  
T B Rogers ◽  
E E el-Fakahany
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Surface ◽  
Muscarinic Receptor ◽  
Muscarinic Receptors ◽  
Receptor Agonist ◽  
Phorbol Esters ◽  
Down Regulation ◽  
Receptor Agonists ◽  
Kinase C

Preincubation with receptor agonists or phorbol esters desensitized muscarinic-receptor-mediated [3H]cyclic GMP responses in mouse neuroblastoma N1E-115 cells. However, desensitization mediated by phorbol esters was heterologous, whereas that effected by receptor agonist was specific towards the muscarinic receptors. In addition, there was no loss of cell surface muscarinic receptors, as measured by the binding of the hydrophilic ligand [3H]N-methylscopolamine, when cells were treated with phorbol esters, but receptor-agonist-induced desensitization was accompanied by a decrease in cell surface receptor density. We examined the role of protein kinase C (PKC) in the desensitization of muscarinic receptors by employing a kinase inhibitor and by down-regulation of PKC by long-term incubation of cells with phorbol esters. Whereas these manoeuvres had marked effects on phorbol-ester-induced desensitization of muscarinic responses, they did not block agonist-induced down-regulation and desensitization of muscarinic receptors. In addition, when phosphoinositide hydrolysis was suppressed, the muscarinic agonist was still capable of mediating receptor sequestration and desensitization. These results suggest that the mechanisms for regulating muscarinic receptor sensitivity could be both PKC-dependent and PKC-independent, being mediated by phorbol esters and receptor agonists respectively.

scholarly journals Aeromonas hydrophila Beta-Hemolysin Induces Active Chloride Secretion in Colon Epithelial Cells (HT-29/B6)

2004 ◽  
Vol 72 (8) ◽  
pp. 4848-4858 ◽  
Author(s):  
H. J. Epple ◽  
J. Mankertz ◽  
R. Ignatius ◽  
O. Liesenfeld ◽  
M. Fromm ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Aeromonas Hydrophila ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Human Colon ◽  
Chloride Secretion ◽  
Ion Secretion ◽  
Kinase C ◽  
Ht 29

ABSTRACT The diarrheal mechanisms in Aeromonas enteritis are not completely understood. In this study we investigated the effect of aeromonads and of their secretory products on ion secretion and barrier function of monolayers of human intestinal cells (HT-29/B6). Ion secretion was determined as a short-circuit current (ISC) of HT-29/B6 monolayers mounted in Ussing-type chambers. Transepithelial resistance (Rt) served as a measure of permeability. A diarrheal strain of Aeromonas hydrophila (strain Sb) added to the mucosal side of HT-29/B6 monolayers induced a significant ISC (39 ± 3 μA/cm2) and decreased the Rt to ∼10% of the initial value. A qualitatively identical response was obtained with sterile supernatant of strain Sb, and Aeromonas supernatant also induced a significant ISC in totally stripped human colon. Tracer flux and ion replacement studies revealed the ISC to be mainly accounted for by electrogenic Cl− secretion. Supernatant applied serosally completely abolished basal ISC. The supernatant-induced ISC was inhibited by the protein kinase C inhibitor chelerythrine, whereas a protein kinase A inhibitor (H8) and a Ca2+ chelator (BAPTA-AM) had no effect. Physicochemical properties indicated that the supernatant's active compound was an aerolysin-related Aeromonas beta-hemolysin. Accordingly, identical ISC and Rt responses were obtained with Escherichia coli lysates harboring the cloned beta-hemolysin gene from strain SB or the aerA gene encoding for aerolysin. Sequence comparison revealed a 64% homology between aerolysin and the beta-hemolysin cloned from Aeromonas sp. strain Sb. In conclusion, beta-hemolysin secreted by pathogenic aeromonads induces active Cl− secretion in the intestinal epithelium, possibly by channel insertion into the apical membrane and by activation of protein kinase C.

scholarly journals Protein Kinase C Activation Promotes Microtubule Advance in Neuronal Growth Cones by Increasing Average Microtubule Growth Lifetimes

2001 ◽  
Vol 152 (5) ◽  
pp. 1033-1044 ◽  
Author(s):  
Nurul Kabir ◽  
Andrew W. Schaefer ◽  
Arash Nakhost ◽  
Wayne S. Sossin ◽  
Paul Forscher
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Fold Increase ◽  
Retrograde Transport ◽  
Growth Cones ◽  
Microtubule Dynamics ◽  
Kinase C ◽  
Microtubule Growth ◽  
Pkc Activation

We describe a novel mechanism for protein kinase C regulation of axonal microtubule invasion of growth cones. Activation of PKC by phorbol esters resulted in a rapid, robust advance of distal microtubules (MTs) into the F-actin rich peripheral domain of growth cones, where they are normally excluded. In contrast, inhibition of PKC activity by bisindolylmaleimide and related compounds had no perceptible effect on growth cone motility, but completely blocked phorbol ester effects. Significantly, MT advance occurred despite continued retrograde F-actin flow—a process that normally inhibits MT advance. Polymer assembly was necessary for PKC-mediated MT advance since it was highly sensitive to a range of antagonists at concentrations that specifically interfere with microtubule dynamics. Biochemical evidence is presented that PKC activation promotes formation of a highly dynamic MT pool. Direct assessment of microtubule dynamics and translocation using the fluorescent speckle microscopy microtubule marking technique indicates PKC activation results in a nearly twofold increase in the typical lifetime of a MT growth episode, accompanied by a 1.7-fold increase and twofold decrease in rescue and catastrophe frequencies, respectively. No significant effects on instantaneous microtubule growth, shortening, or sliding rates (in either anterograde or retrograde directions) were observed. MTs also spent a greater percentage of time undergoing retrograde transport after PKC activation, despite overall MT advance. These results suggest that regulation of MT assembly by PKC may be an important factor in determining neurite outgrowth and regrowth rates and may play a role in other cellular processes dependent on directed MT advance.

Protein kinase C injection into hippocampal pyramidal cells elicits features of long term potentiation

Nature ◽  
1987 ◽  
Vol 328 (6129) ◽  
pp. 426-429 ◽  
Author(s):  
G.-Y. Hu ◽  
Ø. Hvalby ◽  
S. I. Walaas ◽  
K. A. Albert ◽  
P. Skjeflo ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Pyramidal Cells ◽  
Long Term Potentiation ◽  
Kinase C ◽  
Term Potentiation ◽  
Hippocampal Pyramidal Cells
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