Mucin secretion by T84 cells: stimulation by PKC, Ca2+, and a protein kinase activated by Ca2+ ionophore

1993 ◽  
Vol 264 (6) ◽  
pp. G1096-G1102 ◽  
Author(s):  
G. Forstner ◽  
Y. Zhang ◽  
D. McCool ◽  
J. Forstner
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Mucin Secretion ◽  
T84 Cells ◽  
Kinase C ◽  
Protein Kinase Inhibition ◽  
Adenocarcinoma Cells ◽  
Pkc Activation ◽  
Dependent Pathway

T84 adenocarcinoma cells were stimulated to secrete mucin by the phorbol ester phorbol 12-myristate 13-acetate (PMA) and Ca2+ ionophores A23187 and ionomycin. In Ca(2+)-containing media, maximal stimulation by PMA was significantly inhibited by staurosporine, but maximal A23187-stimulated secretion was not affected. Downregulation of protein kinase C (PKC) reduced maximal PMA-stimulated secretion without affecting the response to A23187. Thus PKC activation is not required for maximal Ca(2+)-mediated mucin secretion. PMA stimulated secretion in low-Ca2+ media, with and without intracellular chelation of Ca2+ by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Surprisingly, Ca2+ ionophores also stimulated secretion under the same circumstances. Persistent A23187-stimulated secretion was strongly inhibited by the protein kinase inhibitors staurosporine and H-7. Secretion in Ca(2+)-containing media was also inhibited at submaximal levels of Ca(2+)-ionophore stimulation. These results indicate that PKC and Ca2+ stimulate mucin exocytosis independently. Ca2+ ionophores also stimulate secretion via a protein-kinase dependent pathway. Enhancement of protein kinase inhibition at lower Ca2+ concentrations suggests that the response could be mediated by a Ca2+ ionophore-induced depletion of an intracellular Ca2+ pool.

Regulation of mucin secretion in T84 adenocarcinoma cells by forskolin: relationship to Ca2+ and PKC

1994 ◽  
Vol 266 (4) ◽  
pp. G606-G612 ◽  
Author(s):  
G. Forstner ◽  
Y. Zhang ◽  
D. McCool ◽  
J. Forstner
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Mucin Secretion ◽  
T84 Cells ◽  
Independent Manner ◽  
A 23187 ◽  
Adenocarcinoma Cells ◽  
High Doses ◽  
The Relationship

The relationship between the adenosine 3',5'-cyclic monophosphate-mediated protein kinase A (PKA)-dependent stimulatory pathway for mucin secretion and Ca(2+)-mediated and protein kinase C (PKC)-mediated secretion was studied in T84 cells, using the postreceptor secretagogues forskolin, A-23187, and phorbol 12-myristate 13-acetate (PMA), the protein kinase inhibitors staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), high- and low-Ca2+ media, and the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Staurosporine (10(-5) M) inhibited both PMA and forskolin at their maximally effective concentrations, whereas H-7 (5 x 10(-5) M) inhibited only PMA. Stimulation of mucin secretion by forskolin (5 x 10(-5) M) was not significantly affected by the reduction of medium Ca2+ to 47 and 129 nM, equivalent to published values for intracellular Ca2+ concentration ([Ca2+]i). Stimulation by forskolin was reduced by preloading cells with BAPTA, but to a much smaller extent than Ca(2+)-dependent stimulation by A-23187. A-23187-mediated mucin secretion from BAPTA-loaded cells was augmented by high doses of forskolin. Similar concentrations of forskolin had no effect on A-23187-stimulated secretion in calcium-replete cells. Our results indicate that forskolin does not stimulate mucin secretion by increasing Ca2+ entry or releasing Ca2+ from intracellular stores. Forskolin can stimulate mucin secretion in a Ca(2+)-independent manner but is apparently inhibited by high levels of intracellular Ca2+ induced by Ca2+ ionophores in 1.0 mM Ca2+ media.(ABSTRACT TRUNCATED AT 250 WORDS)

Genistein and tyrphostin 47 stimulate CFTR-mediated Cl- secretion in T84 cell monolayers

1995 ◽  
Vol 269 (6) ◽  
pp. G874-G882 ◽  
Author(s):  
C. L. Sears ◽  
F. Firoozmand ◽  
A. Mellander ◽  
F. G. Chambers ◽  
I. G. Eromar ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Secretory Pathway ◽  
Kinase Inhibitors ◽  
T84 Cells ◽  
Cl Secretion ◽  
Cyclic Monophosphate ◽  
3T3 Fibroblasts ◽  
Kinase C

The involvement of tyrosine phosphorylation in the regulation of epithelial cell Cl- secretion is unknown. Therefore, the purpose of these studies was to determine if tyrosine kinase activation was involved in the regulation of Cl- secretion, using the tyrosine kinase inhibitors, genistein and tyrphostin 47, and human intestinal epithelial cells (T84 cells) as an intestinal Cl- secretory model. Genistein rapidly but reversibly stimulated sustained apical Cl- secretion in monolayers of T84 cells without increasing intracellular cyclic nucleotides or Ca2+ levels. Tyrphostin 47 also stimulated Cl- secretion in T84 monolayers, although it was short-lived. Transfection experiments in 3T3 fibroblasts and IEC-6 intestinal cells utilizing wild-type cystic fibrosis transmembrane conductance regulator (CFTR) showed that genistein and tyrphostin 47 stimulated 125I efflux only in CFTR-transfected cells and not in CFTR-negative cells. Thus genistein- and tyrphostin 47-stimulated Cl- secretion involved CFTR. Genistein also acted synergistically with the Ca(2+)- and protein kinase C-dependent acetylcholine analogue, carbachol, to stimulate Cl- secretion in T84 monolayers. However, the Cl- secretory response to saturating concentrations of the adenosine 3',5'-cyclic monophosphate (cAMP) agonist, forskolin, or the guanosine 3',5'-cyclic monophosphate (cGMP) agonist, Escherichia coli heat-stable enterotoxin, was not further enhanced by genistein. Although the mechanism of activation of Cl- secretion is unclear, these data suggest that tyrosine kinase activity limits basal Cl- secretion in T84 cells and that inhibition of T84 cell tyrosine kinase(s) stimulates apical membrane Cl- secretion, most likely through activation of the CFTR-Cl- channel. Moreover, genistein does not itself act through cAMP or cGMP elevation but appears to share a common Cl- secretory pathway with cyclic nucleotide-dependent agonists, whereas it augments the secretory responses to a Ca(2+)- and protein kinase C-dependent agonist.

Down-regulation of protein kinase C and kinase inhibitors dissociate short- and long-term enhancement produced by one-trial conditioning of Hermissenda

1993 ◽  
Vol 69 (2) ◽  
pp. 636-641 ◽  
Author(s):  
T. Crow ◽  
J. Forrester
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Cellular Plasticity ◽  
Short Term ◽  
Down Regulation ◽  
Kinase C ◽  
Short And Long Term

1. The visual system of Hermissenda has been studied extensively as a site of cellular plasticity produced by classical conditioning. Previous research has shown that one-trial conditioning, consisting of light paired with serotonin (5-HT) results in short- and long-term enhancement of light-elicited generator potentials in identified type B-photoreceptors. Recent evidence suggests that 5-HT exerts its effects on the induction of short-term enhancement by activation of protein kinase C (PKC), a Ca(2+)-activated and phospholipid-dependent protein kinase. However, the contribution of protein kinases in general, and specifically PKC in long-term enhancement has not been established. 2. The protein kinase inhibitors H-7 and sphingosine blocked the induction of short-term enhancement when applied before one-trial conditioning. However, the conditions that are sufficient to block the induction of short-term enhancement do not block long-term enhancement. Sphingosine and H-7 do not block the induction and expression of long-term enhancement when applied before one-trial conditioning. 3. Pretreatment before conditioning with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which leads to down-regulation of PKC, also did not block long-term enhancement. Down-regulation by itself did not produce enhancement, although the transient peak of light-elicited generator potentials was reduced by pretreatment with TPA. 4. The results suggest that the induction of short- and long-term enhancement involve parallel processes, and thus the expression of long-term cellular plasticity produced by one-trial conditioning does not depend on the induction or expression of short-term enhancement.

scholarly journals Protein kinase C regulation of p-glycoprotein-mediated xenobiotic secretion in renal proximal tubule

1998 ◽  
Vol 275 (5) ◽  
pp. F785-F795 ◽  
Author(s):  
David S. Miller ◽  
Caroline R. Sussman ◽  
J. Larry Renfro
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Proximal Tubule ◽  
Kinase Inhibitors ◽  
Primary Cultures ◽  
Renal Proximal Tubule ◽  
Protein Kinase Inhibitors ◽  
Drug Accumulation ◽  
Kinase C ◽  
P Glycoprotein

Fluorescence microscopy, fluorescent substrates [daunomycin and a fluorescent cyclosporin A (CSA) derivative] and digital image analysis were used to examine the role of protein kinase C (PKC) in the control of p-glycoprotein in killifish renal proximal tubules. PKC activators, phorbol ester (phorbol 12-myristate 13-acetate, PMA) and dioctylglycerol, reduced luminal drug accumulation, and protein kinase inhibitors, staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), increased luminal accumulation; a PMA analog that does not activate PKC was without effect. PMA effects were blocked by staurosporine. The increase in luminal fluorescence caused by staurosporine was blocked by the p-glycoprotein substrate, CSA, indicating that this component of transport was indeed mediated by p-glycoprotein. Neither PMA, dioctylglycerol, nor protein kinase inhibitors altered cellular drug accumulation. Finally, in primary cultures of flounder proximal tubule cells, PMA decreased transepithelial [3H]daunomycin secretion. This pharmacological approach demonstrates that in teleost renal proximal tubule, p-glycoprotein-mediated xenobiotic secretion is negatively correlated with changes in PKC activity, a finding that conflicts with results from studies using mammalian tumor cells that express p-glycoprotein.

6-Dimethyl amino purine and 2-amino purine inhibit the induction of expresion of milk protein genes by prolactin

1992 ◽  
Vol 12 (3) ◽  
pp. 189-197 ◽  
Author(s):  
Mahasti Bayat-Sarmadi ◽  
Rachel Maliénou-Ngassa ◽  
Claudine Puissant ◽  
Louis-Marie Houdebine
Keyword(s):  
Protein Kinase ◽  
Milk Protein ◽  
Kinase Inhibitors ◽  
Specific Protein ◽  
Protein Kinase Inhibitors ◽  
Experimental Conditions ◽  
Kinase C ◽  
Milk Protein Genes ◽  
Actin Mrna ◽  
First Time

Two protein kinase-inhibitors, 6-dimethyl amino purine and 2-amino purine inhibited induction of β-casein synthesis by prolactin when added to the culture medium of rabbit mammary explant and cells. The accumulation of the mRNA for αs1- and β-caseins and for whey acidic protein did not take place in the presence of the inhibitors whereas β-actin mRNA concentration was not altered. In the same experimental conditions, H7, an inhibitor of protein kinase C and, to a lower extent, of protein kinase A did not prevent prolactin from acting. These data suggest for the first time that specific protein kinases are involved in the transduction of the prolactin signal to milk protein genes.

scholarly journals Odd chromosome movement and inaccurate chromosome distribution in mitosis and meiosis after treatment with protein kinase inhibitors

1993 ◽  
Vol 104 (4) ◽  
pp. 961-973 ◽  
Author(s):  
R.B. Nicklas ◽  
L.E. Krawitz ◽  
S.C. Ward
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Protein Kinase Inhibitors ◽  
Chromosome Movement ◽  
Chromosome Distribution ◽  
Protein Kinase Inhibition ◽  
Spindle Microtubules ◽  
Mitosis And Meiosis ◽  
Normal Mitosis

Errors in chromosome orientation in mitosis and meiosis are inevitable, but normally they are quickly corrected. We find that such errors usually are not corrected in cells treated with protein kinase inhibitors. Highly inaccurate chromosome distribution is the result. When grasshopper spermatocytes were treated with the kinase inhibitor 6-dimethylaminopurine (DMAP), 84% of maloriented chromosomes failed to reorient; in anaphase, both partner chromosomes were distributed to the same daughter cell. These chromosomes were observed for a total of over 60 h, and not a single reorientation was seen. In contrast, in untreated cells, maloriented chromosomes invariably reoriented, and quickly: in 10 min, on average. A second protein kinase inhibitor, genistein, had exactly the same effect as DMAP. DMAP affected PtK1 cells in mitosis as it did spermatocytes in meiosis: improper chromosome orientations persisted, leading to frequent errors in distribution. We micromanipulated chromosomes in spermatocytes treated with DMAP to learn why maloriented chromosomes often fail to reorient. Reorientation requires the loss of improper microtubule attachments and the acquisition of new, properly directed kinetochore microtubules. Micromanipulation experiments disclose that neither the loss of old nor the acquisition of new microtubules is sufficiently affected by DMAP to account for the indefinite persistence of malorientations. Drug treatment causes a novel form of chromosome movement in which one kinetochore moves toward another kinetochore. Two kinetochores in the same chromosome or in different chromosomes can participate, producing varied, dance-like movements executed by one or two chromosomes. These kinetochore-kinetochore interactions evidently are at the expense of kinetochore-spindle interactions. We propose that malorientations persist in treated cells because the kinetochores have numerous, short microtubules with a free end that can be captured by a second kinetochore. Kinetochores capture each other's kinetochore microtubules, leaving too few sites available for the efficient capture of spindle microtubules. Since the efficient capture of spindle microtubules is essential for the correction of errors, failure of capture allows malorientations to persist. Whether the effects of DMAP actually are due to protein kinase inhibition remains to be seen. In any case, DMAP reveals interactions of one kinetochore with another, which, though ordinarily suppressed, have implications for normal mitosis.

Protein kinase C does not participate in carbachol's secretory action in T84 cells

1992 ◽  
Vol 263 (1) ◽  
pp. C140-C146 ◽  
Author(s):  
R. P. Lindeman ◽  
H. S. Chase
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Current Response ◽  
T84 Cells ◽  
Cl Secretion ◽  
Kinase C ◽  
Secretory Action ◽  
Pkc Activation

We investigated the role of protein kinase C (PKC) in mediating carbachol's stimulation of transepithelial Cl- secretion in T84 cells. Direct PKC activation with phorbol 12-myristate 13-acetate (PMA) stimulated transepithelial Cl- transport (measured as the short-circuit current), demonstrating that PKC could interact with the secretory apparatus. Carbachol stimulated PKC activity, suggesting that the enzyme might participate in the hormone's action. Diacylglycerol metabolism inhibitors (DMIs), known to augment hormone-stimulated increases in diacylglycerol levels, potentiated the short-circuit current response to carbachol. The effect of DMIs was not due to amplification of carbachol-induced increases in PKC activity, however; PKC activity during carbachol stimulation was no higher in the presence of DMIs than in their absence. Augmentation of carbachol's action by DMIs appeared to be due to the direct activation of PKC which, like PMA, stimulated the Cl- conductance of the apical membrane (GCl). The effects of DMIs and carbachol on GCl were additive. Carbachol itself stimulated GCl but not by activating PKC; staurosporine did not blunt the effect of carbachol on GCl. Nor did staurosporine reduce the effect of carbachol on transepithelial Cl- secretion. These observations demonstrate that PKC does not participate in the secretory action of carbachol in T84 cells and suggest that direct PKC activation with DMIs and PMA stimulates an apical pool of PKC that is not accessible to carbachol applied to the basolateral membrane.

scholarly journals Protein kinase regulates tumor necrosis factor mRNA stability in virus-stimulated astrocytes.

1990 ◽  
Vol 172 (3) ◽  
pp. 989-992 ◽  
Author(s):  
A P Lieberman ◽  
P M Pitha ◽  
M L Shin
Keyword(s):  
Protein Kinase ◽  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Tumor Necrosis Factor Mrna ◽  
The Stability ◽  
Dependent Pathway ◽  
Cytokine Mrnas ◽  
Necrosis Factor

Infection of astrocytes with Newcastle disease virus stimulated the production of 1,2-diacylglycerol, and resulted in the kinase-dependent expression of mRNAs encoding tumor necrosis factor (TNF), interferon alpha and beta, and interleukin 6. The half-life of TNF mRNA was significantly decreased in the presence of protein kinase inhibitors H-7 and staurosporine, but not in the presence of HA1004. In contrast to the decay of TNF mRNA, the half-lives of other cytokine mRNAs were only minimally affected by the kinase inhibitors. These data indicated that the stability of TNF mRNA was regulated through a novel, kinase-dependent pathway.

scholarly journals Protein kinase C regulation of organic anion transport in renal proximal tubule

1998 ◽  
Vol 274 (1) ◽  
pp. F156-F164 ◽  
Author(s):  
David S. Miller
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Kinase Inhibitors ◽  
Organic Anion ◽  
Anion Transport ◽  
Protein Kinase Inhibitors ◽  
Organic Anion Transport ◽  
Kinase C ◽  
Tissue Fluorescence

Fluorescence microscopy and digital image analysis were used to examine the role of protein kinase C (PKC) in the control of organic anion (fluorescein, FL) transport in killifish renal proximal tubules. Phorbol ester (1–100 nM) reduced cellular and luminal accumulation of FL, and protein kinase inhibitors [staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, 10–1,000 nM] increased cellular and luminal accumulation. Phorbol ester effects were blocked by staurosporine. The increases in tissue fluorescence caused by staurosporine were blocked by p-aminohippurate, indicating that they represent increased FL transport on the organic anion system. Neither phorbol ester nor staurosporine had any effects on the cell-to-lumen transport of a fluorescent organic anion that was generated intracellularly from a nonfluorescent, uncharged precursor. Finally, studies with a fluorescent PKC inhibitor showed that phorbol ester caused PKC translocation from cytoplasm to the plasma membrane. Together, these findings indicate that renal organic anion transport is negatively correlated with PKC activity and that PKC directly or indirectly controls the basolateral step in transport.

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