Stimulation of de novo Synthesis of Prostaglandin H Synthetase (PHS) in Rat Tracheal Epithelial Cells by TPA Involves Activation of Protein Kinase C

Regulatory and stimulatory mechanisms of H2O2 release from guinea pig tracheal epithelial cells were investigated. Cells in primary culture maintained in a previously described air-liquid interface system released H2O2 to the extracellular space only from the apical side of the cells. The rate of release was 0.044 +/- 0.003 nmol.min-1.mg protein-1. H2O2 release could be stimulated significantly during a 30-min incubation period with phorbol myristate acetate (PMA) and platelet-activating factor (PAF). A stimulatory effect of PAF was achieved at concentrations greater than 100 nM and with PMA at concentrations greater than 10 ng (16 nM). When protein kinase C was inactivated with staurosporine, the responses to both PAF and PMA were abolished, whereas the cyclooxygenase inhibitor, indomethacin, did not affect H2O2 generation. When guinea pig tracheal epithelial cells were exposed to sublethal concentrations of extracellular H2O2 (30 microM), H2O2 was detoxified from both apical and basal sides, H2O2 removal being significantly more rapid from the apical side of the cells. These results suggest that tracheal epithelial cells can be stimulated to generate reactive oxygen species into the airway lumen and that this occurs in response to inflammatory mediators that act through protein kinase C. Luminal H2O2 release may have developed as a defense mechanism against microbes, and, similarly, luminal detoxification of H2O2 could represent an important mechanism of modulation of airway inflammation in response to oxidant stress.

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Selected Contribution: PKC activation inhibits Ca2+ signaling in tracheal epithelial cells kept in simulated microgravity

Journal of Applied Physiology ◽

10.1152/jappl.2000.89.2.855 ◽

2000 ◽

Vol 89 (2) ◽

pp. 855-864 ◽

Cited By ~ 4

Author(s):

Jennifer A. Felix ◽

Ellen R. Dirksen ◽

Michael L. Woodruff

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Simulated Microgravity ◽

Tracheal Epithelial Cells ◽

Kinase C ◽

Tracheal Epithelial ◽

Epithelial Sheets ◽

Pkc Activation ◽

Stimulation Of ◽

In Cells

Microgravity has been shown to alter protein kinase C (PKC) activity; therefore, we investigated whether microgravity influences mechanically stimulated Ca2+signaling and ATP-induced Ca2+ oscillations, both of which are modulated by PKC. Rabbit tracheal epithelial outgrowth cultures or suspended epithelial sheets were rotated in bioreactors to simulate microgravity. Mechanical stimulation of a single cell increased the cytosolic Ca2+ concentration in 35–55 cells of both outgrowth cultures and epithelial sheets kept at unit gravity (G) or in simulated microgravity (sμG). In outgrowth cultures, 12- O-tetradecanoylphorbol-13-acetate (TPA; 80 nM), a PKC activator, restricted Ca2+ “waves” to about 10 cells in unit G and to significantly fewer cells in sμG. TPA only slightly reduced the spread of Ca2+ waves in epithelial sheets kept in sμG but did not inhibit Ca2+ waves of sheets kept in unit G. In both cell preparations from both conditions, TPA inhibited ATP-induced Ca2+ oscillations; however, the effect was more pronounced in cells kept in sμG. These results suggest that PKC activation is more robust in cells subjected to sμG.

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Involvement of protein kinase C in mucus secretion by hamster tracheal epithelial cells in culture

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1994.267.5.l526 ◽

1994 ◽

Vol 267 (5) ◽

pp. L526-L530 ◽

Cited By ~ 6

Author(s):

H. Kai ◽

K. Yoshitake ◽

Y. Isohama ◽

I. Hamamura ◽

K. Takahama ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Epithelial Cells ◽

Intracellular Signaling ◽

Collagen Gel ◽

Mucus Secretion ◽

Tracheal Epithelial Cells ◽

Cells In Culture ◽

Kinase C ◽

Tracheal Epithelial

Abnormal regulation of airway mucus secretion may underlie many pulmonary diseases. The exact evidence for the involvement of intracellular signaling mechanisms in mucus secretion has not been fully elucidated to date. The purpose of this study is to clarify the involvement of protein kinase C in the secretion of high-molecular-weight glycoconjugates (HMWG) by hamster tracheal epithelial cells in culture, which elute in the void volume on Sepharose CL-4B column chromatography. HMWG were secreted by the cells cultured on the thick collagen gel, but not on the plastic plate. Two known activators of protein kinase C, 4 beta-phorbol 12 alpha-myristate 13-acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol (OAG), stimulated HMWG secretion. In contrast, 4 alpha-phorbol 12,13-didecanoate, a biologically inactive phorbol, did not influence HMWG secretion. D-Sphingosine, an inhibitor of protein kinase C, suppressed the maximal PMA- (10(-8) M) and OAG- (200 microM) stimulated HMWG secretion. PMA induced protein kinase C translocation from cytosol to membrane. These data indicate that protein kinase C is involved in HMWG secretion in hamster tracheal epithelial cells in culture.

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CFTR gene transfer corrects defective glycoconjugate secretion in human CF epithelial tracheal cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1995.269.6.l855 ◽

1995 ◽

Vol 269 (6) ◽

pp. L855-L864 ◽

Cited By ~ 7

Author(s):

M. Mergey ◽

M. Lemnaouar ◽

D. Veissiere ◽

M. Perricaudet ◽

D. C. Gruenert ◽

...

Keyword(s):

Cystic Fibrosis ◽

Protein Kinase ◽

Gene Transfer ◽

Epithelial Cells ◽

De Novo ◽

Tracheal Epithelial Cells ◽

Normal Human ◽

Cftr Protein ◽

Tracheal Epithelial ◽

And Control

We demonstrate that in immortalized normal human tracheal epithelial cells (NT-1 and 56FHTE8o-) 14C-labeled glycoconjugate secretion may be regulated independently by agonists of the protein kinase A (PKA) and protein kinase C (PKC) signaling pathways. In contrast, in immortalized cystic fibrosis (CF) human tracheal epithelial cells (CFT-1 and CFT-2), regulation is defective for agonists specific for the PKA but not for the PKC pathway. To characterize the involvement of the cystic fibrosis transmembrane conductance regulator (CFTR) in regulated glycoconjugate secretion, we examined the effect of adenovirus-mediated gene transfer of CFTR to CF and control cells. Forty-eight hours after infection, at a multiplicity of infection of 50 plaque-forming units per cell, high levels of CFTR mRNA were detected by reverse transcription-polymerase chain reaction, and de novo synthesis of CFTR protein was demonstrated by immunoblotting. Gene transfer to CF cells restored defective adenosine 3',5'-cyclic monophosphate (cAMP)-dependent secretion not only of chloride but also of glycoconjugates. Taken together, these results argue for a role for CFTR in cAMP-mediated glycoconjugate secretion.

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PDGF and IL-1 induce and activate specific protein kinase C isoforms in mesangial cells

AJP Renal Physiology ◽

10.1152/ajprenal.1996.271.1.f108 ◽

1996 ◽

Vol 271 (1) ◽

pp. F108-F113 ◽

Cited By ~ 6

Author(s):

M. B. Ganz ◽

B. Saksa ◽

R. Saxena ◽

K. Hawkins ◽

J. R. Sedor

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

De Novo ◽

Mesangial Cells ◽

De Novo Synthesis ◽

Kinase C ◽

Pkc Isoforms ◽

Alpha Beta ◽

Pkc Alpha

In vitro and in vivo data suggest a remarkable plasticity in the differentiated phenotype of intrinsic glomerular cells, which after injury express new structures and functions. We have shown that a protein kinase C (PKC) isoform, beta II, is expressed in diseased but not normal glomeruli. Since intrarenal cytokine synthesis has been implicated in the pathogenesis of progressive glomerular injury, we have hypothesized that these mediators induce a change in isoform profile. To test this hypothesis in vitro, we have determined whether platelet-derived growth factor (PDGF) and interleukin-1 (IL-1) alter the expression or activation of PKC isoforms in cultured mesangial cells (MCs). By immunoblot and ribonuclease (RNase) protection assays, both PDGF and IL-1 induce as early as 2 h de novo synthesis of PKC-beta II. Since MCs constitutively express PKC-alpha, -beta I, and -zeta, we also determined whether IL-1 or PDGF alter the activity of these isoforms. PDGF maximally induced translocation of PKC-alpha (10 min), -beta I (90 min), -epsilon (120 min), and -zeta (120 min) from the cytosolic to the membrane fraction. IL-1, in contrast, did not alter the distribution of alpha, beta I, or epsilon at any time measured but did induce PKC-zeta translocation. These data suggest inflammatory mediators regulate PKC isoform activity in diseased glomeruli both by de novo synthesis of unexpressed isoforms and by activation of constitutively expressed PKC isoforms.

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Oxidants affect permeability and repair of the cultured human tracheal epithelium

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1995.268.2.l284 ◽

1995 ◽

Vol 268 (2) ◽

pp. L284-L293 ◽

Cited By ~ 5

Author(s):

M. Yamaya ◽

K. Sekizawa ◽

T. Masuda ◽

M. Morikawa ◽

T. Sawai ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cell Growth ◽

Epithelial Cells ◽

Short Circuit ◽

Electrical Conductance ◽

Epithelial Barrier ◽

Barrier Functions ◽

Kinase C ◽

Tracheal Epithelial

To examine the effects of oxidants on the airway epithelial barrier functions, human tracheal epithelial cells were cultured on porous filter membrane. Glucose oxidase (GO; 10 U/ml), hydrogen peroxide (H2O2; 4 x 10(-3) M), and xanthine (5 x 10(-4) M) plus xanthine oxidase (20 mU/ml) (X-XO) significantly increased electrical conductance across epithelial membrane (G), short-circuit current (Isc) measured with Ussing's chamber methods, and [3H]mannitol flux through the cultured epithelium. Increases in G and Isc induced by oxidants were significantly inhibited by catalase (1,000 U/ml) and the protein kinase C inhibitor staurosporine (10(-7) M), but superoxide dismutase (SOD; 100 U/ml) was without effect. GO, H2O2, and X-XO inhibited the epithelial cell growth, [3H]thymidine incorporation by the cells, and epithelial repair of artificially produced focal epithelial defects (1–2 mm diam) on plastic vessels. Catalase also inhibited effects induced by oxidants on cell growth and proliferation. These results suggest that oxidants reduce tracheal epithelial barrier functions by damaging tight junctions and inhibiting cell proliferation, and these effects of oxidants on epithelial cells may be mediated by H2O2 rather than superoxide anion and by activation of protein kinase C.

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Insulin's effect on protein kinase C and diacylglycerol induced by diabetes and glucose in vascular tissues

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1994.267.3.e369 ◽

1994 ◽

Vol 267 (3) ◽

pp. E369-E379 ◽

Cited By ~ 22

Author(s):

T. Inoguchi ◽

P. Xia ◽

M. Kunisaki ◽

S. Higashi ◽

E. P. Feener ◽

...

Keyword(s):

Smooth Muscle ◽

Protein Kinase C ◽

Protein Kinase ◽

Smooth Muscle Cells ◽

Insulin Treatment ◽

De Novo ◽

De Novo Synthesis ◽

Glucose Levels ◽

Kinase C ◽

Elevated Glucose

We have reported that membranous protein kinase C (PKC) activities and total diacylglycerol (DAG) levels are increased in the heart and aorta of diabetic rats, which cannot be easily reversed by euglycemic control. However, insulin treatment, which achieved euglycemia, can prevent the increase in PKC activities and DAG levels. Chronic exposure to elevated glucose levels (5.5 vs. 22 mM) increased DAG levels in cultured bovine and rat aortic endothelial cells and smooth muscle cells by 31, 140, and 143%, respectively, only after 3 days of incubation. Glyceraldehyde, which can stimulate the de novo synthesis of DAG, significantly increased DAG levels by 7.1 +/- 0.6-fold after only 16 h of incubation. Elevated glucose levels did not affect labeled DAG when all of the vascular cells were incubated with [3H]arachidonate, [3H]glycerol, or [3H]phosphatidylcholine, whereas [3H]palmitate- and [3H]oleic acid-labeled DAG levels were significantly increased, indicating that the glucose-stimulated increase in DAG is derived partially from the de novo synthesis pathway. Immunoblotting studies showed increases only in PKC isoform beta II but not alpha in aortic smooth muscle cells. The phosphorylation level of MARCKS protein, an intracellular substrate of PKC, was also increased, consistent with the PKC activity increase. These findings showed that diabetic and hyperglycemia-induced increases in PKC activity and DAG levels in the heart and aorta are preventable by insulin treatment.

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