Activators of protein kinase C inhibit sodium transport in A6 epithelia

To evaluate the role of protein kinase C in sodium transport via amiloride-sensitive sodium channels, we studied the effect of activators of protein kinase C on short-circuit current in epithelia formed by A6 cells in culture. In A6 epithelia, short-circuit current is equivalent to net sodium transport and is blocked by low concentrations of amiloride added to the solution bathing the apical surface. After any of four different activators of protein kinase C [phorbol 12,13-dibutyrate (20 ng/ml), phorbol 12-myristate 13-acetate (20 ng/ml), 1-oleoyl-2-acetylglycerol (50 micrograms/ml), and mezerein (10 ng/ml)] was added to the solution bathing the apical surface, short-circuit current fell, and electrical resistance rose. Nystatin added to the apical solution reversed the inhibition of short-circuit current, indicating that activators of protein kinase C inhibited transport at amiloride-sensitive sodium channels in the apical plasma membrane. Under some conditions, the activators also stimulated amiloride-insensitive short-circuit current. The ion transport represented by the amiloride-insensitive short-circuit current appears to be the result of basal to apical transport of chloride, but this has not been established conclusively.

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Adenosine 3',5'-cyclic monophosphate stimulates chloride secretion in A6 epithelia

AJP Cell Physiology ◽

10.1152/ajpcell.1986.251.5.c810 ◽

1986 ◽

Vol 251 (5) ◽

pp. C810-C814 ◽

Cited By ~ 27

Author(s):

M. Yanase ◽

J. S. Handler

Keyword(s):

Short Circuit ◽

Short Circuit Current ◽

Chloride Secretion ◽

Apical Surface ◽

Cyclic Monophosphate ◽

A6 Epithelia ◽

In Series ◽

Cl Channels ◽

Solution Bathing ◽

Stimulation Of

Basal and aldosterone-stimulated short-circuit current (Isc) of A6 epithelia are known to be equivalent to net apical to basal Na flux and are completely inhibited by 0.05 mM amiloride added to the solution bathing the apical surface of the epithelium. In the absence of amiloride, the Isc stimulated by adenosine 3',5'-cyclic monophosphate (cAMP) is also equivalent to net apical to basal Na flux. However, amiloride does not completely inhibit the cAMP-stimulated Isc. In this study, the cAMP-stimulated, amiloride-insensitive Isc was characterized, using vasopressin or forskolin to raise cell cAMP. After basal Isc is inhibited by amiloride, forskolin stimulates Isc, conductance, and bidirectional 36Cl flux. Stimulation of Isc depends on the presence of both Na and Cl; stimulation of conductance depends on the presence of Cl. 36Cl flux studies showed that the cAMP-stimulated, amiloride-insensitive Isc is equivalent to net Cl flux. It is inhibited by ouabain and by furosemide or bumetanide added to the solution bathing the basal surface of the epithelium. In view of the effect of cAMP in some other epithelia, we suggest that cAMP activates apical membrane Cl channels that are in series with a Na-K-Cl cotransporter in the basolateral plasma membrane.

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

Infection and Immunity ◽

10.1128/iai.72.8.4848-4858.2004 ◽

2004 ◽

Vol 72 (8) ◽

pp. 4848-4858 ◽

Cited By ~ 11

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.

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

AJP Cell Physiology ◽

10.1152/ajpcell.1992.263.1.c140 ◽

1992 ◽

Vol 263 (1) ◽

pp. C140-C146 ◽

Cited By ~ 10

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.

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Regulation of chloride secretion in dog tracheal epithelium by protein kinase C

AJP Cell Physiology ◽

10.1152/ajpcell.1987.253.6.c802 ◽

1987 ◽

Vol 253 (6) ◽

pp. C802-C808 ◽

Cited By ~ 63

Author(s):

R. A. Barthelson ◽

D. B. Jacoby ◽

J. H. Widdicombe

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Short Circuit ◽

Short Circuit Current ◽

Chloride Secretion ◽

Tracheal Epithelium ◽

Base Line ◽

Cl Secretion ◽

Cell Extracts ◽

Kinase C

The effects of stimulating protein kinase C on Cl- secretion across dog tracheal epithelium were investigated. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), and the synthetic diacylglycerol, 1-oleolyl-2-acetylglycerol (OAG), which stimulate protein kinase C (PKC), both stimulated short-circuit current (Isc) with Kd of 10 nM and 1 microM, respectively. In Cl(-)-free solution, the increases in Isc were virtually abolished, suggesting that these compounds stimulate Cl- secretion, a hypothesis confirmed for TPA by measurement of 36Cl- fluxes. The stimulations of Cl- secretion were not sensitive to indomethacin, nor were cAMP levels elevated during stimulation. In addition to its transient stimulatory effect, TPA at high doses caused the eventual lowering of the base-line Isc and a block of subsequent stimulation by cAMP-mediated agonists. This was probably not the result of toxicity or an effect on adenylate cyclase or on cAMP-dependent protein kinase. Cell extracts from both cultured and native dog tracheal epithelial cells showed strong PKC activities. These results suggest that PKC may play a role in regulating Cl- secretion across dog tracheal epithelium.

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Regulation of epithelial transport and barrier function by distinct protein kinase C isoforms

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.2.c649 ◽

2001 ◽

Vol 281 (2) ◽

pp. C649-C661 ◽

Cited By ~ 73

Author(s):

Jaekyung Cecilia Song ◽

Celina M. Hanson ◽

Vance Tsai ◽

Omid C. Farokhzad ◽

Margaret Lotz ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Barrier Function ◽

Epithelial Transport ◽

Short Circuit ◽

Short Circuit Current ◽

Kinase Assay ◽

Bryostatin 1 ◽

Kinase C

The phorbol ester phorbol 12-myristate 13-acetate (PMA) inhibits Cl− secretion (short-circuit current, I sc) and decreases barrier function (transepithelial resistance, TER) in T84 epithelia. To elucidate the role of specific protein kinase C (PKC) isoenzymes in this response, we compared PMA with two non-phorbol activators of PKC (bryostatin-1 and carbachol) and utilized three PKC inhibitors (Gö-6850, Gö-6976, and rottlerin) with different isozyme selectivity profiles. PMA sequentially inhibited cAMP-stimulated I sc and decreased TER, as measured by voltage-current clamp. By subcellular fractionation and Western blot, PMA (100 nM) induced sequential membrane translocation of the novel PKCε followed by the conventional PKCα and activated both isozymes by in vitro kinase assay. PKCδ was activated by PMA but did not translocate. By immunofluorescence, PKCε redistributed to the basolateral domain in response to PMA, whereas PKCα moved apically. Inhibition of I sc by PMA was prevented by the conventional and novel PKC inhibitor Gö-6850 (5 μM) but not the conventional isoform inhibitor Gö-6976 (5 μM) or the PKCδ inhibitor rottlerin (10 μM), implicating PKCε in inhibition of Cl− secretion. In contrast, both Gö-6976 and Gö-6850 prevented the decline of TER, suggesting involvement of PKCα. Bryostatin-1 (100 nM) translocated PKCε and PKCα and inhibited cAMP-elicited I sc. However, unlike PMA, bryostatin-1 downregulated PKCα protein, and the decrease in TER was only transient. Carbachol (100 μM) translocated only PKCε and inhibited I sc with no effect on TER. Gö-6850 but not Gö-6976 or rottlerin blocked bryostatin-1 and carbachol inhibition of I sc. We conclude that basolateral translocation of PKCε inhibits Cl−secretion, while apical translocation of PKCα decreases TER. These data suggest that epithelial transport and barrier function can be modulated by distinct PKC isoforms.

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ANF and bradykinin synergistically inhibit transport in M-1 cortical collecting duct cell line

AJP Renal Physiology ◽

10.1152/ajprenal.1992.263.1.f1 ◽

1992 ◽

Vol 263 (1) ◽

pp. F1-F6 ◽

Cited By ~ 3

Author(s):

B. A. Stoos ◽

O. A. Carretero ◽

J. L. Garvin

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cell Line ◽

Collecting Duct ◽

Short Circuit ◽

Short Circuit Current ◽

Duct Cell ◽

Cortical Collecting Duct ◽

Water Excretion ◽

Kinase C

Previous data suggest that atrial natriuretic factor (ANF) and bradykinin (BK) interact to increase Na+ and water excretion. We propose that this interaction is due to a synergistic action that inhibits Na+ absorption in the distal nephron. We examined the effects of BK and ANF on transport by monolayers of a cortical collecting duct cell line, M-1. BK (10(-8) M) had no effect on short-circuit current (Isc). Similarly, ANF (10(-8) M) did not inhibit Isc. In contrast, Isc decreased by 18% (from 57 +/- 8 to 46 +/- 6 microA/cm2) when BK and ANF were added simultaneously at this concentration (P less than 0.05). Because guanosine 3',5'-cyclic monophosphate (cGMP) and protein kinase C are implicated in the second messenger cascades of ANF and BK, we investigated their potential roles in mediating this interaction. Dibutyryl-cGMP (10(-4) M) inhibited Isc from 33 +/- 4 to 22 +/- 3 microA/cm2 (P less than 0.05) in the presence of BK but not in its absence. Staurosporine and calphostin C, inhibitors of protein kinase C, completely blocked the decrease in Isccaused by simultaneous addition of ANF and BK. cAMP levels in M-1 cells were not affected by either ANF alone or BK alone; however, when cultures were treated with both hormones, cAMP decreased from 856 +/- 56 to 332 +/- 26 fmol/10(6) cells (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

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Serotonin-elicited inhibition of Cl− secretion in the rabbit conjunctival epithelium

AJP Cell Physiology ◽

10.1152/ajpcell.2001.280.3.c581 ◽

2001 ◽

Vol 280 (3) ◽

pp. C581-C592 ◽

Cited By ~ 5

Author(s):

Lawrence J. Alvarez ◽

Helen C. Turner ◽

Aldo C. Zamudio ◽

Oscar A. Candia

Keyword(s):

Signal Transduction ◽

Protein Kinase C ◽

Short Circuit ◽

Short Circuit Current ◽

Transepithelial Resistance ◽

Receptor Subtype ◽

Conjunctival Epithelium ◽

Adrenergic Agonists ◽

Physiological Conditions ◽

Kinase C

The effects of serotonin [5-hydroxytryptamine (5-HT)] on the transepithelial electrical properties of the short-circuited rabbit conjunctiva were examined. With this epithelium, the short-circuit current ( I sc) measures Cl− secretion plus an amiloride-resistant Na+ absorptive process. Apical addition of 5-HT (10 μM) elicited a prompt I screduction from 14.2 ± 1.2 to 10.9 ± 1.2 μA/cm2 and increased transepithelial resistance from 0.89 ± 0.05 to 1.03 ± 0.06 kΩ · cm2(means ± SE, n = 21, P < 0.05). Similar changes were obtained with conjunctivae bathed without Na+ in the apical bath, as well as with conjunctivae preexposed to bumetanide with the Cl−-dependent I sc sustained by the parallel activities of basolateral Na+/H+ and Cl−/HCO[Formula: see text] exchangers. In contrast, the 5-HT-evoked effects were attenuated by the absence of Cl−(Δ I sc = −0.5 ± 0.2, n = 5), suggesting that reduced Cl−conductance(s) is an effect of 5-HT exposure. In amphotericin B-treated conjunctiva and in the presence of a transepithelial K+gradient, 5-HT addition reduced K+ diffusion across the preparation by 13% and increased transepithelial resistance by 4% ( n = 6, P < 0.05), indicating that an inhibition in K+ conductance(s) was also detectable. Significant electrical responses also occurred under physiological conditions when 5-HT was introduced to epithelia pretreated with adrenergic agonists or protein kinase C, phospholipase C, phosphodiesterase, or adenylyl cyclase inhibitors or after perturbation of Ca2+ homeostasis. Briefly, the conjunctiva harbors the only known Cl−-secreting epithelium in which 5-HT evokes Cl− transport inhibition; receptor subtype and signal transduction mechanism were not determined.

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Acceleration of trophoblast differentiation by heparin-binding EGF-like growth factor is dependent on the stage-specific activation of calcium influx by ErbB receptors in developing mouse blastocysts

Development ◽

10.1242/dev.127.1.33 ◽

2000 ◽

Vol 127 (1) ◽

pp. 33-44 ◽

Cited By ~ 2

Author(s):

J. Wang ◽

L. Mayernik ◽

J.F. Schultz ◽

D.R. Armant

Keyword(s):

Protein Kinase C ◽

Growth Factor ◽

Protein Kinase ◽

Intracellular Signaling ◽

Erbb Receptors ◽

Apical Surface ◽

Heparin Binding ◽

Trophoblast Cells ◽

Trophoblast Differentiation ◽

Kinase C

Heparin-binding EGF-like growth factor (HB-EGF) is expressed in the mouse endometrial epithelium during implantation exclusively at sites apposed to embryos and accelerates the development of cultured blastocysts, suggesting that it may regulate peri-implantation development in utero. We have examined the influence of HB-EGF on mouse trophoblast differentiation in vitro and the associated intracellular signaling pathways. HB-EGF both induced intracellular Ca2+ signaling and accelerated trophoblast development to an adhesion-competent stage, but only late on gestation day 4 after ErbB4, a receptor for HB-EGF, translocated from the cytoplasm to the apical surface of trophoblast cells. The acceleration of blastocyst differentiation by HB-EGF was attenuated after inhibition of protein tyrosine kinase activity or removal of surface heparan sulfate, as expected. Chelation of intracellular Ca2+ blocked the ability of HB-EGF to accelerate development, as did inhibitors of protein kinase C or calmodulin. The absence of any effect by a phospholipase C inhibitor and the requirement for extracellular Ca2+ suggested that the accrued free cytoplasmic Ca2+ did not originate from inositol phosphate-sensitive intracellular stores, but through Ca2+ influx. Indeed, N-type Ca2+ channel blockers specifically inhibited the ability of HB-EGF to both induce Ca2+ signaling and accelerate trophoblast development. We conclude that HB-EGF accelerates the differentiation of trophoblast cells to an adhesion-competent stage by inducing Ca2+ influx, which activates calmodulin and protein kinase C. An upstream role for ErbB4 in this pathway is implicated by the timing of its translocation to the trophoblast surface.

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Activation of protein kinase C attenuates prostaglandin E2 responses in a colonic cell line

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1988.255.1.g27 ◽

1988 ◽

Vol 255 (1) ◽

pp. G27-G32 ◽

Cited By ~ 2

Author(s):

G. Warhurst ◽

N. B. Higgs ◽

M. Lees ◽

A. Tonge ◽

L. A. Turnberg

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Adenylate Cyclase ◽

Prostaglandin E2 ◽

Cell Line ◽

Cellular Response ◽

Short Circuit ◽

Regulatory Subunit ◽

Epithelial Cell Line ◽

Kinase C

We examined the possibility that the protein kinase C pathway may interact with the adenosine 3',5'-cyclic monophosphate (cAMP) pathway in intestinal epithelium by studying the influence of phorbol esters on the response to prostaglandin E2 (PGE2) in a colonic epithelial cell line. Pretreatment of T84 cells with 4 beta-phorbol 12,13-dibutyrate (PDB) markedly attenuated the rise in short-circuit current provoked by PGE2, a receptor-mediated cAMP agonist. The EC50 of this effect was 52 nM PDB with a half time of 4-6 min. The responses to nonreceptor-mediated agonists, forskolin and dibutyryl cAMP, were unaffected by phorbol ester. PDB also reduced the ability of PGE2 to stimulate adenylate cyclase activity in these cells. The accumulation of cAMP in response to PGE2 was inhibited by PDB (EC50 38 nM), an effect mimicked by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol. In addition, PGE2 stimulation of adenylate cyclase in membranes from PDB-treated cells was reduced by 30-40%. Inhibition was not mediated via the catalytic or regulatory subunit of the adenylate cyclase, implying an action involving desensitization of PGE2 receptors. These results provide evidence of a complex interrelationship between protein kinase C- and cAMP-mediated pathways that might be important in regulating the cellular response to secretagogues.

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