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MUCOSAL BIOLOGY

Chronic PKC- activation in HT-29 Cl.19a colonocytes prevents cAMP-mediated ion secretion by inhibiting apical membrane current generation

¹Department of Integrative Biology and Department of Pharmacology and Physiology, and ²Division of Gastroenterology, Hepatology, and Nutrition, Department of Internal Medicine, University of Texas Health Science Center Medical School, Houston, Texas

Submitted 28 July 2005 ; accepted in final form 16 March 2006

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
We investigated the effects of PKC-stimulating 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) and phorbol 12-myristate 13-acetate (PMA) phorbol esters on cAMP-dependent, forskolin (FSK)-stimulated, short-circuit Cl^– current (I_SC-cAMP) generation by colonocyte monolayers. These agonists elicited different actions depending on their dose and incubation time; PMA effects at the onset (<5 min) were independent of cAMP agonist and were characterized by transient anion-dependent transcellular and apical membrane I_SCgeneration. DOPPA failed to elicit similar responses. Whereas chronic (24 h) exposure to both agents inhibited FSK-stimulated transcellular and apical membrane I_SC-cAMP, the effects of DOPPA were more complex: this conventional PKC--specific agonist also stimulated Ba²⁺-sensitive basolateral membrane-dependent facilitation of transcellular I_SC-cAMP. PMA did not elicit a similar phenomenon. Prolonged exposure to high-dose PMA but not DOPPA led to apical membrane I_SC-cAMP recovery. Changes in PKC -, ₁-, -, and -isoform membrane partitioning and expression correlated with these findings. PMA-induced transcellular I_SC correlated with PKC- membrane association, whereas low doses of both agents inhibited transcellular and apical membrane I_SC-cAMP, increased PKC-₁, decreased PKC-₂membrane association, and caused reciprocal changes in isoform mass. During the apical membrane I_SC-cAMP recovery after prolonged high-dose PMA exposure, an almost-complete depletion of cellular PKC-₁ and a significant reduction in PKC- mass occurred. Thus activated PKC-₁ and/or PKC- prevented, whereas activated PKC- facilitated, apical membrane I_SC-cAMP. PKC--dependent augmentation of transcellular I_SC-cAMP at the level of the basolateral membrane demonstrated that transport events with geographically distinct subcellular membranes can be independently regulated by the PKC -isoform.

protein kinase C; short-circuit current; cystic fibrosis transmembrance conductance regulator; phorbol ester

The PKC family of serine/threonine kinases is involved in the regulation of many aspects of cell growth, differentiation, and function (8). The role of generalized PKC activation in epithelial fluid transport as well as cation (K⁺ channel)- and anion (CFTR activation and priming)-dependent Cl^– secretion has been the subject of a number of excellent studies at the protein/molecular levels (23, 60). In the context of epithelial chloride secretion, 4-phorbol ester stimulation of PKC isoforms has been implicated in the regulation of both apical and basolateral plasma membrane ion transport. The best-studied basolateral phenomena relate to the ability of PMA to inhibit basolateral K⁺ efflux and thereby reduce the cell's ability to maintain a sustained electrochemical gradient for Cl^– exit through the concerted efforts of basolateral Na⁺-K⁺-ATPase, the Na⁺-2Cl^–-K⁺ cotransporter, and open apical membrane CFTR Cl^– channels (59, 66). Supporting a predominantly inhibitory role for PKC signaling in basolateral membrane transport, other reports have implicated phorbol ester-stimulated PKC isoforms as regulators of these same basolateral transport components at the levels of either protein function and/or plasma membrane expression (10, 15, 19, 40). At the apical plasma membrane, phorbol ester-dependent PKC activation in polarized epithelial cells has been shown to potentiate cAMP-responsive Cl^– current generation via phosphorylation-dependent priming of PKA-mediated channel gating (27, 67), and a subset of CFTR molecules (those isolated from Necturus and Xenopus) has been shown to contain a unique CFTR regulatory domain phosphorylation site, absent in human CFTR, that can directly stimulate channel function (11). However, PKC does not appear to directly stimulate CFTR anion channel function, and chronic exposure to nonisoform-specific PMA has been shown to downregulate cAMP-dependent Cl^– transport in many epithelial cell lines through decreased CFTR mRNA and protein levels (6, 58, 64). A recent coimmunoprecipitation study (35) has shown that PKC- and CFTR are colocalized in a signaling complex through association with the scaffold protein RACK1, suggesting that at least one phorbol ester-sensitive, Ca²⁺-independent, DAG-activated (i.e., novel) PKC isoform participates in the local regulation of CFTR at the apical membrane.

Although individual phorbol ester-sensitive PKC isoforms (8) are also implicated in a variety of basolateral ion transport phenomena (21), electrophysiological transport studies linking PKC isoform participation in the apical plasma membrane to cAMP-dependent secretory current generation have not been performed. We addressed this question in a companion study (9a).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Materials. FBS, DMEM, penicillin-streptomycin, and trypsin-EDTA solutions were procured from GIBCO-BRL (Grand Island, NY). Isoform-specific anti-PKC-, -₁, -₂, and - polyclonal antibodies were purchased from Santa Cruz Biotechnology and Upstate Biotechnology. The conventional PKC-specific activator 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) and inhibitor (Gö6976) along with PMA were obtained from Alexis (San Diego, CA). All other chemicals were purchased from Sigma (St. Louis, MO) and were reagent grade unless otherwise specified.

Cell culture. The human colon carcinoma cell line HT-29 Cl.19A was the generous gift of C. L. Laboisse (3) (Institut National de la Santé et de la Recherche Médicale, Paris, France) and was cultured in DMEM supplemented with 4.5 g/l glucose, 10% FBS, 100 U/ml penicillin, and 100 µg streptomycin. Cells were grown at 37°C in a humidified atmosphere of 5% CO₂-95% air.

Treatment of cells with PKC modulators. Cells were grown to near confluence on 12-mm sterile filters (Millicell-HA, Millipore; Bedford, MA) in DMEM with 10% FBS. The medium was replaced with DMEM without serum for 2 h before each experiment and then replaced with 1 ml of serum-free medium containing (dissolved in 50 µl DMSO) PMA (0.01–1µM), DOPPA (1–500 nM), Gö6976 (1–5 µM), or carrier alone for 10 min to 48 h. To ensure that significant cellular metabolism of DOPPA to the nonspecific activator 12-deoxyphorbol-13-phenylacetate (DOPP) did not occur, we exchanged DOPPA incubation media every 4–6 h (29).

Short-circuit current measurement. The 12-mm filter units containing the HT-29 Cl.19A monolayers were placed into custom-designed Ussing chambers (Iowa City, IA). All experiments were carried out at 37°C. Standard Krebs bicarbonate saline solution containing (in mM) 125 NaCl, 4.7 KCl, 1.13 MgCl₂, 25 NaHCO₃, 1.15 Na₂HPO₄, and 10 glucose (pH 7.2) was added, and the monolayers were gassed with 95% O₂-5% CO₂ by airlift circulators. The transepithelial potential difference was clamped to zero, and the short-circuit current (I_SC) was continuously displayed on a pen recorder. Transepithelial conductance was calculated from the magnitude of the current deflections in response to a bipolar voltage pulse imposed every 60 s with a duration of 0.5 s. For curve fitting results, we employed an iterative regression procedure using the Marquartdt-Levenberg algorithm (Sigmaplot version 4.0, SPSS; Chicago, Il). Dose or time of agonist preincubation versus monolayer I_SC response curves were fitted with a three-parameter single-exponential decay [y = ae^{(b/x + c)}, curve fit 1] function. Agonist concentration versus percent inhibition curves were fitted with a three-parameter logistic sigmoidal [y = a/1 + (x/x_o)^b, curve fit 2] function. In both instances, x and y are horizontal and vertical ordinates, respectively, x_o is the maximal value of x with no inhibition, and a, b, and c are the parameters of amplitude, rate, and inflection, respectively. Identical curve fits were used for related procedures.

PKC abundance measurement by Western blot analysis. Crude homogenates were prepared by homogenization in detergent buffer containing (in mM) 50 Tris·HCl, 250 sucrose, 2 EDTA, 1 EGTA (pH 7.5), and 10 2-mercaptoethanol with 0.5% Triton X-100 plus protease inhibitor cocktail (PIC) followed by a low-speed spin (15,000 g for 15 min). The clear supernatant was saved as the Triton X-100-solubilized total cell extract. For total cellular extracts including the Triton X-100-insoluble fraction, crude homogenates were extracted with the same buffer containing 0.5% SDS. Subcellular membrane and cytosolic fractions were extracted from similar cells by the omission of detergent and by centrifugation at 100,000 g. The resulting pellet was then detergent extracted in buffer containing (in mM) 10 Tris·HCl, 140 NaCl, 25 KCl, 5 MgCl₂, 2 EDTA, and 2 EGTA, with 1% Triton X-100 plus PIC (pH 7.5), and the solubilized membrane fraction was collected by centrifugation. The protein concentration was measured in all fractions before electrophoresis was performed, and mouse brain homogenates were used as positive controls. Individual fractions (30 µg protein/lane) were subjected to 10% SDS-PAGE and electrotransferred to nitrocellulose membranes. The transfer efficiency was checked by back staining gels with Coomassie blue and/or by reversible staining of the nitrocellulose membrane with Ponceau S solution; no variability was noted. Destained membranes were blocked with 5% nonfat dried milk in Tris-buffered saline [TBS; 20 mM Tris·HCl and 137 mM NaCl (pH 7.5)] for 1 h at room temperature and then overnight at 4°C. Immunoantigens were detected by incubating the membranes for 2 h with PKC isoform/CFTR antibodies (0.5–1.0 µg/ml in TBS containing 0.1% Tween 20, Sigma Chemical). After being washed, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or rat anti-mouse IgG and developed using the ECL detection system (Amersham; Arlington Heights, IL) according to the manufacturer's instructions.

Data analysis. All summary results are presented as arithmetic means ± SE. Differences between control and treatment data were analyzed using Student's t-test (Excel, Microsoft; Redmond, WA). The probability of making a type I error <0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
To examine the effect of isoform-specific PKC- activation on CFTR-dependent cellular anion transport, we measured Cl^– secretion in response to elevated cellular cAMP levels in HT-29 Cl.19A monolayers exposed to weak (DOPPA) or strong (PMA) exogenous PKC agonists.

DOPPA inhibited forskolin-stimulated I_SC across intact monolayers in a concentration-dependent manner. In the HT-29 Cl.19A subclone, cAMP-responsive transepithelial I_SC under standard high-extracellular Na⁺ conditions reflects electrogenic Cl^– secretion into the mucosal bath, as demonstrated by Ussing-chamber and patch-clamp measurements (4, 5, 42, 66). DOPPA, a specific PKC- agonist (55) with 40-fold less potency than the unselective parental compound PMA (38), was employed to delineate the role of low level cellular PKC activation on cellular cAMP-regulated Cl^– secretion. Monolayers grown to confluence on filters for 14–20 days were treated for 24 h with 1–500 nM DOPPA, and changes in transcellular cAMP-dependent I_SC (I_SC-cAMP) were monitored in response to the cAMP-generating agonist forskolin (FSK; 10 µM) alone or in combination with the Ca²⁺-mobilizing agonist carbachol (CCh; 50 µM).

In intact monolayers, DOPPA inhibited FSK-stimulated transcellular I_SC-cAMP in a concentration-dependent manner [Fig. 1, A and B (see MATERIALS AND METHODS for curve fit 1)]. These effects were independent of monolayer resistance, which in fact rose slightly between 10 and 100 nM DOPPA before decreasing modestly at DOPPA concentrations of <200 nM (Table 1). The combined CCh and FSK response was biphasic with the intact basolateral membrane providing a Ba²⁺-sensitive component.

View larger version (17K): [in this window] [in a new window]   Fig. 1. A: composite showing monolayer short-circuit secretory current (ISC) measurements after 24-h preincubation with 0–500 nM of the weak phorbol ester deoxyphorbol 13-phenylactetate 20-actetate (DOPPA). ISC responses were elicited by a bilateral bath addition of 10 µM of the cAMP-mobilizing agonist forskolin (FSK; i), followed by the serosal bath addition of 10 µM of the Ca2+-mobilizing agonist carbachol (CCh; ii) and 400 µM bumetanide (Bumet; iii). B: dose-vs.-response curves for Bumet-sensitive ISC constructed from the FSK response alone () and that of FSK combined with CCh () (n = 19–35)

View larger version (17K): [in this window] [in a new window]   Fig. 2. A: enlargement of the agonist-stimulated ISC response after a 24-h preincubation with carrier alone [(–) trace] or with carrier plus 50 nM DOPPA [(+) trace] exposed to 10 µM FSK (i) and subsequently to basolateral bath CCh (ii) and 400 µM Bumet (iii). Irrespective of whether DOPPA preincubation occurred, the net ISC produced by the combined agonists (1 + 2) was identical. In contrast, cAMP-dependent ISC (ISC-cAMP) was clearly attenuated by DOPPA preincubation (1), indicating that larger CCh effects (2) made up this difference during FSK + CCh stimulation. B: when the responses were replotted as the %inhibition of the agonist response in control monolayers, the concentrations of DOPPA required to cause 50% inhibition (ID50) were 69 ± 3 nM for FSK alone () and 98 ± 2 nM for FSK + CCh combined (). At DOPPA concentrations of <30 nM, the mean combined agonist response was larger than that recorded for untreated monolayers but was not significant (n = 19–35).

View larger version (13K): [in this window] [in a new window]   Fig. 3. Effects of DOPPA preincubation on FSK-stimulated apical plasma membrane ISC. A: monolayers preexposed for 24 h with either carrier alone [(–) trace] or carrier plus 100 nM DOPPA [(+) trace] were initially maintained with140 mM NaCl in both serosal and mucosal baths. After an equilibration period, a high (140 mM NaCl) mucosal-to-low (7 mM NaCl) serosal bath anion gradient was imposed (i), and the ISC responses to bilateral bath 10 µM FSK addition were recorded (ii). Nystatin (50 mg/ml) was then added to the serosal bath to permeabilize the basolateral plasma membrane to univalent ions (iii), and apical plasma membrane-generated ISC was recorded. The subsequent addition of 10 µM CCh to the serosal bath (iv) produced small, highly variable responses. Reducing the mucosal [NaCl] to 7 mM, thereby equilibrating bath ion gradients (v), caused the ISC to return to close to zero-urrent values. B: dose-vs.-response curves for FSK-stimulated ISC-cAMP from similar monolayers preincubated for 24 h with 0–500 nM DOPPA ().

When resuslts were replotted as percent inhibition, an ID₅₀ of 65 nM [Fig. 4 (see MATERIALS AND METHODS for curve fit 2)], close to that recorded in intact monolayers, was estimated (Fig. 2B). Apical membrane I_SC-cAMP measured across 24-h DOPPA (>30 nM)-treated apical plasma membranes was much smaller than that recorded in the absence of cellular DOPPA preincubation, and both DOPPA-treated and control monolayers exhibited few combined (FSK + CCh) agonist responses (see Fig. 10). Thus the stimulatory effects of <30 nM DOPPA preincubation on CCh-facilitated transcellular I_SC-cAMP (Fig. 1) were dependent on the presence of an intact basolateral plasma membrane, and, because of the Ba²⁺ sensitivity (16), were predicted to be K⁺ channel dependent.

View larger version (11K): [in this window] [in a new window]   Fig. 4. Inhibition curve detailed in Fig. 3 graphed as percentages of the untreated ISC response, showing that a progressive inhibition occurred at DOPPA concentrations of >30 nM (n = 9–12). DOPPA at 10 nM produced a weak but significant (P < 0.05) percent increase in apical plasma membrane-generated ISC, whereas the ID50 under these conditions was 66 ± 5 nM.

View larger version (26K): [in this window] [in a new window]   Fig. 5. Postactivation status of cellular phorbol ester-sensitive PKC isoform membrane-to-cytoplasmic partitioning in monolayers preincubated for 24 h with 50 nM DOPPA. A: Western blots of PKC isoform expression in detergent-extracted membrane (m) and cytosolic (c) subcellular fractions isolated from control (blot i) or DOPPA-treated (blot ii) monolayers. Little change in the densitometric appearance of membrane PKC-, -1, -, or - or cytosolic PKC-, -1, -, or - was detected, although the cytoplasmic PKC-2 band increased. B: corresponding bar graph of membrane-to-cytosolic ratios (densitometric values relative to -actin) recording an 3-fold decrease in the ratio of PKC-2 membrane to cytosol association. Changes were significant. Open bars, control monolayers; hatched bars, DOPPA-pretreated monolayers (n = 3, P < 0.01).

View larger version (18K): [in this window] [in a new window]   Fig. 6. Acute effects of PMA on ISC generated by basolateral plasma membrane-permeabilized monolayers. A–D: initially, both mucosal and serosal baths contained 140 mM NaCl. In A–D, serosal bath NaCl was reduced to 7 mM (i), followed by a serosal bath addition of Nystatin (50 mg/ml; ii) and a mucosal bath addition of 1 mM diphenylamine-2-carboxylic acid (DPC; iii). In A and B, the mucosal bath NaCl was subsequently lowered to 7 mM (iv). In A, acute 50 nM PMA (P1) resulted in the generation of ISC with a slow onset compared with the bilateral 10 µM FSK (F) addition (B). Mean changes in ISC are shown in the right bar graph (bars 1 and 2 for A and B, respectively). C: the ISC-stimulating effects of PMA and FSK were additive, and 50 nM PMA (P1) followed by a 10 µM FSK (F) bath addition produced a total ISC response similar in magnitude to that reported for FSK alone (right bar graph; crosshatched area of bar 3 = 50 nM PMA and additional open area of bar 3 = PMA + FSK). D: increasing the concentration of PMA to 500 nM (P2) did not affect the overall magnitude of the combined agonist response. Average values were similar in the corresponding bar graph (right, fine crosshatched area of bar 4 = 500 nM PMA; additional open area of bar 4 = PMA + FSK). Thus PMA and FSK effects on apical membrane ISC were not synergistic and were likely to be mediated through the same DPC-sensitive anion channel target, CFTR (n = 6 observations, bar graph values are means ± SD).

View larger version (14K): [in this window] [in a new window]   Fig. 7. Time course comparing the chronic effects of either the weak PKC agonist DOPPA (50 nM; ) or the strong PKC agonist PMA (50 nM; ) on FSK-stimulated ISC-cAMP generated by intact monolayers. A <2-h preincubation with carrier plus PMA reduced ISC to 15% of the untreated response, whereas in monolayers preincubated for 24 h with carrier plus DOPPA, 51% of the untreated ISC response remained (n = 11–23 observations for each time point for DOPPA and 4–6 observations of each time point for PMA).

View larger version (11K): [in this window] [in a new window]   Fig. 8. FSK-generated apical plasma membrane ISC recorded after chronic PMA preincubation. A: monolayers were preincubated for 24 h with either carrier alone (a) or carrier plus PMA (50 nM; b). Initially, both mucosal and serosal baths contained 140 mM NaCl. i, mucosal bath NaCl reduced to 7 mM; ii, serosal bath addition of Nystatin (50 mg/ml); iii, bilateral addition of 10 µM FSK; iv, mucosal bath addition of 1 mM DPC. Chronic PMA stimulation led to a nearly complete inhibition of apical plasma membrane ISC-cAMP. B: bar graph of the DPC-sensitive apical membrane ISC-cAMP inhibitory response after 24-h preincubation with 50 nM PMA. Irrespective of the direction to the prevailing Cl– gradient [140:7 mucosal to serosal (bar i); 140:7 mM serosal to mucosal (bar ii)], near-complete inhibition occurred.

View larger version (25K): [in this window] [in a new window]   Fig. 9. Postactivation status of cellular phorbol ester-sensitive PKC isoform membrane to cytoplasmic partitioning in monolayers preincubated for 24 h with 50 nM PMA. A: Western blots of PKC isoform expression in detergent-extracted membrane and cytosolic subcellular fractions isolated from control (blot i) or PMA-treated (blot ii) monolayers, demonstrating increases in the densitometric appearance of membrane-associated PKC -, 1-, and -isoforms bands. Conversely, both membrane and cytosolic PKC-2 bands appeared to increase. B: corresponding bar graph of membrane-to-cytosolic ratios (densitometric values relative to -actin). All changes were clearly significant. Open bars, control monolayers; hatched bars, PMA-pretreated monolayers (n = 3, P < 0.01).

View larger version (10K): [in this window] [in a new window]   Fig. 10. Effects of chronic (48 h) high-dose PMA (500 nM) preincubation on FSK-stimulated ISC generated by basolateral membrane ion-permeabilized monolayers. A: monolayers were preincubated with carrier alone (a) or carrier plus 500 nM PMA (b). Initially, both mucosal and serosal baths contained 140 mM NaCl. I, change to 7 mM serosal bath NaCl; ii, bilateral addition of 10 µM FSK; iii, serosal bath addition of Nystatin (50 mg/ml); iv, mucosal bath addition of 1 mM DPC. Chronic, high-dose PMA stimulation led to a partial recovery of ISC previously inhibited by lower doses of this agonist (Fig. 8). *Effects of 10 µM CCh on FSK-stimulated apical membrane ISC under these conditions; 26% of all (control, DOPPA, and PMA) monolayers exhibited a small additional current component, and the rest either exhibited no CCh effect (59%) or a slight inhibition (15%) (n = 50). B: DPC-sensitive ISC responses replotted as a bar graph of the %inhibition of the untreated monolayer FSK ISC-cAMP response after 48-h monolayer preincubation with 500 nM PMA (P2). Irrespective of the direction to the prevailing ionic gradient [140:7 mM serosal to mucosal (bar i); 140:7 mM mucosal to serosal (bar ii)], chronic PMA stimulation at the higher dose (P2) led to a 61 ± 3% and 56 ± 5% inhibition, respectively (n = 4, values are means ± SD).

DOPPA affected the membrane translocation status and expression levels of individual PKC- isoforms.

Figure 5 shows a representative Western blot (A) and corresponding mean densitometric ratio (B) for the membrane-to-cytosolic partitioning ratio [R_{(membrane/cytosol)}] for all phorbol ester-sensitive PKC isoforms detected in control and DOPPA-treated monolayers. Incubation for 24 h with 50 nM DOPPA failed to induce membrane translocation of Ca²⁺- and DAG-sensitive PKC-/₁/ or Ca²⁺-insensitive but DAG-sensitive PKC-. However, a significant reduction in the membrane translocation ratio of PKC-₂ was recorded, an effect caused by increased cytosolic PKC-₂ levels without any corresponding change in the size of the membrane-associated pool. When total cellular levels for each immunodetected isoform were determined, DOPPA preincubation selectively and reciprocally affected cellular PKC -isoform expression; a modest decrease (1.4 ± 0.1-fold) in PKC-₁ and increase (1.6 ± 0.1-fold) in PKC-₂ was recorded [see companion study (9a)]. The relative expression levels of the other isoforms did not change relative to -actin (n = 3 separate experimental observations). The percent membrane association [membrane/(cytostol + membrane) x 100] for the PKC-, -₁, -, and - isoforms remained unchanged at 50%, 50%, 28%, and 77%, respectively. The percent membrane association for PKC-₂ decreased from 90% to 70%, demonstrating that a significant fraction remained active when the cytoplasmic PKC-₂ mass increased. Western blots were stripped and reprobed for cytoplasmic -actin, and back staining of SDS gels to check transfer efficiency was routinely performed (see MATERIALS AND METHODS).

The specificity of these effects were consistent with the findings of Khare and colleagues (28), who showed that monolayers exposed acutely (5–20 min) to 200 nM DOPPA exhibited selective PKC- activation with no change in the activation status of other PKC isoforms. Thus, during the 24-h low-dose DOPPA preincubation, the increased cytoplasmic PKC-₂ partitioning predicted an increase in the postactivation status of the PKC -isoforms. We therefore hypothesized that inhibition of cAMP-dependent apical plasma membrane Cl^– current under these conditions was mediated by PKC-₂ or PKC-₁ homolog-specific changes in subcellular signaling function.

PMA, but not DOPPA, acutely stimulated apical plasma membrane-generated I_SC through PKC- or PKC- interactions with CFTR Cl^– channels. Intact or basolateral membrane-permeabilized HT-29 Cl.19A monolayers exposed acutely to either 50 nM DOPPA or 1 µM Gö6976 (a selective PKC-/ inhibitor) did not exhibit any change in I_SC (Table 3). In contrast, 50–500 nM PMA generated significant I_SC in both intact and basolateral membrane-permeabilized monolayers. An example of PMA-induced apical membrane I_SC-cAMP is shown in Fig. 6.

Chronic monolayer exposure to PMA identified both PKC- and PKC- as candidates for PKC-dependent inhibition of apical plasma membrane-generated I_SC-cAMP.

Within 2 h, 50 nM PMA reduced the transcellular I_SC-cAMP by 86% and at 24 h by 89% [n = 24 (see MATERIALS AND METHODS for curve fit 1)] compared with DOPPA, where currents changed by 18% and 41%, respectively. PMA, unlike DOPPA, caused a 25 ± 5% and 41 ± 3% decrease in basal monolayer resistance at these times (n = 12). When apical membrane I_SC-cAMP was measured in 24-h PMA-pretreated monolayers exposed to serosal bath nystatin, a similar steep time-dependent reduction in FSK-stimulated current was recorded (Fig. 8).

Monolayers were bathed in high-Na⁺, bicarbonate-free HEPES-buffered saline in the presence of a 140:7 mM serosal-to-mucosal Cl^– gradient. Nystatin permeabilization of the basolateral plasma membrane under these conditions did not in itself elicit apical membrane I_SC-cAMP. However, when this was followed in control monolayers by a bilateral bath FSK (10 µM) addition, a large apical membrane I_SC-cAMP, reflecting current flow along a mucosal-to-serosal electrochemical gradient, was recorded (Fig. 8A). A 24-h PMA preincubation led to a nearly complete loss of this current (Fig. 8A), which was not dependent on the direction of the imposed anion gradient: similar inhibition (>97%) occurred when I_SC flowed from the mucosal-to-serosal or serosal-to-mucosal baths (Fig. 8B). A previous study (65) in HT-29 Cl.19A monolayers demonstrated that chronic PMA exposure does not effect the ability of FSK to raise cAMP levels. Thus the lack of any additive effects between PMA and FSK on apical membrane I_SC-cAMP generation and a shared DPC sensitivity indicated that the same PKC-sensitive cAMP-responsive anion channel population was being chronically downregulated upon prolonged PMA exposure.

Time-dependent changes in PMA-sensitive PKC membrane partitioning underlie the chronic inhibitory effects of PMA on apical membrane I_SC-cAMP. The acute effects of high-dose PMA on monolayer transcellular I_SC have previously been shown to be mediated by PKC- (66). In this series of experiments, we determined the status of the PMA-sensitive pool after a 24-h low-dose (50 nM) PMA exposure when >97% of apical membrane I_SC-cAMP was inhibited (see Fig. 8). Changes in immunospecific PMA-sensitive PKC expression in both the cytosolic and membrane fractions were determined by Western blot analysis (Fig. 9).

A modest but not significant increase in the PKC- R_{(membrane/cytosol)}, equal to 1.2 ± 0.1 (n = 4, untreated monolayers used for comparison), was recorded. Both PKC-₁ and PKC- exhibited elevated R_{(membrane/cytosol)} values (2.4 ± 0.1 and 1.5 ± 0.3, respectively) and were significantly different (P < 0.001; Fig. 9, A and B). Thus both isoform pools were present in a chronically activated state. In contrast, PKC-₂ membrane association was reduced by 2.7 ± 0.2-fold. This statistically significant (P < 0.001, n = 4) increase in the size of the inactive cytoplasmic PKC-₂ pool mimicked that obtained after a 24-h DOPPA incubation (1.4-fold reduction, P < 0.05, n = 4; Fig. 5B).

When the total cellular levels of each isoform were immunoassayed (data not shown), more pronounced and significant decreases in PKC-₁ (1.4 ± 0.2-fold) and increases in PKC-₂ (1.7 ± 0.2-fold) expression were recorded. Changes in PKC- (1.2 ± 0.1-fold) expression or the relative cellular levels of the other isoforms (PKC- and PKC-) were not significant (P < 0.001, n = 4). The percent membrane association [membrane/(cytosol + membrane) x 100] for the PKC -isoform remained unchanged at 22%, and a modest rise in PKC- membrane association was recorded (from 50% to 59%). Values for the other isoforms either rose from 77% to 90% (PKC-) or from 50% to 71% (PKc-₁) or decreased from 90% to 79% (PKC-₂).

Thus the larger apical membrane I_SC-cAMP inhibitions recorded with the stronger agonist PMA were matched with greater changes in PKC- cell signaling. However, in contrast to DOPPA and reflecting the less-specific effects of this agonist, PKC- activation was also implicated in the chronic inhibitory phenomena.

The downregulation of PKC-₁ and PKC- by PMA was associated with recovery of apical plasma membrane I_SC-cAMP. To address in chronic PMA experiments whether PKC- along with PKC-₁/PKC-₂ affected apical plasma membrane CFTR function, we attempted to selectively downregulate individual isoforms by increasing the dose and/or time of monolayer PMA exposure. This experiment was performed with the assumption that if an isoform was important, then, after its downregulation, a matching increase in cAMP-mediated apical plasma membrane CFTR anion channel function should occur. Given our DOPPA findings and reports indicating that epithelial cell PKC is more resistant to downregulation than nonepithelial cell PKC mass (33), we did not consider using a similar approach with the PKC--specific but weaker agonist DOPPA. Increasing the preexposure time to 50 nM PMA from 24 to 48 h had little effect on apical membrane I_SC-cAMP (data not shown). Similarly, increasing the monolayer PMA dose to 500 nM for 24 h only marginally affected apical membrane I_SC-cAMP (inhibition was 90% of control monolayer response, data not shown). However, increasing both the PMA dose to 500 nM and prolonging the exposure time to 48 h clearly led to less-inhibitory effects on apical membrane I_SC-cAMP in both membrane configurations [shown are results from basolateral plasma membrane-permeabilized monolayers (Fig. 10)].

In Fig. 10, a and b, the monolayers were maintained under symmetrical high (140 mM) NaCl gradients. A reversed 140:7 mM mucosal-to-serosal Cl^– gradient was then established, and apical membrane I_SC-cAMP was recorded. In control monolayers, the addition of Nystatin to the serosal bath reversed the direction of the apical membrane I_SC-cAMP (Fig. 10A). However, unlike the findings shown in Fig. 8, PMA-preincubated monolayers exposed for an extended period at the higher dose exhibited a significant transcellular I_SC-cAMP (Fig. 10b). When changes were expressed as percent inhibition (Fig. 10B), values were 41% of the control untreated monolayer response compared with the <97% inhibition recorded in earlier protocols. No directional effects of anion gradient were present, and inhibitions were 63 ± 5% and 56 ± 2% for 140:7 mM serosal-to-mucosa and 140:7 mM mucosal-to-serosal Cl^– gradients, respectively (n = 8). Thus, compared with 24-h exposure to 50 nM PMA, an 40% apical membrane I_SC-cAMP recovery occurred after 48-h exposure to 500 nM PMA.

A similar partial recovery, but at the level of transcellular I_SC-cAMP, has been reported previously in intact HT-29 Cl.19A monolayers preincubated with high doses of PMA. This was attributed to regained basolateral K⁺ conduction upon cellular PKC- depletion (66). These results differ from our own findings in that we were unable to produce significant changes in the cellular mass of PKC- at 24-h post-PMA exposure. The previous investigations did not determine direct PKC effects at the apical plasma membrane. Following their example, we assayed both total cellular (Triton X-100/SDS solubilized) PKC expression levels in the Triton X-100-solubilized cellular homogenate. The results are shown in Fig. 11.

View larger version (91K): [in this window] [in a new window]   Fig. 11. Effects of chronic 48-h Cl.19A monolayer preexposure to 500 nM PMA on cellular PKC isoforms expression. Shown are Western blots of PKC isoforms detected in cellular extracts from the same monolayers used in Fig. 10. A 48-h preincubation with either carrier alone [(–) lanes] or carrier plus 500 nM PMA [(+) lanes] revealed that the PKC-1 and PKC- bands decreased significantly, whereas PKC- and PKC-2 expression did not change (n = 2, P < 0.05).

	DISCUSSION

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Biophysical studies have demonstrated that CFTR anion channel activity is regulated by PKC-dependent phosphorylation (for a review, see Ref. 22). However, there are few details concerning which PKC isoforms participate in this response. In this study, we sought to determine whether phorbol ester use in vitro could provide us with such information.

Complexity in PKC signaling revealed. Our initial hypothesis was that PKC signaling in Cl^–-secreting gastrointestinal HT-29 Cl.19A monolayers could be broken down into separate components through the use of either weak or strong PKC agonists. This initial assumption proved to be oversimplified. However, we were able to show that chronic (24 h) exposure to the weak PKC--specific agonist DOPPA downregulated apical plasma membrane CFTR-dependent I_SC while failing to induce direct CFTR Cl^– channel activation (Figs. 3 and 4 and Table 3). These effects were accompanied by a shift in the total PKC isoform mass toward postactivation status, leading to increased cellular PKC-₂ expression/cytoplasmic partitioning and decreased cellular PKC-₁ content (Fig. 5). Similar changes were not recorded for other PKC isoforms (either conventional or novel). We also found that chronic exposure to <30 nM DOPPA potentiated CCh effects on transcellular I_SC-cAMP, a phenomena mediated by a Ba²⁺-sensitive transport event localized at the basolateral plasma membrane (Table 1). On the basis of previous studies in this cell line (see the Introduction) and the well-established sensitivity of K⁺ but not Cl^– channels to this divalent cation (21), we attributed this relief from inhibition to be due to PKC--sensitive stimulation of basolateral K⁺ channel and/or accompanying Na⁺-K⁺-2Cl^– cotransport function (21, 22, 24, 25). However, given that this was not our focus, this phenomenon was not investigated further.

The more effective but nonspecific agonist PMA (50 nM) acutely stimulated apical plasma membrane-generated I_SC (Fig. 6) and caused significant downregulation of apical membrane I_SC-cAMP induced by FSK within 2-h preexposure (Fig. 7). These changes were accompanied by activation of a wider range of PKC isoforms (PKC-, -₁, and -; Fig. 9) than with DOPPA. Because 70% of the acute PMA-stimulated current was sensitive to the highly selective PKC-/ antagonist Gö6976 (Table 4), PKC- and/or PKC-₁ were predicted to be major participants in I_SC under these conditions. Moreover, our earlier findings that the -isoform-specific agonist DOPPA did not acutely stimulate apical plasma membrane I_SC (Table 3) further identified PKC- as the most likely candidate for CFTR-mediated current activation.

Compared with DOPPA, the overall effects of chronic (24 h) low-dose (50 nM) PMA on transcellular I_SC-cAMP (Fig. 8) and on cellular PKC-₂ expression/membrane partitioning were similar but potentiated. However, unlike DOPPA, the nonspecificity of this agonist led to the activation of three additional PKC isoforms (PKC-, -₁, and -; Fig. 9) and introduced further complexity into the system. To address the question of multiple cellular inhibitory pathways, differing in their time course of activity, we looked for both chronic effects of PMA on PKC isoform mass/apical membrane I_SC-cAMP inhibition in control and agonist-treated monolayers [and determined the subcellular location of CFTR anion channels; see companion study (9a)]. Exposing monolayers for an extended time period (48 h) to high (500 nM) PMA doses selectively reduced cellular PKC-₁ and PKC- content (Fig. 11) and induced a partial (40%) recovery in apical plasma membrane-generated I_SC-cAMP (Fig. 10). These findings predicted a role for PKC-₁, but not PKC-₂ or PKC-, in uncoupling the FSK-generated cAMP signal from apical plasma membrane CFTR activity. They also highlighted PKC- as an alternative candidate. In the airway-derived cell line Calu-3, PKC- stimulated a cAMP-dependent activation of CFTR-mediated Cl^– efflux (34), whereas, in colonocytes, PKC- has been shown to mediate phorbol ester-induced inhibition of Cl^–/OH^– exchange (56), and, in duodenal mucosal sheets from Swiss-Webster mice, cAMP potentiated bicarbonate transport (63). In the context of CFTR-mediated Cl^– secretion, PKC-₁ is the candidate for chronic phorbol ester-dependent inhibition of apical plasma membrane CFTR-mediated secretory current.

Evidence favoring PKC-₁ as a negative modulator of apical plasma membrane CFTR function. The inhibitory effects of PMA in the intact cell (Fig. 7) and on apical plasma membrane-generated I_SC-cAMP were >80% complete within 2 h. This time course was much faster than that required for brefeldin A (BFA) (42) or DOPPA effects on apical plasma membrane CFTR targeting with the cellular biosynthetic pathway [PKC-₂ dependent; see companion study (9a)]. In addition, the after PMA-induced downregulation of PKC-₁ (Fig. 9), the corresponding recovery in apical plasma membrane-generated I_SC-cAMP (Fig. 10) occurred in monolayers exhibiting CFTR immunostaining below detectable limits within the apical pole of the cell [companion study (9a)]. These facts argue for a separate inhibitory locus at, or below, the apical plasma membrane leaflet.

Three -stands within the C2 region of PKC-, which are also shared with other PKCs, function as the minimal RACK-binding domain (41). Peptides made against these regions inhibit the movement of all C2 domain-containing PKCs and have been shown to prevent phorbol ester-mediated L-type Ca²⁺ channel downregulation in cardiac myocytes (69). PKC activation in these cells leads to both stimulation of Ca²⁺ channel activity and recruitment of Ca²⁺ channels to the plasma membrane (68). The PDZ-binding domain in the COOH-terminus of CFTR is known to interact with cytoskeletal elements and numerous PDZ domain-containing binding partners, which are hypothesized to maintain CFTR localization and function at the apical plasma membrane (32). Of these binding partners, PKC-dependent phosphorylation of EPB50/NHERF has been shown to decrease CFTR channel opening, resulting in inhibition of CFTR currents in airway epithelial cells (53). Both PKC- and PKC- are known to interact with RACK1 (35, 41). PKC- has been proposed to mediate the stimulation of cAMP-dependent CFTR activity, whereas, as we show here, PKC-₁ paradoxically appears to mediate inhibition of cAMP-dependent CFTR activity. Thus we propose that both isoforms may act in concert to reciprocally regulate CFTR through the phosphorylation of target proteins interacting with CFTR close to or at the apical plasma membrane.

Several lines of evidence support a physiological role for the regulation of CFTR by controlled insertion in the apical membrane. CFTR exists in a rapidly recycling endocytic pool (52). Removal of the internalization signals from the COOH terminal of the CFTR increases expression at the cell surface (50). cAMP stimulates activation of the membrane resident pool of the CFTR channel and trafficking of a vesicular pool of CFTR near the apical membrane (62) and may decrease endocytic retrieval of apical membrane-resident CFTR (31, 37, 45, 52). Using a highly sensitive cryoimmunogold labeling technique and electron microscopy, Ameen and colleagues (1) have recently identified CFTR in subapical vesicles and at the apical plasma membrane in rat intestinal crypt cells. cAMP stimulation in the rat proximal small intestine resulted in fluid secretion and apical pole CFTR redistribution (1). Thus, in the latter instance, the PKC dependency to CFTR function at the apical plasma membrane reported in the present study provides a mechanistic basis for the movement of CFTR observed in these ex vivo studies.

Other forms of regulation not involving a direct association of PKC with signaling complexes interacting with channels at the channel membrane can be considered. A BFA-insensitive (nonconstitutive) exocytotic mechanism is utilized by neuronal cells to functionally upregulate N-type Ca²⁺ channel activity through mechanisms involving vesicle delivery to the plasma membrane (65). Syntaxin-1 and associated modulator proteins (13) have been shown to functionally upregulate a variety of plasma membrane proteins including 1) adipocyte and smooth muscle insulin-regulated glucose uptake mediated by the glucose transporter GLUT4 (49); 2) a variety of neurotransmitter transporters (14, 25); and 3) CFTR expression in oocytes (47), in cultured tracheal epithelial cells, and in T84 gastrointestinal epithelial cells (46). Both GLUT4 and CFTR/syntaxin-1 association in vitro are inhibited by the modulator protein Munc-18, which reverses syntaxin-dependent inhibition of both insulin-stimulated glucose transport (for a review, see Ref. 49) and cAMP-regulated anion channel current (47), respectively. Interestingly, PKC phosphorylation of Munc-18 in cell-free systems results in prevention of its interaction with syntaxin-1 (21) and has been shown in vitro to increase the availability of syntaxin for GABA neurotransmitter transporter binding and subsequent functional downregulation (7).

Fluid-secreting HT-29 Cl.19A monolayers do not exhibit high levels of PKC-regulated apical plasma membrane exocytosis (43). However, taken together, these facts may provide a mechanistic basis for the PKC-₁ inhibitory effects on transcellular I_SC-cAMP: PKC-₁ acting through phosphorylation of a related modulatory protein within late stages the constitutive biosynthetic pathway could prevent or stabilize a subapical plasma membrane pool of CFTR undergoing membrane insertion. Our finding that the PKC inhibitor Gö6976 transiently protected a pool of CFTR not already associated with the apical plasma membrane (Table 3) may relate to this phenomena.

The hypothesized role of PKC-₁ in plasma membrane-resident cAMP signal/CFTR coupling or subplasma membrance vesicle fusion would not be mutually exclusive.

Evidence for a possible role for PKC- in the cellular regulation of apical plasma membrane-generated I_SC. Finally, our results do not exclude a possible role for PKC- in both the stimulation and inhibition of apical plasma membrane-generated CFTR Cl^– current because PKC-, like PKC-₁, was downregulated after a high-dose PMA exposure (Fig. 10) in monolayers exhibiting partial recovery of apical plasma membrane I_SC-FSK (Fig. 11). Actin-binding PKC- (51) has been implicated in CCh-stimulated apical membrane exocytosis in rabbit lacrimal acini (15) as well as the regulated endocytotic retrieval of Shaker K⁺ channels in oocytes (9), in basolateral plasma membrane endocytosis (61), and in the inhibitory effects of EGF on CCh-mediated basolateral K⁺ channel stimulation in T84 monolayers (12). In both later instances (17), the authors surmised that elevated basolateral membrane internalization could account for the PMA-dependent inhibition of monolayer anion secretion. A cellular locus for PKC-'s effects may involve the actin-binding protein coronin_se (48), which binds ezrin in vitro and is also a cellular target for PKC- phosphorylation. Thus, although PKC- modulation of goblet epithelial cell mucin exocytosis (26) and cAMP-regulated Cl^– efflux in cultured Calu-3 airway epithelial cells is established (34), we presently favor the hypothesis that, in colonocytes, the primarily locus of PKC- regulation is the basolateral plasma membrane.

	GRANTS

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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-59550 (to A. P. Morris).

	FOOTNOTES

 

Address for reprint requests and other correspondence: A. P. Morris, Div. of Gastroenterology, Depts. of Integrative Biology and Internal Medicine, University of Texas Health Science Center, Rm. 4.236, Medical School Bldg., 6431 Fannin, Houston, TX 77030 (e-mail: Andrew.P.Morris{at}uth.tmc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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