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 (ISC-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 ISCgeneration. DOPPA failed to elicit similar responses. Whereas chronic (24 h) exposure to both agents inhibited FSK-stimulated transcellular and apical membrane ISC-cAMP, the effects of DOPPA were more complex: this conventional PKC-β-specific agonist also stimulated Ba2+-sensitive basolateral membrane-dependent facilitation of transcellular ISC-cAMP. PMA did not elicit a similar phenomenon. Prolonged exposure to high-dose PMA but not DOPPA led to apical membrane ISC-cAMP recovery. Changes in PKC α-, β1-, γ-, and ε-isoform membrane partitioning and expression correlated with these findings. PMA-induced transcellular ISC correlated with PKC-α membrane association, whereas low doses of both agents inhibited transcellular and apical membrane ISC-cAMP, increased PKC-β1, decreased PKC-β2membrane association, and caused reciprocal changes in isoform mass. During the apical membrane ISC-cAMP recovery after prolonged high-dose PMA exposure, an almost-complete depletion of cellular PKC-β1 and a significant reduction in PKC-ε mass occurred. Thus activated PKC-β1 and/or PKC-ε prevented, whereas activated PKC-α facilitated, apical membrane ISC-cAMP. PKC-β-dependent augmentation of transcellular ISC-cAMP at the level of the basolateral membrane demonstrated that transport events with geographically distinct subcellular membranes can be independently regulated by the PKC β-isoform.
- protein kinase C
- short-circuit current
- cystic fibrosis transmembrance conductance regulator
- phorbol ester
viral and bacterial toxins that mobilize intracellular Ca2+ have been shown to stimulate downstream signaling pathways linked to PKC (30, 57). In the case of rotavirus infection, Ca2+ mobilization by virus entry (54) or by phospholipase C stimulation through extracellular exposure to the viral enterotoxin NSP4 (18) has been proposed to effect cellular PKC signaling (30, 44). Hence, Ca2+- and diacyglycerol (DAG)-sensitive PKC activation may participate in the mucosal cell anion permeability and ion transport changes recorded in virally infected mucosal cells (44) and in mucosal sheets treated with NSP4 (36). To further address this hypothesis in a simplified in vitro assay, we undertook studies in the colonocyte epithelial cell line HT-29 Cl.19A to determine whether the activation status of specific PKC isoforms directly or indirectly promoted cAMP-regulated Cl− secretion. cAMP-dependent Cl− secretion in this and other gastrointestinal models is dependent on the apical membrane expression of the anion channel CFTR (2). The CFTR transport protein is characterized by two roughly similar halves, each incorporating six putative transmembrane α-helixes followed by an intracellular nucleotide-binding domain. Unique to CFTR is a large intracellular regulatory domain containing multiple sites for phosphorylation by PKA and PKC (22), which facilitate changes in channel gating.
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, 4β-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-ε 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
FBS, DMEM, penicillin-streptomycin, and trypsin-EDTA solutions were procured from GIBCO-BRL (Grand Island, NY). Isoform-specific anti-PKC-α, -β1, -β2, and -γ 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.
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 (ISC) 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 ISC 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/xo)b, curve fit 2] function. In both instances, x and y are horizontal and vertical ordinates, respectively, xo 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.
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.
To examine the effect of isoform-specific PKC-β 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 ISC across intact monolayers in a concentration-dependent manner.
In the HT-29 Cl.19A subclone, cAMP-responsive transepithelial ISC 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-β 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 ISC (ISC-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 ISC-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.
The inhibitory effects of DOPPA on transcellular ISC-cAMP are shown more clearly in Fig. 2A. When the results were replotted as percent inhibition of the untreated response (Fig. 2B), the ID50 under these conditions was 71 nM (see materials and methods for curve fit 2). In paired monolayers exposed consecutively to FSK and CCh, a biphasic concentration versus transcellular ISC-cAMP response was recorded (Fig. 1B). At low (<30 nM) DOPPA concentrations, any reduction in transcellular ISC-cAMP was compensated for in the combined agonist response by an increase in transcellular ISC-cAMP promoted by the subsequent addition of CCh (Fig. 2A). In this concentration range, responses were not significantly different from non-DOPPA-treated monolayers (P > 0.05). The effect of DOPPA on CCh-mediated transcellular ISC-cAMP synergism was Ca2+ dependent, because chelation of bath Ca2+ with 2 mM EGTA for 5 min before the agonist addition abolished the potentiated current (data not shown, n = 8). At DOPPA concentrations >50 nM, a rightward shift in the combined FSK plus CCh agonist concentration versus response curve was recorded, producing an ID50value of 98 nM [Fig. 2B; n = 29–36 observations made for each data point (see materials and methods for curve fit 2)]. The transcellular ISC-cAMP responses of both agonists alone or in combination were inhibited by the serosal bath addition of bumetanide (400 μM; Figs. 1A and 2A), by reducing serosal bath Cl−, by adding the anion channel inhibitor diphenylamine-2-carboxylic acid (DPC, 2 mM; Ref. 20) to the mucosal bathing media, or by adding serosal bath Ba2+ (Table 1).
Identical studies were performed in age-matched monolayers exposed on their serosal bath surface (basolateral plasma membrane) to the univalent ion pore-forming antibiotic nystatin (50 μg/ml; Fig. 3 and Table 2).
Monolayers exhibiting basal transepithelial resistance within the 300–500 Ω/cm2 range were selected for these studies (Table 2). After 24 h of incubation with either carrier alone (control) or 1–500 nM DOPPA, monolayers were then bathed in standard high-Na+-Krebs saline with a 140:7 mM mucosal-to-serosal Cl− gradient (Fig. 3A). An initial challenge with 10 μM FSK resulted in a small ISC-cAMP against the prevailing anion gradient, which inverted after nystatin permeabilization of the basolateral plasma membrane into a large absorptive current [Fig. 3A, (−) trace]. Under these conditions, current flow across the still-intact apical plasma membrane reflected cAMP-stimulated Cl− conduction along a counterluminal (mucosal to serosal) electrochemical gradient. A 24-h incubation with 50 nM DOPPA significantly reduced the magnitude of this response [Fig. 3A, (+) trace]. When the change in apical membrane ISC-cAMP was plotted against agonist concentration, 1–500 nM DOPPA stepwise inhibited up to 80% of the apical membrane-generated cAMP-dependent Cl− current [Fig. 3B; n = 9–11 observations for each data point (see materials and methods for curve fit 1)]. DOPPA incubation did not significantly affect basal transepithelial resistance values until DOPPA concentrations >200 nM were used (Table 2).
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 ISC-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 ISC-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.
DOPPA affected the membrane translocation status and expression levels of individual PKC-β isoforms.
To validate that DOPPA was affecting PKC function in our experimental assay, Western blot analysis was used to measure changes in immunospecific phorbol ester-sensitive PKC expression in both cytosolic (non-Triton X-100 extracted) and membrane-associated (Triton X-100 extracted) subcellular fractions. It is widely accepted that PKC exists in an inactive conformation in the cytosol, and, after activation, the enzyme translocates from the cytosol to membranes. The active state is usually held for a short period (minutes to hours for endogenous signaling pathways; much shorter periods when exogenous agents are used) before the kinase is once again inactivated and returns to the cytosol (8). Thus, in long-term experiments, the results report on an equilibrium between cellular PKC isoform pools in inactive (cytosolic >> membrane), active (membrane >> cytosolic), and postactivation (cytosolic > membrane) status.
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-α/β1/γ or Ca2+-insensitive but DAG-sensitive PKC-ε. However, a significant reduction in the membrane translocation ratio of PKC-β2 was recorded, an effect caused by increased cytosolic PKC-β2 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 β-isoform expression; a modest decrease (1.4 ± 0.1-fold) in PKC-β1 and increase (1.6 ± 0.1-fold) in PKC-β2 was recorded [see companion study (9a)]. The relative expression levels of the other isoforms did not change relative to β-actin (n = 3 separate experimental observations). The percent membrane association [membrane/(cytostol + membrane) × 100] for the PKC-α, -β1, -γ, and -ε isoforms remained unchanged at 50%, 50%, 28%, and 77%, respectively. The percent membrane association for PKC-β2 decreased from 90% to 70%, demonstrating that a significant fraction remained active when the cytoplasmic PKC-β2 mass increased. Western blots were stripped and reprobed for cytoplasmic β-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-β 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-β2 partitioning predicted an increase in the postactivation status of the PKC β-isoforms. We therefore hypothesized that inhibition of cAMP-dependent apical plasma membrane Cl− current under these conditions was mediated by PKC-β2 or PKC-β1 homolog-specific changes in subcellular signaling function.
PMA, but not DOPPA, acutely stimulated apical plasma membrane-generated ISC through PKC-α or PKC-β 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-α/β inhibitor) did not exhibit any change in ISC (Table 3). In contrast, 50–500 nM PMA generated significant ISC in both intact and basolateral membrane-permeabilized monolayers. An example of PMA-induced apical membrane ISC-cAMP is shown in Fig. 6.
Confirming previous reports (4, 5, 65, 66), PMA acted as a potent agonist (Fig. 6A). Compared with FSK (Fig. 6B), PMA-induced ISC exhibited a slower time course for activation [initial rates (R) for PMA and FSK (R = ΔISC/ΔT), where T is time, were 0.43 ± 0.4 and 2.8 ± 0.5 μA/s, respectively; n = 15 monolayers/experimental condition)]. The overall magnitude of the apical membrane ISC response was also smaller (Fig. 6A and right bar graph). Increasing the PMA dose from 50 to 500 nM did not significantly (P > 0.5) increase the rate constant of the stimulated apical membrane ISC (R = 0.52 ± 0.4 μA/s) or affect the total magnitude of the combined phorbol ester- and FSK-generated apical membrane ISC-cAMP response (Fig. 6, C, D, and right bar graph). We found that the acute apical membrane ISCstimulated by PMA was largely mediated by PKC-α/β activation because 70% of the PMA-evoked DPC-sensitive ISC was blocked by prior monolayer exposure to the highly selective PKC inhibitor Gö6976 (39) (Table 3). This was not the case for FSK, which exhibited nearly normal current levels after chronic (>60 min) Gö6976 preincubation. However, we did find that 1-min to 2-h Gö6976 monolayer preincubation before FSK inhibited 10–15% of apical membrane ISC-cAMP (Table 4), whereas more prolonged treatment (2–24 h) of monolayers failed to affect apical membrane ISC-cAMP generation, similar to Gö6976 application post-FSK challenge (Table 3). From these findings, we hypothesized that 1) PMA, unlike DOPPA, stimulated apical plasma membrane CFTR function, 2) whereas Gö6976 preexposure significantly reversed PMA-dependent activation of apical plasma membrane resident CFTR, and short-term (<15 min) but not long-term Gö6976 preexposure before FSK challenge prevented the acute stimulation of a much smaller PKC-sensitive CFTR pool. This pool was sensitive to cellular cAMP levels but not initially associated with the apical plasma membrane (see discussion). The PKC-β specificity for DOPPA within these monolayers (Fig. 5) and in other colonocyte preparations (28) indicates that Gö6976-sensitive PKC-α activation was responsible for the acute stimulation of apical membrane ISCby FSK and PMA.
Chronic monolayer exposure to PMA identified both PKC-β and PKC-ε as candidates for PKC-dependent inhibition of apical plasma membrane-generated ISC-cAMP.
Time-dependent effects of PMA stimulation on transcellular ISCcAMP were investigated in intact 12-day-old monolayers. The potency of this agonist was compared with DOPPA by creating a time course of drug incubation versus change in transcellular ISC-cAMP (Fig. 7).
Within 2 h, 50 nM PMA reduced the transcellular ISC-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 ISC-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 ISC-cAMP. However, when this was followed in control monolayers by a bilateral bath FSK (10 μM) addition, a large apical membrane ISC-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 ISC 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 ISC-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 ISC-cAMP.
The acute effects of high-dose PMA on monolayer transcellular ISC have previously been shown to be mediated by PKC-α (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 ISC-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-α R(membrane/cytosol), equal to 1.2 ± 0.1 (n = 4, untreated monolayers used for comparison), was recorded. Both PKC-β1 and PKC-ε 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-β2 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-β2 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-β1 (1.4 ± 0.2-fold) and increases in PKC-β2 (1.7 ± 0.2-fold) expression were recorded. Changes in PKC-ε (1.2 ± 0.1-fold) expression or the relative cellular levels of the other isoforms (PKC-α and PKC-γ) were not significant (P < 0.001, n = 4). The percent membrane association [membrane/(cytosol + membrane) × 100] for the PKC γ-isoform remained unchanged at 22%, and a modest rise in PKC-α membrane association was recorded (from 50% to 59%). Values for the other isoforms either rose from 77% to 90% (PKC-ε) or from 50% to 71% (PKc-β1) or decreased from 90% to 79% (PKC-β2).
Thus the larger apical membrane ISC-cAMP inhibitions recorded with the stronger agonist PMA were matched with greater changes in PKC-β cell signaling. However, in contrast to DOPPA and reflecting the less-specific effects of this agonist, PKC-ε activation was also implicated in the chronic inhibitory phenomena.
The downregulation of PKC-β1 and PKC-ε by PMA was associated with recovery of apical plasma membrane ISC-cAMP.
To address in chronic PMA experiments whether PKC-ε along with PKC-β1/PKC-β2 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-β-specific but weaker agonist DOPPA. Increasing the preexposure time to 50 nM PMA from 24 to 48 h had little effect on apical membrane ISC-cAMP (data not shown). Similarly, increasing the monolayer PMA dose to 500 nM for 24 h only marginally affected apical membrane ISC-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 ISC-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 ISC-cAMP was recorded. In control monolayers, the addition of Nystatin to the serosal bath reversed the direction of the apical membrane ISC-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 ISC-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 ISC-cAMP recovery occurred after 48-h exposure to 500 nM PMA.
A similar partial recovery, but at the level of transcellular ISC-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-α depletion (66). These results differ from our own findings in that we were unable to produce significant changes in the cellular mass of PKC-α 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.
We found that both PKC-β1 and PKC-ε exhibited decreased overall cellular expression (relative to control conditions, densitometry readings were 13% and 39% of untreated monolayer values, respectively, n = 2). The cellular levels of the other isoforms (PKC-α and PKC-β2) were not significantly altered (Fig. 11A). Thus, while clearly demonstrating that PKC activation was required for the inhibition of apical membrane ISC-cAMP, the sensitivity of PKC-β1 and PKC-ε to chronic downregulation by PMA was too similar to determine whether an individual isoform or both isoforms were responsible. However, given that 70% of the acute PMA-sensitive current could be protected by the highly selective PKC inhibitor Gö6976 (Table 3), we propose that along with PKC-α, PKC-β1/β2 plays a major role in positively and negatively regulating cAMP-dependent secretory current generated by this membrane, respectively.
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-β-specific agonist DOPPA downregulated apical plasma membrane CFTR-dependent ISC 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-β2 expression/cytoplasmic partitioning and decreased cellular PKC-β1 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 ISC-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-β-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 ISC (Fig. 6) and caused significant downregulation of apical membrane ISC-cAMP induced by FSK within 2-h preexposure (Fig. 7). These changes were accompanied by activation of a wider range of PKC isoforms (PKC-α, -β1, and -ε; Fig. 9) than with DOPPA. Because 70% of the acute PMA-stimulated current was sensitive to the highly selective PKC-α/β antagonist Gö6976 (Table 4), PKC-α and/or PKC-β1 were predicted to be major participants in ISC under these conditions. Moreover, our earlier findings that the β-isoform-specific agonist DOPPA did not acutely stimulate apical plasma membrane ISC (Table 3) further identified PKC-α 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 ISC-cAMP (Fig. 8) and on cellular PKC-β2 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-α, -β1, and -ε; 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 ISC-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-β1 and PKC-ε content (Fig. 11) and induced a partial (∼40%) recovery in apical plasma membrane-generated ISC-cAMP (Fig. 10). These findings predicted a role for PKC-β1, but not PKC-β2 or PKC-α, in uncoupling the FSK-generated cAMP signal from apical plasma membrane CFTR activity. They also highlighted PKC-ε as an alternative candidate. In the airway-derived cell line Calu-3, PKC-ε stimulated a cAMP-dependent activation of CFTR-mediated Cl− efflux (34), whereas, in colonocytes, PKC-ε 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-β1 is the candidate for chronic phorbol ester-dependent inhibition of apical plasma membrane CFTR-mediated secretory current.
Evidence favoring PKC-β1 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 ISC-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-β2 dependent; see companion study (9a)]. In addition, the after PMA-induced downregulation of PKC-β1 (Fig. 9), the corresponding recovery in apical plasma membrane-generated ISC-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 β-stands within the C2 region of PKC-β, 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-β and PKC-ε are known to interact with RACK1 (35, 41). PKC-ε has been proposed to mediate the stimulation of cAMP-dependent CFTR activity, whereas, as we show here, PKC-β1 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-β1 inhibitory effects on transcellular ISC-cAMP: PKC-β1 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-β1 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-ε in the cellular regulation of apical plasma membrane-generated ISC.
Finally, our results do not exclude a possible role for PKC-ε in both the stimulation and inhibition of apical plasma membrane-generated CFTR Cl− current because PKC-ε, like PKC-β1, was downregulated after a high-dose PMA exposure (Fig. 10) in monolayers exhibiting partial recovery of apical plasma membrane ISC-FSK (Fig. 11). Actin-binding PKC-ε (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-ε's effects may involve the actin-binding protein coroninse (48), which binds ezrin in vitro and is also a cellular target for PKC-ε phosphorylation. Thus, although PKC-ε 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-ε regulation is the basolateral plasma membrane.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-59550 (to A. P. Morris).
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