Modulation of Na-K-2Cl cotransport by intracellular Clminus and protein kinase C-delta in Calu-3 cells

Carole M. Liedtke, Robert Papay, and Thomas S. Cole

W. A. Bernbaum Center for Cystic Fibrosis Research, Departments of Pediatrics and Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4948


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we tested the hypothesis that intracellular Cl- (Cl<UP><SUB>i</SUB><SUP>−</SUP></UP>) regulates the activity of protein kinase C (PKC)-delta and thus the activation of Na-K-Cl cotransport (NKCC1) in a Calu-3 cell line. The alpha 1-adrenergic agonist methoxamine (MOX) and hypertonic sucrose increased Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and increased or decreased intracellular volume, respectively, without changing Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> concentration ([Cl-]i). Titration of [Cl-]i from 20-140 mM in nystatin-permeabilized cell monolayers did not affect the baseline activity of PKC-delta , PKC-zeta , or rottlerin-sensitive NKCC1. At 200 mM Cl-, rottlerin-sensitive NKCC1 was activated, and PKC isotypes were localized predominantly to a particulate fraction. MOX induced a biphasic increase in NKCC1 activity and PKC-delta in activity and particulate localization of PKC-delta and -zeta . Activity of NKCC1 and PKC-delta decreased with increasing Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> from 20 to 80 mM Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> then increased at 140-200 mM Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> apparently as an additive effect to high [Cl-]i levels. Rottlerin inhibited the effects of MOX, which indicates that PKC-delta was required for activation of NKCC1. The results indicate that, in airway epithelial cells, a Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> electrochemical gradient alone is not sufficient to stimulate NKCC1 activity; rather, elevated activity of PKC-delta is necessary. Further, high Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> levels induce a subcellular redistribution of PKC-delta , which results in increased enzyme activity.

alpha -adrenergic; methoxamine; volume; hyperosmotic stress; bumetanide; shrinkage; cystic fibrosis; rottlerin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NA-K-2CL COTRANSPORTERS (NKCCS) comprise a family of Cl--dependent cation cotransporters that are expressed in many animal cells (24). This widespread distribution suggests a fundamental role for NKCCs in physiological functions. In most cells that have been well studied, NKCCs are activated by cell shrinkage and thus play a potentially critical role in cell-volume regulation. Epithelia NKCCs serve secretory and absorptive functions depending on the localization of the NKCC to the basolateral or apical plasma membrane, respectively. Absorptive functions of NKCCs are best observed in the absorptive epithelium of the thick ascending limb of Henle's loop of mammalian kidney. Secretory functions are well studied in a number of epithelia including those of the colon, trachea, small intestine, and salivary gland (24).

Bumetanide-sensitive NKCC1 is localized to the basolateral plasma membrane of epithelia and participates in homeostatic control of cell volume and secretion of fluids and electrolytes. In epithelia of the colon and trachea, for example, Cl- secretion is rapidly elicited by agents that act on cAMP. cAMP-mediated activation of apical Cl- channels is generally modeled as the major regulatory event to elicit secretion. However, basolateral NKCC1 is necessary for secretion because it supplies Cl- for secretion. The intracellular Cl- concentration ([Cl-]i) itself has been linked to regulation of NKCC1 activity in secretory epithelia and avian erythrocytes (22, 24) as well as in dialyzed squid axon (1). In general, a reduction in intracellular Cl- (Cl<UP><SUB>i</SUB><SUP>−</SUP></UP>) appears to stimulate NKCC1, whereas elevation of Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> appears to inhibit NKCC1. Models based on these findings depict Cl- secretion as a coordinated control of Cl- exit at the apical membrane through Cl- channels and entry at the basolateral membrane through NKCC1. However, the mechanism by which [Cl-]i regulates NKCC1 activity is not well understood.

One mechanism that is thought to control activity of NKCC1 is NKCC1 phosphorylation through the action of serine and threonine protein kinases. NKCC1 isoforms from mammalian epithelia share similar putative sites for phosphorylation by protein kinases A and C (PKA and PKC; Ref. 24). Yet elevations in PKA alone do not fully explain the activation of NKCC1 in shark rectal gland cells and human tracheal epithelial cells (9). Rather, we have shown that activation of NKCC1 in human tracheal epithelial cells and in a Calu-3 cell line can be isolated from activation of Cl- channels through stimulation with an alpha 1-adrenergic agonist (9, 10, 12). Our studies also demonstrate a signaling mechanism in which PKC-delta is required for alpha 1-adrenergic activation of NKCC1 (11). An earlier study (8) on rabbit tracheal epithelial cells showed that NKCC1 is activated by hyperosmotic stress, which causes a rapid loss of water and leads to cell shrinkage and subsequent compensatory uptake of ions via NaCl or NKCC.

In this study, we investigated the sensitivity of human airway epithelial NKCC1 activity and PKC-delta activity to [Cl-]i in Calu-3 cells. A Calu-3 epithelial cell line has been shown by others and by us to express NKCC1 mRNA and hormone-mediated activation of NKCC1 (4, 10). We first identified experimental conditions that allow alterations in Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> while minimizing changes in cell volume. The results show that cell volume in Calu-3 cells is differentially altered by hormone stimulation with methoxamine (an alpha 1-adrenergic agonist) and by hyperosmolarity induced using sucrose. Next, we studied the effects of varying [Cl-]i on the activity of NKCC1 and on the activity and subcellular distribution of PKC-delta and -zeta . The results indicate that NKCC1 is quiescent over a range of [Cl-]i until stimulated by hormone. Peak net activity of NKCC1 and PKC-delta occurs at 20 mM Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and declines with increasing Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> levels. Increasing Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> levels, however, promotes translocation of PKC-delta to a particulate fraction. Surprisingly, at very high Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> levels of 140-200 mM, NKCC1 activity increases significantly and is further stimulated by basolateral addition of an alpha 1-adrenergic agonist. Agonist stimulation of NKCC1 is sensitive to rottlerin (an inhibitor of PKC-delta ). Therefore, PKC-delta is necessary for activation of NKCC1 even at very high [Cl-]i. Thus, in human airway epithelial cells, a Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> electrochemical gradient is not sufficient for stimulation of NKCC1 function; rather, PKC-delta activity is necessary.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Calu-3 cells were grown in cell culture in Earle's balanced salt solution supplemented with 2.4 mM L-glutamine and 10% fetal bovine serum on 0.4-µm pore size Transwell-Clear polyester filter inserts (Corning Costar, Cambridge, MA). For measurement of cell volume and transepithelial flux, cells were seeded at a density of 2.0 × 106 cells onto filters with a growth area of 4.52 cm2. Cells were incubated in a humidified CO2 incubator at 37°C. Culture medium was changed at 48-h intervals until confluence was reached. Confluence was assessed by microscopic examination of the cell monolayer and by measurement of electrical resistance across the cell monolayer. Transepithelial resistance was quantitated using chopstick electrodes and an epithelial voltohmmeter (EVOM, World Precision Instruments, New Haven, CT). Values were corrected for background resistance of the filter alone bathed in medium. Cell monolayers were serum deprived for 18 h before the experiments.

Measurement of Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and intracellular volume. The equilibrium distribution of [14C]urea was used to measure intracellular water space, and that of 36Cl was used to measure intracellular content. After incubation overnight in serum-free culture medium, cell monolayers were washed twice in Hanks' balanced salt solution containing 10 mM HEPES with pH 7.4 (HPSS) and were incubated with HPSS containing 0.4 µCi of [14C]urea and 1 µCi of 36Cl for 1 h. Cells were exposed to vehicle, 10 µM methoxamine, or sufficient sucrose to raise medium osmolarity to 500 mosmol/kgH2O for the time intervals indicated in the table and figure legends. Radiotracer flux was terminated by washing the cell monolayer rapidly four times in ice-cold isotonic sucrose (100 mM MgSO4 and 137 mM sucrose) (12). Intracellular radioactivity was extracted by incubating cells in 0.1 N NaOH. Radioactivity in an aliquot of extract was determined by scintillation counting, and protein content was measured by the Bradford protein assay using bovine serum albumin as the standard. The intracellular water space Vi (in µl/mg of protein) was calculated as Vi = (VoAi)/(Aom), where Ai and Ao are the radioactivity of [14C]urea in the cell lysate and incubation medium, respectively, m is the protein content in the cell lysate, and Vo is the volume of incubation medium (in µl) used for Ao. Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> levels (in nmol/mg of protein) were calculated as Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> = (CloAi)/(Aom), where Ai and Ao are the radioactivity values of 36Cl in the cell lysate and incubation medium, respectively; Cl<UP><SUB>o</SUB><SUP>−</SUP></UP> is the amount of Cl- (in nmol) in an aliquot of incubation medium; and m is the protein content in the cell lysate. [Cl-]i (in mM) was calculated as Cl<UP><SUB>i</SUB><SUP>−</SUP></UP>/Vi.

The sensitivity of urea as a marker for intracellular volume was evaluated in preliminary experiments using [14C]urea as a marker for intracellular water space and [3H]polyethylene glycol ([3H]PEG) as a marker for extracellular space. Four rapid washes with ice-cold isotonic buffer were sufficient to remove extracellular [3H]PEG. Retention of extracellular marker amounted to 0.26% of added radioactivity. Water loss in control cell monolayers was 0.067 µl · mg protein-1 · s-1.

Measurement of NKCC1 activity. NKCC1 activity was measured as bumetanide-sensitive, basolateral-to-apical, unidirectional flux of 86Rb, a congener of K, as previously described (13). Cell monolayers were preincubated for 10 min at 37°C with HPSS or HPSS supplemented with 10 µM bumetanide or 10 µM rottlerin in a basolateral bathing solution. Cells were permeabilized at the apical membrane using 175 U/ml nystatin in an apical cytosolic medium containing (in mM) 20 Na+, 110 K+, 0.4 Mg2+, 4.2 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and 10 HEPES at pH 7.5 with gluconate replacing Cl- at concentrations <132 mM. For concentrations >132 mM, sufficient N-methyl-D-glucamine was added to achieve the desired concentration. Basolateral [Cl-] was held constant at 136 mM. To assure that treatment with nystatin altered Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> levels without affecting Vi, Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and Vi were measured in cell monolayers treated for 15 min with nystatin and apical bathing medium of varying [Cl-]. Treatment with nystatin did not significantly alter Vi measurements (Table 1). [Cl-]i (expressed in mM) was calculated from values for Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and Vi for each apical bathing solution. [Cl-]i was directly proportional to [Cl-] in the apical bathing medium, which indicates that treatment with nystatin allowed movement of Cl- between the cytosol and extracellular medium.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Intracellular volume and Cl- measurements in nystatin-permeabilized Calu-3 cell monolayers

To initiate transepithelial flux, 1 µCi of 86Rb was added to the basolateral solution. The apical bathing medium was collected for radioactive counts at 2.5-min intervals for 10 min and replaced with an equal volume of nystatin-supplemented cytosolic medium. At 10 min, methoxamine was added to the basolateral solution to a final concentration of 10 µM, and sampling continued at 2.5-min intervals for 10 min. After the last sampling, cell monolayers were washed in 1% PBS and extracted with 0.5 ml of 0.1 N NaOH. Aliquots of the cell extract were assayed for protein content. The accumulation of 86Rb in the apical compartment was calculated (as nmol/mg of protein) over time.

Immunoprecipitation, PKC activity assay, and Western blot analysis of PKC isotypes. Calu-3 cells were grown on filter inserts before being serum deprived and preequilibrated with an apical perfusate containing 225 U of nystatin/ml, 20 mM Na+, 110 mM K+, 0.4 mM Mg2+, 4.2 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and 10 mM HEPES at pH 7.5 and varying [Cl-] as described (see Measurement of NKCC1 activity). Cells were then stimulated with methoxamine or, as a control, with vehicle of HPSS. Cells were rapidly immersed in ice-cold PBS to halt the stimulation and were harvested in 1 ml of lysis buffer consisting of 100 mM NaCl, 50 mM NaF, 50 mM Tris · HCl, pH 7.55, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, and the protease inhibitors as described. Lysates were clarified by microcentrifugation at 4°C for 20 min at 12,000 g and incubated with antiserum against a specific PKC isotype as previously described (13). Immune complexes were recovered using protein G agarose beads that were prewashed and resuspended in lysis buffer. Western blot analysis of immunoprecipitated protein was used to titrate antiserum and select an optimal antiserum concentration (13). Kinase activity of PKC isotypes was measured by taking immunoprecipitates to a final volume of 50 µl in assay mixture {50 mM Tris · HCl, pH 7.5, 10 mM beta -mercaptoethanol, 10 mM MgSO4, 40 µg/ml phosphatidylserine, 0.1 µM phorbol 12-myristate 13-acetate (PMA), 50 µM ATP, 10 µg/ml histone III, 3 µCi of [gamma -32P]ATP, 1 mM sodium orthovanadate, and protease inhibitors} and incubating at 30°C for 15 min. The reaction was terminated by addition of 30 µl of glacial acetic acid. A 40-µl aliquot was spotted on P-81 phosphocellulose paper, washed, and counted for radioactivity using Cerenkov counting.

For studies on the distribution of PKC isotypes, cells were grown to confluence on filter inserts and incubated overnight in serum-free media. [Cl-]i was varied as described. After treatment with vehicle (for 1 or 10 min), methoxamine (for 1 min), or sucrose (for 10 min), cell monolayers were immediately washed twice with ice-cold PBS, harvested in 1 ml of ice-cold hypotonic buffer, and fractionated as previously described (11). Aliquots of subcellular fractions were solubilized in SDS-Laemmli buffer and subjected to gel electrophoresis on 4-15% gradient slab gels. Protein bands were transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Bedford, MA) and immunoblotted with polyclonal antibodies to specific PKC isotypes. Immunoreactive protein bands were detected using enhanced chemiluminescence and quantitated by laser densitometry. Preliminary experiments showed that antibody to PKC-delta did not recognize PKC-zeta and vice versa.

NKCC1 was immunoprecipitated according to the method of D'Andrea and colleagues (3) with modifications. In brief, Calu-3 cells were lysed in 1 ml of ice-cold 10 mM HEPES buffer (pH 7.4) supplemented with 3.5 mM MgCl2, 150 mM NaCl, 1 mM benzamide, and protease inhibitors. The lysate was clarified by microcentrifugation for 10 min at 4°C. A 0.5-ml aliquot of supernatant was incubated with 1.1% SDS for 1 h at room temp. The SDS-solubilized lysates were combined with 1.4 ml of 3.0% Triton X-100 in lysis buffer and incubated for 1 h on ice. NKCC1 was immunoprecipitated by overnight incubation at 4°C with 1:2,500 dilution of T4 monoclonal antibody (15). Immune complexes were recovered using protein G agarose beads that had been prewashed and resuspended in lysis buffer. The immune complexes were recovered by centrifugation, washed five times, and subjected to gel electrophoresis on 4-15% gradient slab gels. NKCC1 and coimmunoprecipitated proteins were detected by Western blot analysis using specific antibodies and enhanced chemiluminescence as previously described (10, 11).

Data analysis. Data are reported as means ± SE. Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and Vi values are expressed in some figures as a ratio of the experimental values for stimulated cells divided by the values for unstimulated cells. To determine the level of significance, ANOVA or unpaired t-tests were performed using GraphPad Prism 3.0 software.

Materials. 86Rb (sp act 154 Bq/g of Rb; 4,200 Ci/g of Rb) and an enhanced chemiluminescence kit were purchased from Amersham Life Science, 36Cl (sp act 260 MBq/g of Cl, 7.5 mCi/g of Cl) was purchased from ICN Radiochemical, and [14C]urea (sp act 37 GBq/mmol) was purchased from NEN. Transwell-Clear filter inserts were purchased from Fisher Scientific. Polyclonal anti-PKC isotype-specific antibodies were obtained from Santa Cruz Biotechnology, and recombinant PKC isotypes were from Calbiochem (La Jolla, CA). T4 monoclonal antibody was obtained from the Developmental Studies Hybridoma Bank. Methoxamine-HCl, bumetanide, and nystatin were purchased from Sigma Chemical and rottlerin was from Research Products International (Natick, MA). All other chemicals were reagent grade.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Modulation of Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and Vi levels by alpha -adrenergic stimulation. To measure changes in Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and Vi levels, the respective values were measured by equilibrium distribution of 36Cl and [14C]urea. Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and Vi were expressed as a ratio of values from experimentally treated cells and untreated cells within the same experiment to minimize the effects of interexperimental variations. A comparison of ratios for Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and Vi values in cells treated with vehicle or with the alpha 1-adrenergic agonist methoxamine is presented in Fig. 1. We also calculated means for cumulative data, which are presented in Table 2. Stimulation of Calu-3 cells with methoxamine resulted in a time-dependent increase in the ratios of Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and Vi, thereby indicating elevated Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and Vi levels. The data of Fig. 1 show that hormone stimulation rapidly increased the ratios of Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and Vi to attain maximal levels after 1 min of stimulation. Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and Vi then declined and reached baseline levels after 5 min of stimulation. At maximal levels, Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> increased 32.2 ± 9.5% (n = 4) and Vi increased 31.2 ± 7.0% (n = 4). When cumulative data were examined, we found that methoxamine treatment also increased mean Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and Cli values (as shown in Table 2). A Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> value of 485.0 ± 13.3 nmol/mg of protein increased 32.8% to 644.2 ± 59 (P < 0.02) nmol/mg of protein. A Vi value of 11.2 ± 1.9 µl/mg of protein increased by 27.7% to 14.3 ± 1.2 µl/mg of protein. These increases are consistent with increases in the ratios of paired values (see Fig. 1), which suggests a pattern of increased Vi and Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> values.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1.   Time-dependent fluctuations in intracellular Cl- (A) and volume (B) induced by methoxamine. Calu-3 cells were preincubated for 60 min with 0.4 µCi of [14C]urea and 1.0 µCi of 36Cl to radioisotopic equilibrium and then pretreated where indicated with 10 µM bumetanide (black-triangle) for 30 min. Methoxamine (, black-triangle) was added to the basolateral bathing solution at a final concentration of 10 µM at time 0. Stimulation with methoxamine was terminated at the specified times and intracellular radioactive tracer was quantitated as described in MATERIALS AND METHODS. Data for stimulated cells were calculated as a ratio of Cl- and Vi in stimulated cells divided by Cl- and Vi in unstimulated cells, respectively. Values are means ± SE for 4 separate filters. *P < 0.05; **P < 0.01, significantly different from a ratio of 1.0.


                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Calu-3 cells differentially alter Vi and Cli in response to alpha 1-adrenergic agonist and to hyperosmotic stress

Pretreatment of cell monolayers with bumetanide, an inhibitor of NKCC1, blocked the methoxamine-induced increase in the ratios of Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and Vi (Fig. 1) and increased Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and Vi (Table 2). These results indicate that NKCC1 is required for the effects of methoxamine on Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and Vi.

Modulation of Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and Vi values by hyperosmotic stress. The results observed with methoxamine suggest that activation of NKCC1 results in elevated Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> values and cell swelling associated with elevated Vi values. Vi is also theoretically regulated, independent of Cl<UP><SUB>i</SUB><SUP>−</SUP></UP>, by environmental stimuli such as hyperosmotic stress. In an earlier study (8), we reported that rabbit tracheal epithelial cells responded to hyperosmotic stress with elevated NKCC1 activity. Here we investigated the response to hyperosmotic stress on Vi in human airway epithelial cells by adding sufficient sucrose to Calu-3 cells to increase medium osmolarity to 500 mosmol/kgH2O. Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and Vi levels were measured over a 30-min time frame as described in the Fig. 1 and Table 2 legends. After 1 min of exposure to sucrose, Vi values decreased 9.8% to 10.1 ± 0.6 µl/mg of protein and Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> values increased 17.7% to 530.2 ± 53.6 nmol/mg of protein (Table 2). Elevations in Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> reached a maximum of 18.3% after 5 min of stimulation and remained elevated at 10 min (Fig. 2A and Table 2). Figure 2B shows that basolateral addition of sucrose significantly decreased Vi levels for periods up to 10 min. The ratios of Vi and Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> returned to baseline levels after 30 min of exposure to hyperosmotic medium. Bumetanide pretreatment blocked hyperosmotic-induced elevations in Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and prolonged the return of Vi values to baseline levels (Fig. 2B). These results indicate that NKCC1 activity is necessary for the observed increase in Cl<UP><SUB>i</SUB><SUP>−</SUP></UP>. In addition, the data demonstrate that the Calu-3 cells still retained the ability to shrink after addition of sucrose.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2.   Hyperosmotic induced alterations in Cl- (A) and Vi (B) in Calu-3 cells. Calu-3 cells were preincubated for 60 min with 0.4 µCi of [14C]urea and 1.0 µCi of 36Cl then (where indicated) with bumetanide (black-triangle) for 30 min. At time 0, sufficient sucrose (, black-triangle) was added to the basolateral bathing solution to increase osmolarity to 500 mosmol/kgH2O. Radioactive fluxes were terminated at the specified times and radioactive tracer was quantitated. Data for stimulated cells were calculated as a ratio of Cl- and Vi in stimulated cells divided by Cl- and Vi in unstimulated cells, respectively. Values are means ± SE for 4-5 separate filters. *P < 0.03; **P < 0.001, significantly different from a ratio of 1.0.

To gain more insight into the response to hyperosmotic stress, we examined mean values for Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and Vi from the set of experiments depicted in Fig. 2. These data are presented in Table 2. In the presence of bumetanide, Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> and [Cl-]i levels do not significantly change, but there is a further decrease in Vi to 9.1 ± 1.1 µl/mg of protein. These results suggest that Calu-3 cells preserve [Cl-]i levels by adjusting Vi quantities (Table 2). Treatment with sucrose alone for >1 min resulted in elevated Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> values. After 5 and 10 min of treatment, Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> remained elevated at 517.2 ± 30.1 (n = 4) and 508.9 ± 38.9 nmol/mg of protein (n = 5), respectively. After prolonged treatment for 30 min, Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> decreased to baseline levels of unstimulated cells of 450.7 ± 45.8 nmol/mg of protein (n = 4). Over this same time frame, Vi values declined to 9.7 ± 0.4 (n = 4) and 8.2 ± 0.5 µl/mg of protein (n = 5) at 5 and 10 min of treatment with sucrose, respectively. After 30 min of exposure to sucrose, Vi was 10.2 ± 0.7 µl/mg of protein (n = 4), a value that is not significantly different from the baseline Vi value in unstimulated cells. These results indicate that hyperosmotic stress, induced with sucrose, transiently decreases Vi and elevates Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> values in Calu-3 cells.

Modulation of NKCC1 activity by [Cl-]i. To evaluate the effects of [Cl-]i on cell function in the absence of cell volume changes, we measured NKCC1 activity as bumetanide-sensitive, 86Rb-unidirectional, basolateral-to-apical flux in nystatin-permeabilized Calu-3 cell monolayers grown on filter inserts. The apical perfusion medium was adjusted to match intracellular Na+, K+, and Mg2+ concentrations, and the [Cl-] in a basolateral perfusion medium was held constant at 136 mM. The data of Fig. 3 demonstrate that NKCC1 activity was detected when [Cl-]i was as low as 10 mM and as high as 200 mM in cells that were treated with vehicle of HPSS. NKCC1 activity levels measured at [Cl-]i from 10 to 140 mM were not significantly different, which indicates that an electrochemical gradient across the basolateral membrane is not sufficient for stimulation of NKCC1 function. Surprisingly, at [Cl-]i of 200 mM, bumetanide-sensitive K+ flux increased significantly to 281 nmol · mg protein-1 · 10 min-1 (P < 0.001; ANOVA), which accounts for 35.8% of total K+ flux. Bumetanide-insensitive K+ flux did not significantly change with increasing [Cl-]i, which indicates that elevated K+ flux at 200 mM could be attributed to NKCC1 activity. A comparison of NKCC1 activity at 200 and 40 mM Cl<UP><SUB>i</SUB><SUP>−</SUP></UP>, which mimics in vivo endogenous [Cl-]i of 45 mM (Table 2), revealed a difference in bumetanide-sensitive baseline K+ flux. At 40 mM Cl<UP><SUB>i</SUB><SUP>−</SUP></UP>, baseline NKCC1 activity was 40.6 ± 11 nmol · mg protein-1 · 10 min-1 (n = 4) and comprised 9.1% of total K+ flux. In comparison, baseline NKCC1 activity at 200 mM Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> increased 7.0-fold and the portion of total flux attributed to NKCC1 increased 4.0-fold.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of Cl- on activity of Na-K-Cl cotransporters (NKCC1). Apical surface of Calu-3 cells was permeabilized in medium containing 225 U of nystatin/ml and an ionic composition that matched the cytosol except that the Cl- concentration ([Cl-]i) varied from 10 to 200 mM. [Cl-]i of the basolateral perfusion medium was held constant at 136 mM. Paired cell monolayers were pretreated on the basolateral surface with HEPES at pH 7.4 (HPSS) or with 10 µM bumetanide for 20 min before basolateral addition of 86Rb. After a baseline basolateral-to-apical flux was established, cells were stimulated by addition of methoxamine (solid bars) or as a control, HPSS (open bars) to the basolateral medium. Sampling of apical medium continued for 10 min. Data were calculated as differences in paired values for K+ flux in cells treated with or without 10 µM bumetanide and are reported as means ± SE of 3-7 separate experiments. *P < 0.05, paired comparison of cells treated with vehicle or methoxamine; #P < 0.001, compared with vehicle-treated cells only.

Methoxamine significantly increased activity of NKCC1 in a biphasic manner with peak stimulated K+ flux at 20-40 and 140-200 mM Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> (Fig. 3). Increasing Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> from 20 to 80 mM still supported agonist-stimulated NKCC1 function; however, net activity, defined as methoxamine-stimulated NKCC1 minus unstimulated NKCC1, decreased with increasing Cl<UP><SUB>i</SUB><SUP>−</SUP></UP>. When NKCC1 activity was calculated as a percentage of total K+ flux, the contribution of NKCC1 activity increased 2.8-fold at 20 mM Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> from 19.5 to 55.1%, 2.5-fold at 40 mM Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> from 14.5 to 36.7%, and 2.2-fold at 140 mM Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> from 22.5 to 48.7%. We also note that K+ flux at 200 mM Cl<UP><SUB>i</SUB><SUP>−</SUP></UP>, although significantly elevated by high Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> levels alone, increased 1.6-fold after the addition of methoxamine. This high Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> value has been reported to prevent bumetanide-sensitive K+ influx (6), yet our results suggest an additive response to a combination of high [Cl-]i and methoxamine.

We previously demonstrated (11) that activation of NKCC1 by alpha 1-adrenergic stimulation requires an increase in the activity of PKC-delta , a PKC isotype that is selectively inhibited by rottlerin. The sensitivity of methoxamine-stimulated K+ flux to rottlerin was examined at apical [Cl-]i of 20, 140, or 200 mM. Pretreatment with rottlerin blocked methoxamine-stimulated K+ flux at each [Cl-]i tested. In this series of experiments, a rottlerin-sensitive K+ flux of 147.7 ± 27 nmol/mg of protein (n = 4) at 20 mM accounted for 32.2 ± 6% (n = 4) of stimulated flux, or ~60% of bumetanide-sensitive K+ flux. At a [Cl-]i of 140 mM, rottlerin blocked 72.6 ± 15 nmol/mg of protein (n = 5) of methoxamine-stimulated K+ flux. The rottlerin-sensitive flux accounted for 44.5 ± 7% (n = 5) of stimulated flux and was comparable to a 34.3 ± 12% (n = 3) of bumetanide-sensitive K+ flux. Rottlerin-sensitive K+ flux at a [Cl-]i of 200 mM was 498.4 ± 66 nmol/mg of protein (n = 7), which is 36.2 ± 6% (n = 7) of stimulated K+ flux. At this high Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> value, the rottlerin-sensitive methoxamine-stimulated flux was not significantly different from a bumetanide-sensitive methoxamine-stimulated flux of 345.8 ± 50 nmol/mg of protein (n = 5), which represented 49.6 ± 3% (n = 5) of stimulated K+ flux. These results support our earlier conclusion that activity of PKC-delta is necessary for activation of NKCC1 (13).

Modulation of in vivo PKC activity by [Cl-]i. We have previously shown (11-13) that hormone stimulation rapidly increases activity of PKC-delta and -zeta and that the increased activity of PKC-delta is necessary for activation of NKCC1. Our results from this study indicated that methoxamine also increases Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> (see Fig. 1), which suggests the possibility that activity of PKC-delta might be sensitive to Cl<UP><SUB>i</SUB><SUP>−</SUP></UP>. To evaluate this possibility, [Cl-]i was varied in permeabilized cell monolayers using nystatin as described (see Measurement of NKCC1 activity). Increasing apical [Cl-]i from 10 to 200 mM did not significantly affect baseline activity of PKC-delta in cells that were treated with vehicle (Fig. 4).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of [Cl-]i on activity of protein kinase C (PKC)-delta . Apical surfaces of Calu-3 cells were permeabilized in medium containing 225 U of nystatin/ml and an ionic composition that matched the cytosol except that [Cl-]i varied from 10 to 200 mM. To stimulate cells, methoxamine (solid bars) or HPSS (vehicle; open bars) was added to the basolateral medium. After a 5-min incubation, stimulation was terminated by rapid immersion of filters in ice-cold PBS. PKC-delta was immunoprecipitated, and kinase activity of immune complexes was measured. Numbers above bars represent the differences between methoxamine-stimulated activity and vehicle. Data are reported as means ± SE of 3-5 separate experiments. *P < 0.05; **P < 0.01; ***P < 0.004, compared with cells treated with vehicle.

Basolateral application of the alpha 1-adrenergic agonist methoxamine induced a biphasic increase in activity of PKC-delta (Fig. 4). Peak levels were reached at [Cl-]i values of 20 and 140-200 mM. At a [Cl-]i of 20 mM, methoxamine-stimulated PKC-delta activity was significantly different compared with other Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> values (P < 0.03). At this Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> level, methoxamine increased the activity of PKC-delta by 2.4-fold, which corresponded to a 2.8-fold increase in NKCC1 activity. Indeed, for each increase in Cl<UP><SUB>i</SUB><SUP>−</SUP></UP>, which supported a significant increase in agonist-stimulated activity of PKC-delta , there was also a significant increase in agonist-stimulated activity of NKCC1. The results indicate that, in the absence of cell shrinkage, there is a parallel stimulation of PKC-delta and NKCC1.

Subcellular distribution of PKC isotypes in Calu-3 cells. Our previous studies (11-13) of PKC-delta distribution in human tracheal epithelial cells localized PKC-delta to both cytosol and a Triton X-100 soluble particulate fraction. We examined the subcellular distribution of PKC-delta and, because its activity also increases after alpha 1-adrenergic stimulation (10), we also investigated the distribution of PKC-zeta in Calu-3 cells. The results are illustrated in Fig. 5. At the endogenous [Cl-]i of 47 mM, 54.5% of PKC-delta and 37.6% of PKC-zeta were localized to a particulate fraction. Decreasing Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> to 20 mM decreased the particulate localization of both PKC isotypes, and increasing Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> to 200 mM significantly increased particulate PKC-delta and -zeta levels. Methoxamine increased particulate PKC-delta at 20 mM Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> to 76.1% and PKC-zeta to 70.9%. Similarly, at 47 mM Cl<UP><SUB>i</SUB><SUP>−</SUP></UP>, particulate PKC-delta and -zeta levels increased after methoxamine stimulation to 81.4 and 62.1%, respectively. At 200 mM Cl<UP><SUB>i</SUB><SUP>−</SUP></UP>, the amount of each isotype localized to a particulate fraction did not change after methoxamine stimulation, which indicates that maximal translocation of a PKC isotype to a particulate fraction had already occurred.


View larger version (46K):
[in this window]
[in a new window]
 
Fig. 5.   Subcellular distribution of PKC-delta and -zeta in Calu-3 cells. Apical surface of Calu-3 cells was permeabilized with nystatin in perfusion medium of varying [Cl-]i as indicated on the x-axis. Cell monolayers were harvested in hypotonic buffer and fractionated into cytosol and a particulate Triton X-100 soluble fraction. Proteins from a 20-µg aliquot of cell fraction were separated by gel electrophoresis on 4-15% gradient slab gels and transferred to Immobilon-P paper. A: immunoblots of cytosol (C) and particulate (P) fractions were probed with polyclonal antibodies to PKC-delta or -zeta . Exposed X-ray films were analyzed by laser densitometry. Percent particulate mass was calculated from arbitrary units as 100 × particulate/(cytosol + particulate). B: distribution of PKC-delta (hatched bars) and PKC-zeta (solid bars) is reported as means ± SE for experiments performed on 4 separate cell cultures. Addition of 10 µM methoxamine (open bars) increased the percent particulate mass of PKC-delta and -zeta at [Cl-]i of 20 and 47 mM. *P < 0.02; **P < 0.003, compared with [Cl-]i of 47 mM.

Simultaneous alpha -adrenergic activation of NKCC1 and PKC-delta at a specific Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> level suggests that PKC-delta and NKCC1 might be closely associated. To examine this possibility, we immunoprecipitated NKCC1 and probed the immune complexes for PKC-delta and vice versa using Western blot analysis. The results, shown in Fig. 6, demonstrate that at least seven protein bands ranging from 32 to 190 kDa are detected in the immune complexes of NKCC1. NKCC1 is detected as a broad band between 130 and 170 kDa. The lower molecular mass band might represent an incompletely glycosylated protein. The higher molecular mass band is thought to represent a fully glycosylated cotransporter delivered to the plasma membrane (20). A predominant band at 75 kDa corresponds to the molecular mass of most PKC isotypes. Indeed, PKC-delta was detected in immunoprecipitates of NKCC1 (Fig. 6). More importantly, NKCC1 was detected in immunoprecipitates of PKC-delta . In these experiments, coimmunoprecipitation of PKC-delta and NKCC1, taken together with previous reports of alpha -adrenergic effects on PKC-delta activity and subcellular localization that coincide with alpha -adrenergic stimulation of NKCC1 function (11, 13), indicates a physiologically relevant association.


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 6.   PKC-delta coimmunoprecipitates with NKCC1. Cell monolayers were grown to confluence on filter inserts and were serum deprived overnight. Total cell lysates were prepared in NKCC1 lysis buffer. PKC-delta and NKCC1 immunoprecipitated from equal aliquots of total cell lysate. Immune complexes were subjected to SDS-PAGE on 4-15% gradient slab gels. Protein stain was performed using a silver stain kit. Western blot analyses for NKCC1 and PKC-delta in immune complexes (IP) and total cell lysates (LYS) are typical for 3 independent experiments. In the absence of primary antibody, there were no detectable protein bands that were immunoreactive with antisera. Pretreatment of total cell lysates with agarose beads before addition of primary antibody to reduce nonspecific binding yielded the same results.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

[Cl-]i has been linked to regulation of NKCC1 in a number of cells and tissues including secretory epithelia, avian erythrocytes, and squid axon (1, 6, 24). This study of Calu-3 airway epithelial cells demonstrates for the first time that a Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> electrochemical gradient alone is not sufficient to stimulate NKCC1 activity; rather, elevated activity of PKC-delta is necessary. Increasing [Cl-]i, independent of changes in cell volume, correlates with shifts in the subcellular localization of PKC-delta and -zeta (see Fig. 5) and in the activity and regulation of PKC-delta and NKCC1 by the alpha 1-adrenergic agonist methoxamine (see Figs. 3 and 4). Titration of [Cl-]i from 10 to 140 mM in permeabilized Calu-3 cells does not alter the baseline activity of NKCC1 except at [Cl-]i of 200 mM. At this high [Cl-]i level, baseline NKCC1 activity increased significantly. This response differs from other reports, which describe cotransport as strongly dependent on [Cl-]i (6, 21). In these other systems, the dependence on [Cl-]i is biphasic and shows stimulation with increasing [Cl-]i from 0 to 40 mM followed by inhibition. The response of Calu-3 cells cannot be explained by a change in cell volume (see Table 1) or by an increase in PKC-delta activity (see Fig. 4). One explanation could be the sensitivity of NKCC1-regulated K+ flux to intracellular electrolyte concentration, or conversely water content, which has been postulated to reflect the dependence of thermodynamic activity of intracellular proteins on total protein concentration or macromolecular crowding (18). Macromolecular crowding decreases the dissociation rate constants of protein complexes leading to prolonged formation of complexes (23) such as F actin (14) and kinases/phosphatases (18) that regulate electrolyte exchange and cotransport pathways. This could explain, for example, an increase in rottlerin-sensitive baseline NKCC1 activity of almost sixfold at a [Cl-]i of 200 mM. Rottlerin blocks methoxamine-stimulated NKCC1 regardless of [Cl-]i, which indicates that PKC-delta is required for activation of NKCC1 and that hormonal activation is additive to the effects of high [Cl-]i alone.

Elevating [Cl-]i to 200 mM also enhanced translocation of PKC-delta and -zeta from the cytosol to the cell periphery (see Fig. 5). Translocation did not, however, lead to a significant increase in enzyme activity, nor did it prevent a methoxamine-induced increase in the activity of PKC-delta (see Fig. 4). These results indicate that Calu-3 cells resemble human tracheal epithelial cells (13) and a CF/T43 airway epithelial cell line (12), which exhibit an approximately even distribution of PKC-delta between the cytosol and a particulate fraction and a predominantly cytosolic localization of PKC-zeta . With Calu-3 cells, we speculate that macromolecular crowding at high [Cl-]i promotes protein-protein interactions, which lead to the association of PKC with a particulate subcellular fraction with slight or no increase in PKC activity. One indication of a protein-protein interaction is the coimmunoprecipitation of PKC-delta with NKCC1 and vice versa (see Fig. 6). Hormonal stimulation at varying [Cl-]i produced distinct effects on PKC-delta activity. At [Cl-]i of <140 mM, methoxamine increased the PKC association with a particulate fraction and rottlerin-sensitive NKCC1 activity and, in addition, increased the activity of PKC-delta . However, at [Cl-]i of >140 mM, the response to methoxamine is additive to the effects of high [Cl-]i and retains a sensitivity to rottlerin, which suggests a PKC-delta -dependent activation of membrane-associated NKCC1 or a separate pool of NKCC1. Aspects of this model have yet to be tested in airway epithelial cells.

The [Cl-]i in Calu-3 cells was 47.4 mM, as measured by radioisotopic equilibrium distribution of 36Cl (see Table 2). This is comparable to the [Cl-]i values reported for other secretory epithelia, including shark rectal gland (49 mM, Ref. 5), dog tracheal epithelial cells (47.2 mM, Ref. 25), and submandibular acinar cells (56 mM, Ref. 28). In these cells, stimulation with cAMP-generating agents increased transepithelial flux, which resulted in secretion. cAMP treatment of shark rectal gland decreased [Cl-]i from 49 to ~40 mM (5) and reduced dog tracheal epithelial cell [Cl-]i from 47.2 to 32.2 mM (25). However, when activity of Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> is accounted for, the actual decrease in [Cl-]i is much less than needed to activate NKCC1. A similar finding by Robertson and Foskett (22) using a rat salivary acinar cell preparation was consistent with the conclusion that a decrease in Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> is necessary but not sufficient for activation of NKCC1. In our studies, we isolated the activation of NKCC1 from cAMP-dependent secretion by using a NKCC1-selective activator, methoxamine. Our results indicate, for the first time, that alpha 1-adrenergic activation of NKCC1 is sensitive to a fall in [Cl-]i, but requires stimulation of PKC-delta activity. The results have important implications for diseases such as cystic fibrosis in which the apparent secretory capacity of airway epithelium is defective. Studies by Slotki and colleagues (27) using CFPAC cells, which are derived from pancreatic adenocarcinoma of a cystic fibrosis patient, demonstrate that secretory capacity can be limited by low cotransporter activity. This implies that genetic approaches to correct cystic fibrosis transmembrane conductance regulator levels to improve secretory capacity will depend, for success, on optimal functioning of NKCC1. Understanding intracellular mechanisms leading to activation of NKCC1 is thus critical for successful development of new therapeutics to correct secretion in cystic fibrosis.

From previous studies (9, 10), we predicted agonist-induced increases in Vi and Cl<UP><SUB>i</SUB><SUP>−</SUP></UP> due to increased influx via NKCC1. The data of Fig. 1 and Table 2 support this prediction and show in addition that NKCC1 expressed in Calu-3 cells can be activated by hyperosmotic stress (see Fig. 2), which is a response shared by epithelial cells of alveolus (2), kidney (26), colon (16), and pancreas (17). In addition, the data show that Calu-3 cells act as osmometers but lose water slowly. Inhibition of NKCC1 caused a prolonged recovery of Vi (see Fig. 2); hence, [Cl-]i appears to be necessary for recovery from hyperosmotic stress. In cystic fibrosis and other pulmonary diseases, an increased osmolarity of a mucociliary fluid layer, due to lack of sufficient fluid secretion, hypersecretion of proteins or mucus, bacterial infection, or an enhanced inflammatory response, might induce alterations in epithelial [Cl-]i that limit the level of NKCC1 activity with consequences for the ability of epithelial cells to mount a defense through increased fluid secretion. Successful therapeutic approaches to restore or stimulate secretory capacity must take into account any sensitivity toward Cl<UP><SUB>i</SUB><SUP>−</SUP></UP>-sensitive regulation of NKCC1 to achieve optimal secretion.


    ACKNOWLEDGEMENTS

The authors thank Dr. Calvin Cotton for helpful discussions.


    FOOTNOTES

This research was supported by National Institutes of Health Grant HL-58598.

Address for reprint requests and other correspondence: C. M. Liedtke, Pediatric Pulmonology, Case Western Reserve Univ., BRB, Rm. 824, 2109 Adelbert Rd., Cleveland, OH, 44106-4948 (E-mail: cxl7{at}po.cwru.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.

First published December 14, 2001;10.1152/ajplung.00143.2001

Received 25 April 2001; accepted in final form 11 December 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Breitwieser, GE, Altamirano AA, and Russell JM. Osmotic regulation of Na+-K+-Cl- cotransport in squid giant axon is [Cl-]i dependent. Am J Physiol Cell Physiol 258: C749-C753, 1990[Abstract/Free Full Text].

2.   Clerici, C, Couette S, Loiseau A, Herman P, and Amiel C. Evidence for Na-K-Cl cotransport in alveolar epithelial cells: effect of phorbol ester and osmotic stress. J Membr Biol 147: 295-304, 1995[ISI][Medline].

3.   D'Andrea, LC, Lytle C, Matthews JB, Hofman P, Forbush B, III, and Madara JL. Na/K/2Cl cotransporter protein of intestinal epithelial cells: surface distribution, immunoprecipitation as a protein complex, and surface expression in response to cAMP. J Biol Chem 271: 28969-28976, 1996[Abstract/Free Full Text].

4.   Devor, DC, Singh AK, Lambert LC, DeLuca A, Frizzell RA, and Bridges RJ. Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells. J Gen Physiol 113: 743-760, 1999[Abstract/Free Full Text].

5.   Greger, R, Schlatter E, Wang F, and Forrest JN, Jr. Mechanism of NaCl secretion in rectal gland tubules of spiny dogfish (Squalus acanthias). III. Effects of simulation of secretion by cyclic AMP. Pflügers Arch 402: 376-384, 1984[ISI][Medline].

6.   Haas, M, and McBrayer DG. Na-K-Cl cotransport in nystatin-treated tracheal cells: regulation by isoproterenol, apical UTP, and [Cl-]i. Am J Physiol Cell Physiol 266: C1440-C1452, 1994[Abstract/Free Full Text].

7.   Haas, M, McBrayer D, and Yankaskas J. Dual mechanisms for Na-K-Cl cotransport regulation in airway epithelial cells. Am J Physiol Cell Physiol 264: C189-C200, 1993[Abstract/Free Full Text].

8.   Liedtke, CM. Bumetanide-sensitive Na+ and Cl- uptake in rabbit tracheal epithelial cells is stimulated by neurohormones and hypertonicity. Am J Physiol Lung Cell Mol Physiol 262: L621-L627, 1992[Abstract/Free Full Text].

9.   Liedtke, CM. Role of protein kinase C in alpha -adrenergic regulation of NaCl(K) cotransport in human airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 268: L414-L423, 1995[Abstract/Free Full Text].

10.   Liedtke, CM, Cody D, and Cole TS. Differential regulation of Cl- transport proteins by PKC in Calu-3 cells. Am J Physiol Lung Cell Mol Physiol 280: L739-L747, 2001[Abstract/Free Full Text].

11.   Liedtke, CM, and Cole T. Antisense oligodeoxynucleotide to PKC-delta blocks alpha 1-adrenergic activation of Na-K-2Cl cotransport. Am J Physiol Cell Physiol 273: C1632-C1640, 1997[Abstract/Free Full Text].

12.   Liedtke, CM, and Cole T. PKC signalling in CF/T43 cell line: regulation of NKCC1 by PKC-delta . Biochim Biophys Acta 1495: 24-33, 2000[ISI][Medline].

13.   Liedtke, CM, Cole T, and Ikebe M. Differential activation of PKC-delta and PKC-zeta by alpha 1-adrenergic stimulation in human airway epithelial cells. Am J Physiol Cell Physiol 273: C937-C943, 1997[Abstract/Free Full Text].

14.   Lindner, RA, and Ralston GB. Macromolecular crowding: effects on actin polymerisation. Biophys Chem 66: 57-66, 1997[ISI][Medline].

15.   Lyttle, C, Xu JC, Biemesderfer D, and Forbush B, III. Distribution and diversity of Na-K-Cl cotransport proteins: a study with monoclonal antibodies. Am J Physiol Cell Physiol 269: C1496-C1505, 1995[Abstract/Free Full Text].

16.   Matthews, JB, Smith JA, Mun EC, and Sicklick JK. Osmotic regulation of intestinal epithelial Na+-K+-Cl- cotransport: role of Cl- and F-actin. Am J Physiol Cell Physiol 274: C697-C706, 1998[Abstract/Free Full Text].

17.   Miley, HE, Holden D, Grint R, Best L, and Brown PD. Regulatory volume increase in rat pancreatic beta -cells. Eur J Physiol 435: 227-230, 1998[ISI][Medline].

18.   Parker, JC, Dunham PB, and Minton AP. Effects of ionic strength on the regulation of Na/H exchange and K-Cl cotransport in dog red blood cells. J Gen Physiol 105: 677-699, 1995[Abstract].

19.   Payne, JA, and Forbush B, III. Molecular characterization of the epithelial Na-K-Cl cotransporter isoforms. Curr Opin Cell Biol 7: 493-503, 1995[ISI][Medline].

20.   Payne, JA, Xu JC, Haas M, Lytle CY, Ward D, and Forbush B. Primary structure, functional expression, and chromosomal localization of the bumetanide-sensitive Na-K-Cl cotransporter in human colon. J Biol Chem 270: 17977-17985, 1995[Abstract/Free Full Text].

21.   Putney, LK, Vibat CRT, and O'Donnell ME. Intracellular Cl regulates Na-K-Cl cotransport activity in human trabecular meshwork cells. Am J Physiol Cell Physiol 277: C373-C383, 1999[Abstract/Free Full Text].

22.   Robertson, MA, and Foskett JK. Na+ transport pathways in secretory acinar cells: membrane cross talk mediated by [Cl-]i. Am J Physiol Cell Physiol 267: C146-C156, 1994[Abstract/Free Full Text].

23.   Rohwer, JM, Postma PW, Kholodenko BN, and Westerhoff HV. Implications of macromolecular crowding for signal transduction and metabolite channeling. Proc Natl Acad Sci USA 95: 10547-10552, 1998[Abstract/Free Full Text].

24.   Russell, JM. Sodium-potassium-chloride cotransport. Physiol Rev 80: 211-276, 2000[Abstract/Free Full Text].

25.   Shorofsky, SR, Field M, and Fozzard HA. Mechanism of Cl secretion in canine trachea: changes in intracellular chloride activity with secretion. J Membr Biol 81: 1-8, 1984[ISI][Medline].

26.   Simmons, NL, and Tivey DR. The effect of hyperosmotic challenge upon ion transport in cultured renal epithelial layers (MDCK). Pflügers Arch 421: 503-509, 1992[ISI][Medline].

27.   Slotki, IN, Breuer WV, Greger R, and Cabantchik ZI. Long-term cAMP activation of Na+-K+-Cl- cotransporter activity in HT-29 human adenocarcinoma cells. Am J Physiol Cell Physiol 264: C857-C865, 1993[Abstract/Free Full Text].

28.   Zeng, W, Lee MG, and Muallem S. Membrane-specific regulation of Cl- channels by purinergic receptors in rat submandibular gland acinar and duct cells. J Biol Chem 272: 32956-32965, 1997[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 282(5):L1151-L1159
1040-0605/02 $5.00 Copyright © 2002 the American Physiological Society