Studies on the mechanism of short-term regulation of pulmonary artery endothelial cell Na/K pump activity

Studies on the mechanism of pulmonary artery endothelial

short-term regulation of cell Na/K pump activity

AMOS CHARLES, DOLORETTA D. DAWICKI, EBEN OLDMIXON, CHARLES KUHN, MICHAEL CUTAIA, and SHARON ROUNDS

PROVIDENCE, RHODE ISLAND

The Na/K pump is critically important in maintenance of cell homeostasis in the face of injury. Little is known about the regulation of endothelial cell Na/K-pump activity. We previously reported that short-term (30-minute) oxidant-induced endothelial cell perturbation increased Na/K-pump activity in intact monolayers of bovine pulmonary artery endothelial cells (BPAECs). In this study we investigated the mechanism of oxidant-induced increases in endothelial Na/K-pump activity, focusing on short-term modulation of ~1-pump subunit. By using immunofluorescence microscopy and confocal scanning laser microscopy, we found =i subunit on both apical and basal aspects of BPAECs without polarized distribution. Short-term (30-minute) incubation of PAEC monolayers with H202 (I mmol/L) did not change the relative amounts of =~ subunit in membrane fractions, as assessed by immunoblotting. Phosphorylation of the =~ subunit also was not affected by H202 treatment. Because protein kinases have been reported to alter Na/K-pump activity in several tissues and because H202 has been reported to increase PKC activity of endothelial cells, we determined the effects of inhibition and activation of protein kinase C (PKC) on Na/K-pump activity quantitated as ouabain-inhibitable uptake of 86Rb. We also determined the effects of PKC activation and inhibition on H202-induced increases in Na/K-pump activity. Inhibitors of PKC increased Na/K-pump activity over a 30-minute period in intact monolayers. Inhibition or depletion of PKC did not prevent H202-induced increases in pump activity. These results indicate that PKC is an endogenous regulator of pulmonary artery endothelial cell Na/K-pump activity but that the effects of H202 are not mediated by activation of PKC or by changes in the expression or phosphorylation of =~ subunit. (J Lab Clin Med 1997; 130:157-68)

Abbreviations: ATP = adenosine triphosphate; BPAEC = bovine pulmonary artery endothelial cells; D1T : dithiothreitol; ECL = enhanced chemiluminescence; H7 = 1-(5-isoquinolinylsulfo- nyl)-2-methylpiperazine; HPAEC = human pulmonary artery endothelial cells; MEM = minimal essential medium; Na/K pump = sodium/potassium adenosine triphosphatase; PBS = phosphate-buffered saline; PDBU = phorbol-12,13-dibutyrate; PKC = protein kinase C; PMSF = phenylmethylsulfonyl fluoride; SDS-PAGE = sodium dodecylsulfate-polyacrylamide gel electrophoresis

From the Pulmonary and Critical Care Medicine Section, De- partments of Medicine and Pathology, Brown University School of Medicine, Providence Veterans Affairs Medical Center, and Memorial Hospital of Rhode Island, Providence.

Supported by Merit Review grants from the Department of Vet- erans Affairs (S. Rounds and M. Cutaia), by HL-34009 and HL-26863 from the National Heart Lung and Blood Institute of the National Institutes of Health, by a research grant from the Cystic Fibrosis Foundation (S. Rounds), and by a grant from the Dean, Brown University School of Medicine.

Presented in preliminary form at the May 1994 annual meeting of the American Thoracic Society and published in abstract form (Am J Respir Crit Care Med 1994;149:A296).

Submitted for publication Jan. 23, 1997; revision submitted Feb. 20, 1997; accepted March 26, 1997.

Reprint requests: Sharon Rounds, Pulmonary and Critical Care Medicine Section, Providence VA Medical Center, 830 Chalk- stone Ave., Providence, RI 02908.

0022-2143/97 $5.00 + 0 5/1/82137

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J Lab Clin Med 158 Charles et al. August 1997

Maintenance of normal intracellular/extracellular Na ÷ and K ÷ gradients is of vital importance to all mammalian cells. The Na ÷ gradient provides energy for Na+-coupled transport of nutrients into cells, including transport of glucose and amino acids. Gra- dients of Na + and H + govern the movement of other ions, such as Ca 2÷, across cell membranes. Osmotic balance and cell volume are dependent on normal ion gradients. The Na/K pump (E.C. 3.6.1.3) is primarily responsible for maintenance of Na + and K ÷ gradients. 1 Thus function of the Na/K pump is critically important in cell homeostasis in the face of injury. Maintenance of cell homeostasis is a crucial component of endogenous mechanisms of resis- tance to cell injury.

The Na/K pump is a glycosylated heterodimer that traverses the plasma membrane of nearly all mammalian cells. 1 The pump functions in a cyclic manner, transporting two moles of K + intracellu- larly for every three moles of Na + moved extracel- lularly, a process requiring Mg 2÷ as a cofactor and the hydrolysis of ATP. 1 During its cycle the pump is phosphorylated at aspartate376 of the e~ subunit (catalytic site phosphorylation). The pump is a heterodimer consisting of e~ and [3 subunits, which have been cloned and sequenced. 2'3 The oL subunit exists in %, %, and % isoforms and is the site of cation and ouabain binding and of catalytic-site phosphorylation during activation. 3 The glycosylated, smaller [~ subunit may be important in stabilization of the pump in the cell membrane. 2 A y subunit of un- known function also has been reported. 3

The activity of the Na/K pump can be controlled in both long- and short-term fashions. Long-term control involves regulation of tissue expression of subunit isoforms, such as that occurring in type II epithelial cells under hyperoxic conditions. 4'5 Short- term regulation may be caused by changes in intracellular Na + concentration. 6'7 In addition, short- term (-<30 minute) alterations in Na/K-pump activity may be mediated by changes in subcellular distribution of pump units, by reversible noncatalytic-site phosphorylation of the e~ subunit, or by changes in association of the pump with cytoskeletal structures. 6'v Factors reported rapidly to modulate Na/K-pump activity in various cells include phorbol esters and a variety of hormones. 6'7

Little is known about the function or modulation of Na/K-pump activity in vascular endothelial cells. We found that oxidant-induced endothelial cell perturbation increased Na/K-pump activity in intact monolayers of cultured BPAECs, an effect observed after only 30 minutes of exposure to H202 or to xanthine/xanthine oxidase. 8 This effect was associated with in-

creased 22Na influx, with increased cycling of the pump, and with a decrease in Bma x for ouabain binding. Elliott and Schilling 9 reported similar results with the tert-butyl hydroperoxide model of endothelial cell injury, as did Robison and Kim 1° with the NO2 model of airway epithelial injury. Thus the H202 model of endothelial cell injury provides a convenient model for assessing short-term regulation of NaN-pump activity. This model of endothelial cell injury is relevant to pathophysiologic states in that oxidant-induced endothelial injury is thought to be important in the pathogenesis of acute lung injury mediated by leukocytes, such as that caused by sepsis.

Because little is known regarding the distribution of the vascular endothelial Na/K pump, we used the a subunit as an immunologic marker for the Na/K pump to characterize the distribution of the pump in cultured pulmonary vascular endothelial cells. In preliminary studies, we did not detect the ~2 subunit in cultured endothelial cells by ECL/Western blotting, so subsequent studies focused on the % subunit. In these studies we assessed the distribution of the % subunit on endothelial cell membrane and cytoplasm by using immunofluorescence microscopy and confocal scanning laser microscopy.

We hypothesized that H202 increased Na/K- pump activity by short-term alterations in membrane expression or % subunit phosphorylation or both. We investigated whether incubation with H202 changed the cell-membrane expression of the % subunit by comparing immunoblots of cell-membrane preparations. To determine whether H202 altered phosphorylation of the % subunit, we compared phosphorylation of % subunit immunoprecipitates from monolayers that had been incubated with H202.

Because protein kinases have been reported to alter Na/K-pump activity in several tissues, 6'7 we determined the effects of inhibition and activation of PKC on Na/K-pump activity, as assessed by ouabain-inhibitable uptake of S6Rb by intact monolayers. Finally we determined whether the effects of H202 on Na/K-pump activity might be the result of effects on PKC by assessing the effects of PKC activation and inhibition on the H202-induced increase in ouabain-inhibitable S6Rb uptake.

METHODS

Materials. Minimal essential medium and endothelial cell culture additives were obtained from GIBCO (Gaith-

An editorial relevant to this article appears on p. 119 of this issue of the Journal.

J Lab Clin Med Volume 130, Number 2 Charles et al, 159

ersburg, Md.), unless otherwise noted. Ouabain, staurosporine, PDBU, and Cy3 were obtained from Sigma Chemical Co., St. Louis, Mo. The PKC inhibitor H7 was obtained from LC Laboratories, Woburn, Mass., and calphostin C was purchased from Calbiochem, La Jolla, Calif. Reagent grad e H202 was purchased from Aldrich Chemical, Milwaukee, Wis. Protein A conjugated to agarose was obtained from Pierce, Rockford, Ill. Immobilon was obtained from Millipore, Bedford, Mass. a6RbC1 and H332po4 were purchased from New England Nuclear, Boston, Mass. Polyclonal rabbit anti-rat antibody to cq subunit was obtained fi'om Upstate Biotechnologies, Lake Placid, N.Y., and mouse anti-dog monoclonal cq subunit antibody was a gift from Dr. Michael Caplan of Yale University.

Cell cultures. BPAECs were isolated from freshly slaughtered animals by using a scraping technique, which we described previously. 8 Cells were grown to confluence in minimal essential medium with 10% fetal bovine serum, penicillin G (100 U/ml), streptomycin (100 Ixg/ml), and amphotericin-B (0.25 ixg/ml).

HPAECs were obtained from Clonetics Corp., San Di- ego, Calif., and were maintained in defined endothelial cell growth medium containing 2% fetal bovine serum, obtained from Clonetics Corp. Endothelial cell growth medium contains the following additives: human epider- mal growth factor (10 ng/ml), hydrocortisone (1 ixg/ml), gentamicin (50 Ixg/ml), amphotericin-B (50 ng/ml), and bovine brain extract (12 ~g/ml).

Endothelial cells were passaged with 0.05% trypsin/0.5 mmol/L EDTA and grown to confluence in 100 mm plastic dishes or 16 mm culture wells of 24-well plates. Several different cell lines were used. There were no differences among the results from using cells of differing passage numbers or cell lines.

Endothelial cells were identified by typical cobblestone structure by using phase-contrast microscopy, by uptake of fluorescein-labeled acetylated low-density lipoprotein, and by display of factor VIII antigen by using immunofluorescence microscopy.

Immunofluorescence of ~1 subunit on BPAECs. Mona- layers of BPAECs were grown on glass coverslips coated with Cell-Tak (4 ixg/slide; Collaborative Biomedical Prod- ucts), permeabilized and fixed in methanol for 15 minutes at - 20 ° C, washed with phosphate buffered saline (PBS), and incubated overnight at 4 ° C with mouse monoclonal antibody to dog cq subunit. The staining characteristics of this antibody have been described previously. 11 Cultures were then incubated for 1 hour with goat anti-mouse secondary antibody, conjugated to the fluorescent probe, Cy3. Control cultures were incubated with mouse immunoglobulin G. Cultures were examined with a Nikon Op- tiphot microscope equipped with a super-high-pressure mercury vapor lamp for epifluorescence.

Confocal laser scanning microscopy of oh subunit on

BPAECs. Confocal laser scanning microscopy was used to assess the distribution of the cq subunit on the apical and basal aspects of cultured endothelial cells. BPAEC mona-

layers were fixed and immunolabeled, as described previously, and then examined by using a Molecular Dynamics confocal laser scanning microscope with Nikon Optics. A ×60 objective was used, giving 512 × 512 pixel images; the vertical advance between serial sections was 0.2 Ixm. Thus data voxels were 0.17 × 0.17 × 0.20 txm along the x, y, and z axes, respectively.

The datasets obtained comprised 30 serial sections spanning the entire thickness of the fixed cell monolayers and some of the supporting microscope slide. The data were visualized in two ways. First, the entire dataset was viewed along the z axis, giving an en face view of the monolayer. Next, bands of 30 × 512 voxels were selected from the same locations in each of 25 serial sections, reconstructed in three dimensions, and viewed from the side to give a lateral view of a 5 Ixm-thick section through the monolayer.

Effect of H202 on membrane distribution of ~I subunit. To determine whether H20 2 changed the membrane expression of the cq subunit, we compared immunoblots of SDS-PAGE-resolved membrane proteins. BPAECs and HPAECs were grown to confluence in 100 mm dishes and then incubated with H20 2 (1 mmol/L) in MEM for 30 minutes. In all experiments with H202, the concentration of H20 2 was verified spectrophotometrically by absor- bance at 240 nm (e = 43.6 M/cm). Control cultures were incubated with MEM alone.

At the end of the incubation period the monolayers were washed twice with ice-cold PBS, the cells harvested with a rubber policeman, suspended in ice-cold PBS, and centrifuged at 600 g for 5 minutes at 4 ° C. The cell pellets were resuspended in homogenization buffer containing 10 mmol/L TRIS, pH 7.5, with EDTA (5 mmol/L), EGTA (5 real/L), NaF1 (10 mol/L), PMSF (1 real/L), leupeptin (0.1 mol/L), Na vanadate (0.1 real/L), and aprotinin (3 ~mol/ L). The cell suspensions were homogenized in a Dounce homogenizer with 30 to 40 strokes. The homogenates were centrifuged at 1000 g for 10 minutes at 4°C to remove nuclei and unbroken cells. The supernatants were centrifuged at 100,000 g at 4 ° C for 90 minutes to separate the total membrane fraction from the soluble cytoplasmic fraction. The membrane fractions were washed twice with homogenization buffer, resuspended, and centrifuged at 100,000 g for 30 minutes.

The membrane protein fractions were solubilized in 1% SDS/62.5 mol/L TRIS, pH 6.9/1 mol/L EDTA, and protein contents were determined by using the BioRad Detergent Compatible protein assay. Samples were prepared for SDS-PAGE in 10% sucrose, 40 mol/L DTT, and subjected to electrophoresis by using a 7.5% polyacrylamide gel according to Laemmli, with equal loading of protein (50 Ixg) into all lanes. 12 The proteins were electrophoretically transferred to Immobilon, as described by Tobin et al. 13 The cq subunit of the Na/K pump was identified by ECL- Western immunoblotting.

ECL-Western immunoblotting. The Western blot was soaked in 100% methanol for 1 minute, rinsed 3 times with deionized water, and incubated for 1 hour at 37 ° C in

J Lab Clin Med 160 Charles et al, August 1997

blocking buffer, pH 7.5, containing TRIS HC1 (20 mol/L), NaC1 (150 mol/L), Tween 20 (0.1% vol/vol), and bovine serum albumin (5%). The blot was rinsed 3 times with rinsing buffer containing TRIS HC1 (20 mol/L), NaC1 (150 mol/L), Tween 20 (0.5% vol/vol), and bovine serum albumin (0.1%), and incubated at room temperature for 2 hours with rabbit anti-rat Na/K ATPase % subunit polyclonal antibody. The antibody was diluted 1:500 in rinsing buffer containing 5% nonfat dry milk. At the end of that incubation period, the blot was again rinsed with rinsing buffer and reincubated for 2 hours in a solution containing protein A conjugated to horseradish peroxidase diluted 1:2000 with rinsing buffer. Protein A detection was done by the ECL method, as described by the manufacturer (Amersham, Arlington Heights, Ill.).

Effect of H202 on phosphorylation of ~i subunit of BPAECs. PAECs were grown to confluence in 100 mm plastic dishes. The confluent monolayers were washed with phosphate-free MEM and incubated for 2 hours at 37°C in 95% air and 5% CO 2 in phosphate-flee MEM containing 32p-labeled phosphoric acid (250 ~Ci/dish) to label ATP. The radiolabeled medium was then aspirated, and the cell monolayers were washed twice with phosphate- containing MEM and reincubated for 30 minutes in MEM in the absence (control) or presence of 1 mmol/L H202.

At the end of the incubation period, the monolayers were washed twice with ice-cold PBS, the cells harvested with a rubber policeman, suspended in ice-cold PBS, and centrifuged at 600 g for 5 minutes at 4 ° C. The cell pellet was resuspended in lysing buffer containing 1% Triton X-100, 10 mol/L TRIS HC1, pH 7.5, with EDTA (5 retool/ L), EGTA (5 mmol/L), NaF1 (10 mmol/L), PMSF (1 mmol/L), leupeptin (0.1 mmol/L), Na vanadate (0.1 mmol/L), and aprotonin (3 ixmol/L). Immunoprecipitates of the % subunit, prepared as described subsequently, were compared by using autoradiography.

Immunoprecipitation of Na/K-pump eq subunit. The cell lysate was centrifuged at 15,000 g for 5 minutes, and the supernatant was incubated for 2 hours at 4 ° C with 10 pA of nonspecific rabbit serum immunoglobulin G. A 50 ~1 aliquot of a 50% solution of protein A-agarose beads was added to cell lysate-immunoglobulin G mixture, incubated for 90 minutes at 4°C on a rocker mixer, and centrifuged at 15,000 g for 1 minute at 4 ° C. The pellet was discarded, and the supernatant was mixed with 10 p~l of rabbit anti-rat Na/K-ATPase % subunit antiserum and incubated overnight at 4 ° C on a rocker. A 50 p~l aliquot of a 50% solution of protein A-agarose beads was added to this mixture, incubated for 90 minutes, and centrifuged at 15,000 g for 1 minute at 4 ° C, and the resulting supernatant and pellet were separated. The pellet, which constituted the precipi- tated % protein bound to the anti-c% antibody and protein A on agarose beads, was washed with lysing buffer. The immunoprecipitates were solubilized in SDS-PAGE sample buffer and subjected to electrophoresis by using a 7.5% polyacrylamide gel according to Laemmli. a2 The proteins were electrophoretically

transferred to Immobilon. Autoradiographs were obtained at - 8 0 ° C with an enhancing screen, and ECL Western immunoblotting was done, as described previously.

Effects of PKC inhibitors on Na/K-pump activity. Na/K- pump activity in control and HaO2-treated cultures was assessed by measurement of ouabain-inhibitable 86Rb uptake (see the following) after incubation of monolayers with inhibitors of PKC. BPAEC monolayers in 24-well plates were incubated for 30 minutes at 37 ° C with S6RbC1 with or without 0.1 mmol/L ouabain in MEM alone (control) or MEM containing calphostin (5 ~mol/L), staurosporine (0.3 ixmol/L), or H7 (20 p.mol/L). These concentrations were chosen based on literature indicating effective inhibition of PKC. 14'15 As a positive control for pump activation, other monolayers were incubated with H20 2 (1 retool/L) alone. Other monolayers were coincubated with PKC inhibitors plus 1 mmol/L HaO 2 to determine whether the inhibitors prevented the H2Oz-induced increase in pump activity. After a 30-minute incubation, monolayers were washed and ouabain-inhibitable 86Rb uptake determined, as described subsequently. The effi- cacy of PKC inhibition was assessed by measurement of PKC activity by using endothelial cell lysates, as described subsequently.

Effects of PDBU on Na/K-pump activity. As an alterna- Live method of determining the effects of PKC on endothelial Na/K-pump activity, we also assessed the effect of PKC activation with short-term (30-minute) incubation with the phorbol ester PDBU and PKC depletion with 24-hour incubation with PDBU. BPAEC monolayers in 24-well plates were preincubated with MEM alone or PDBU (1 ~mol/L) in MEM for 30 minutes at 37 ° C before assessing Na/K-pump activity, as described subsequently. In other experiments, monolayers were incubated with MEM alone or PDBU (1 ixmol/L) in MEM for 24 hours. At the end of the 24-hour incubation, the monolayers were washed and incubated for 30 minutes with MEM alone or MEM plus PDBU with 86RbC1 in the presence or absence of 0.1 mmol/L ouabain, and Na/K-pump activity was determined, as described subsequently.

The effects of PKC activation with PDBU on HaO2- induced changes in pump activity were assessed by 30- minute coincubation of monolayers with PDBU (1 txmol/L) in the presence or absence of H20 2 (1 retool/L). The effects of PKC depletion were determined by incubation of monolayers with PDBU (1 p~mol/L) for 24 hours, followed by a 30-minute incubation with either MEM alone or containing 1 mmol/L H202, with S6RbC1 in the presence or absence of 0.1 mmol/L ouabain. The Na/K- pump activity was determined as described subsequently.

Measurement of Na/K-pump activity. Cellular Na/K- pump activity was measured in confluent monolayers in 24-well plates, by using ouabain-inhibitable S6Rb uptake, as we previously described in detail. 8 Monolayers were incubated for 30 minutes at 37 ° C in 95% air/5% CO2 in MEM containing S6RbC1 (2 ixCi/ml; specific activity, 0.648 Ci/mmol; effective [86Rb], 0.0076 mEq/L) in the presence

J Lab Clin Med Volume 130, Number 2

or absence of 0.1 mmol/L ouabain. Uptake of 86Rb was converted to K ÷ uptake based on the assumption of pro- portional behavior and by using the equation

[Ki] = [Ko] × (86Rbi/S6Rbo)

where the subscripts i and o indicate the intra- and extracellular concentrations, respectively, and Ko was measured to be 5.4 mEq/L. Ouabain-inhibitable K + uptake, a measure of Na/K-pump activity, was determined by sub- tracting the K + uptake in the presence of 0.1 mmol/L ouabain from the K ÷ uptake in the absence of ouabain. Uptake of K + in three wells was averaged to yield an N of 1. A fourth well, exposed to the same conditions, was used for determination of adherent cell count by using a hema- tocytometer, and the data were expressed as nmoles of K + uptake per 10 6 cells per 30 minutes. Uptake of 86Rb by monolayers of BPAECs is linear for at least 30 minutes.*

Preparation of cells for PKC assays. Confluent monolayers of BPAECs in 24-well plates were incubated with MEM alone (control) or with PKC inhibitors, as described previously. After 30 minutes, monolayers were washed twice with PBS and lysed with lysing buffer (10 mmol/L TRIS, pH 7.5, 0.1% Triton X 100, 2 mmol/L EDTA, 2 mmol/L EGTA, 2 mmol/L DTT, 1 mmol/L PMSF, 3 ~mol/L aprotinin, and 0.1 mmol/L leupeptin) for 30 minutes on ice. Lysates (10 Ixl) were then used in PKC assays. Results were compared with controls and expressed as percentage inhibition of PKC activity.

In other experiments, membrane and cytosol fractions were compared with respect to PKC activity. Confluent monolayers of BPAECs in 100 mm dishes were incubated with MEM alone or containing PDBU (1 txmol/L) for 10, 30, or 60 minutes or 24 hours at 37 ° C in 95% air/5% COa. Monolayers were rinsed twice with PBS, scraped from the dishes, and centrifuged at 1000 g for 10 minutes at 4 ° C. Cell pellets were resuspended in sonication buffer containing 10 mmol/L TRIS, pH 7.5, with EDTA (2 mmol/L), EGTA (2 mmol/L), DTT (2 mmol/L), NaF1 (10 mmol/L), PMSF (1 mmol/L), leupeptin (0.1 mmol/L), and aprotinin (3 p.mol/L). The samples were sonicated at 4 ° C and then centrifuged at 1000 g for 10 minutes at 4 ° C to remove unbroken cells. Supernatants were removed and centrifuged at 12,000 g for 30 minutes at 4°C to pellet cell membranes. The supernatant cytosolic fractions were removed and saved for PKC assays. The membrane pellets were solubilized with lysing buffer for 30 minutes on ice. Triton-insoluble material was removed by centrifugation (12,000 g for 5 minutes at 4 ° C), and the soluble membrane material was used in PKC assays. Protein in the cytosolic and membrane fractions was determined by using the standard Bio-Rad protein assay.

PKC assays. Phosphorylation of histone IllS (25 p~g) was assessed in a reaction mixture (50 ~xl) containing 25 mmol/L TRIS/HC1, pH 7.5, 5 mmol/L MgC12, 1 mmol/L DTT, 5 Ixg phosphatidylserine, 0.5 Ixg diolein, 1 mmol/L CaC12, 50 ixmol/L [,/-3:P]ATP (200 to 500 cpm/pmol) and 10 I~1 or 5 I~g BPAEC protein. Background reactions contained 3 mmol/L EGTA without CaC12, phosphatidyl-

Charles et al. 161

serine, and diolein. Phosphorylation occurred for 3 to 5 min at 30 ° C. Reactions were terminated by the addition of ice-cold 1 mmol/L ATP. Aliquots (40 ~1) of the reactions were spotted onto 2 cm squares of Whatman 3 mm filter paper in a grid composed of the necessary number of squares. The grid was washed twice with ice-cold 10% trichloroacetic acid containing 10 mmol/L sodium pyro- phosphate (8 ml/square, 10 rain/wash), twice with ice-cold 5% trichloroacetic acid containing 10 mmol/L pyrophos- phate, rinsed with acetone, air dried, and cut into individ- ual squares. Radioactivity associated with each square was quantitated by liquid scintillation counting. Protein kinase activity was expressed as pmoles Pi transferred. 16

Statistical analysis. All data are expressed as mean _+ SEM. Comparisons involving three or more groups were analyzed by one-way analysis of variance, followed by the least significant difference multiple-comparison test (Stat- view). Differences between means were considered significant at p < 0.05.

RESULTS

Fig. 1 shows a representative immunofluorescence photomicrograph of BPAECs incubated with monoclonal antibody to the dog oq subunit of the Na/K pump and with a secondary goat anti-mouse antibody conjugated to Cy3. Staining is apparent in a punctate pattern, most intense over the cytoplasm as compared with the nucleus. The o[ 1 subunit is not localized to the lateral edges of the cells. Control cultures revealed no staining.

Fig. 2 is a photomicrograph obtained by using confocal laser scanning microscopy of a confluent BPAEC monolayer grown on a glass coverslip and incubated with mouse monoclonal antibody to the dog ~1 subunit of the Na/K pump, with a secondary goat anti-mouse antibody conjugated to Cy3. The upper panel shows an en face view with intense staining appearing dark. The most intense stain is perinuclear. The lower panel shows the lateral view through the area denoted by dotted lines on the upper panel. Staining of the o h subunit is apparent over both the apical and basal aspects of the cells. Thus unlike cultured epithelial cells, 17 cultured pulmonary vascular endothelial cells do not exhibit basolateral membrane localization of the Na/K pump, as assessed by the s t subunit.

We previously reported that short-term (30- minute) incubation of with H202 or xanthine/xanthine oxidase increased Na/K pump activity. 8 We investigated the effects of short-term incubation with H 2 0 2 on membrane expression of the a 1 subunit in BPAECs and HPAECs by using ECL-West- ern blotting after SDS-PAGE of membrane fractions. Fig. 3 shows that there was no difference in electrophoretic mobility or amount of 97 kDa ed


Fig. 1. Confluent cultures of bovine main pulmonary artery endothelial cells, grown on Cell-Tak-coated glass coverslips, were fixed in methanol at -20°C and incubated with mouse anti-dog % subunit monoclonal antibody and goat anti-mouse secondary antibody, conjugated to Cy3. Cultures were examined with a phase fluorescent microscope at excitation k 554 nm and emission k 580 nm. Shown are representative results from one experiment of three. Magnification ×1400.

subunit of Na/K pump in membrane fractions from control and HzO2-treated cells. These results indicate that H20 2 does not increase endothelial cell Na/K-pump activity by increasing expression of the pump on the cell membrane.

We assessed the effects of short-term incubation with H20 2 on phosphorylation of the immunoprecipitated % subunit in intact BPAECs. Fig. 4 shows an autoradiograph and immunoblot of the oL 1 subunit immunoprecipitated from monolayers incubated with MEM alone or with 1 mmol/L H20 2 for 30 minutes. The immunoblot shows the expected dense % subunit band at 97 kDa. Another band at 50 to 55 kDa probably represents protein A reacting with the heavy chain of the anti-% subunit antibody. The intensities of the 97 kDa bands were similar in control and H202-treated immunoblots, indicating that approximately equivalent amounts of the % subunit were immunoprecipitated. The autoradiograph of the Western blot shows phosphorylation of 97 kDa material in both control and H202-incubated monolayers. There were no dramatic H202- induced changes in phosphorylation at this molecular mass. Thus it is unlikely that noncatalytic-site phosphorylation of the % subunit is the mechanism

of H202-induced increases in endothelial cell Na/K- pump activity.

As previously reported, short-term (30-minute) incubation with H202 increased Na/K-pump activity of intact monolayers of BPAECs (Fig. 5). We found that the PKC inhibitors (calphostin [5 ~xmol/L], staurosporine [0.3 txmol/Ll, and H7 [20 ~mol/L]) also increased pulmonary artery endothelial cell Na/K- pump activity, as assessed by ouabain-inhibitable S6Rb uptake (Fig. 5). The effect of the PKC inhibitors was similar in magnitude to that of H20 2. Co- incubation of cultures with H202 and the PKC inhibitors (calphostin [5 ixmol/L], H7 [20 ~mol/L], and staurosporine [0.3 ixmol/L]) resulted in increased Na/K-pump activity of a magnitude similar to that induced by H20 2 or inhibitors alone (Fig. 5). Fig. 5 shows that the effects of H20 2 and PKC inhibitors on Na/K-pump activity were not synergis- tic or additive. These results suggest that there may be a maximal magnitude of stimulation of endothelial pump activity under the conditions of these experiments.

Under these conditions, calphostin (5 ixmol/L), H7 (20 ~mol/L), and staurosporine (0.3 ~mol/L) inhibited endothelial PKC activity compared with

J Lab Clin Med Volume 130, Number 2 Charles et al. 163

Fig. 2. Confluent cultures of bovine main pulmonary artery endothelial cells grown on Cell-Tak-coated glass coverslips were fixed in methanol at -20 ° C and incubated with mouse anti-dog %-subunit monoclonal antibody and goat anti-mouse secondary antibody, conjugated to Cy3. Cultures were examined with a confocal laser scanning microscope at magnification x 1529. Upper panel, En face view of a confluent monolayer. Intense stain appears dark. N1 and N2, nuclei. Lower panel, Lateral view through the area denoted by dotted lines on A. Shown are representative results from one experiment of three.

control cultures (inhibition: calphostin, 27% _+ 7%, n = 4; H7, 12% _+ 4%, n = 4; and staurosporine, 44% _+ 2%, n = 3). Short-term (-<60 minutes) incubation with PDBU increased cell-membrane PKC activity compared with cytosol, and 24 hour incubation with PDBU decreased both membrane and cytosol PKC activity (Table I).

Short-term incubation of BPAEC monolayers with the phorbol ester PDBU did not change Na/K- pump activity (Fig. 6). These results suggest that activation of PKC does not alter endothelial Na/K- pump activity. However, 24-hour incubation with PDBU increased Na/K-pump activity (Fig. 6). These

results are consistent with the effects of PKC inhibitors on Na/K-pump activity (Fig. 5) and indicate that depletion of PKC by PDBU increases endothelial Na/K-pump activity.

We assessed the effects of PKC activation and depletion on H20: - induced increases in Na/K- pump activity. Fig. 6 shows that coincubation of BPAEC monolayers with PDBU and H 2 0 2 for 30 minutes prevented H202-induced increases in Na/ K-pump activity. On the other hand, 24-hour t reatment of cultures with PDBU did not prevent subsequent H202-induced increases in pump activity (Fig. 6).


M e m b r a n e s

1 m MH202 - + - +

97 £Da, >

HPAEC BPAEC

Fig. 3. Human (HPAEC) and bovine (BPAEC) pulmonary artery endothelial cells were incubated for 30 minutes in the absence (-) or presence (+) of 1 mmol/L H202. Membrane fractions were subjected to SDS-PAGE (50 }xg protein/lane), and Western blots were examined for % subunit by using the ECL technique. Shown are representative results from one experiment of three.

D I S C U S S I O N

The results of these studies indicate that the Na/K pump, as assessed by % subunit localization, was present on both apical and basal aspects of main PAECs, without polarized subcellular distribution. Perturbation of endothelial cells with H202 did not change the relative amounts of OL 1 subunit in membrane fractions of endothelial cells, nor did it change the phosphorylation of the % subunit in whole-cell lysates. Inhibitors of PKC increased Na/K-pump activity in intact monolayers, as assessed by ouabain- inhibitable S6Rb uptake. Depletion of PKC did not prevent H202-induced increases in pump activity. However, activation of PKC with brief exposures to the phorbol ester PDBU did prevent H202-induced increases in activity.

There is surface localization of Na/K-pump mol- ecules on the basolateral surface of type II epithelial cells and in other epithelial tissues. 4'17'18 This surface localization is important in determining the direction of net Na flux across epithelial membranes, in that Na enters cells via apical Na channels and is extruded by the pump into the basolateral environment. In disease states, the polarity of the Na/K pump may be disturbed. In the renal epithe- lium, ischemic injury decreases renal tubular sodium

1 mM H202

kDa

9 7 -

66-

Autoradiograph Immunoblot - + +

45-

Fig. 4. Bovine pulmonary artery endothelial cells were labeled with H332po 4 and then incubated for 30 minutes in the absence (-) or presence (+) of 1 mmol/L HzO2. The subunit of the Na/K pump was immunoprecipitated from whole-cell lysates, subjected to SDS-PAGE, and autoradiographs and immunoblots of % subunit obtained.

reabsorption, probably because of redistribution of the Na/K pump from the basolateral to apical epithelial cell surface. 19 Little is known about the surface distribution of the Na/K pump in endothelium. Indeed no pump was observed on lung capillary endothelial cells in earlier studies. 4'1s However, these studies may have been limited in sensitivity by fixation or pH of cytochemical reactions. By using a unique method of harvesting apical and basal endothelial cell membranes, Stolz and Jacobson 2° reported increased Na/K-ATPase activity in basal membranes of BAECs in a small number of experiments and presented data suggestive of polarization of other endothelial cell-surface functions. 2° Betz et al. a~ reported basolateral polarization of the Na/K pump on brain capillary endothelial cells. Others reported polarization of cultured endothelial cells with respect to a variety of other membrane mark- ers. 22-24 The lack of polarization of the % subunit of the Na/K pump that we observed indicates that there is no cell-surface polarization of the pump on cultured main PAECs. This suggests that the pump is not used for vectorial ion transport across large- vessel endothelial monolayers. The lack of localization may be the result of tissue-culture conditions. It is possible also that localization of the pump is polarized on lung microvascular endothelial cells.

Similar lack of polarized distribution of the oL subunit was reported on hippocampal neurons in vivo and in culture, n The mechanism of differences

J Lab Clin Med Vo lume 130, Number 2 Charles et al. 165

, l

> , n

g E

O

O

¢,O o

E + v

o

e- Z v

100

80

60

40

20

0 Control H202 Calphostin H7 Staurosporine

[ ] No H202

[ ] + H202

Fig. 5. Confluent cultures of BPAECs were incubated for 30 minutes with MEM alone (control), 1 ixmol/L H202, 5 p.mol/L calphostin, 20 ~mol/L H7, or 0.3 txmol/L staurosporine (solid bars). Other monolayers were coincubated with 1 mmol/L H202 plus inhibitors (hatched bars). Na/K-pump activity was assessed by ouabain-inhibitable S6Rb uptake. Numbers in bars are numbers of experiments. *Significant difference from control at p < 0.05.

in localization of Na/K-ATPase between epithelial and other tissues is not known but could be caused by differences in the cellular machinery for sorting of proteins, n47

We assessed the expression of the pump % subunit on membrane among the possible mechanisms of H202-induced short-term modulation of Na/K-pump activity. In several tissues, insulin stimulates Na/K-pump activity within 30 minutes of exposure. 6 The mechanism of this effect appears to involve translocation of the ~2 subunit to the plasma membrane in the skeletal muscle. 25'26 We did not find H202-induced changes in the expression of the % subunit on membrane fractions of main PAECs. These results are consistent with our previous finding that H20 2 decreased the Bma x for ouabain binding to PAECs. 8 These results suggest that increased expression of the % subunit on cell membrane was not responsible for H2Oz-induced increases in Na/K-pump activity of intact monolayers. However, it is possible that H202 shifted %-subunit binding from intracellular membranous structures (e.g., smooth endo- plasmic reticulum) to plasma membrane. Such shifts might not be detected by comparing immunoblots of total membrane preparations. 27

In preliminary studies we did not observe the ~2 subunit in membrane preparations of cultured main PAECs. Similarly, Zahler et al. 28 did not observe the % or % subunits on large-vessel endothelial cells. However, those authors did observe the % subunit

Table I. Effect of phorbol dibutyrate (PDBU) on protein kinase C (PKC) activity of pulmonary artery endothelial cells

PKC activity of cell fraction Incubation Incubation medium time Cytosol Membrane

MEM

PDBU (1 Fmol/L)

10 rain 4.46 2.36 30 min 4.40 3.35 60 min 4.58 5.50 24 hr 3.70 4.61 10 min 3.36 8.51 30 min 2.85 19.10 60 rnin 3.14 16.99 24 hr ND 1.25

Effects of PKC activation or depletion by PDBU were assessed by coincubation of monolayers with PDBU for varying times, followed by assessment of PKC activity in cytosol and membrane fractions. PKC activity is expressed as picomoles of Pi transferred/3 min/5 I~g protein. Each time point represents one experiment. ND, None detected.

on cardiac small-vessel endothelium. 28 Thus it is possible that H202-induced increases in NaN-pump activity are caused by shifts of the e~ 2 subunit from intracellular membrane-bound sites to plasma membrane. Such shifts would involve ~2 subunits unde- tectable with our methods.

The short-term regulation of Na/K-pump activity has been extensively studied in epithelial tissues. 6'7 Although there are significant differences among cell types and species, a pattern has emerged to suggest that in intact cells, a number of intracellular


100

8O

E

I..- ~ 60

~" + 40

Z o E = 20

[ ] Mean(3Omin)

[ ] Mean(24hrs)

MEM PDBU P D B U / H 2 0 2

Fig. 6. Confluent cultures of BPAECs were incubated for 30 minutes (solid bars) or 24 hours (hatched bars) with MEM alone or i ixmol/L PDBU. Other cultures were incubated with 1 txmol/L PDBU plus H20 2 for 30 minutes or with 1 ~mol/L PDBU for 24 hours, followed by 30-minute incubation with 1 mmol/L HzO a. Numbers in bars are numbers of experiments. *Significant difference from control atp < 0.05.

second-messenger systems modulate pump activity. Among these are cyclic adenosine monophosphate- PKA, diacylglycerol-activated phospholipid and calcium dependent-PKC, intracellular calcium, lyso- phospholipids, and arachidonic acid and other eicosanoids. 6'7 Stimulation of PKC has been reported to have different effects on Na/K-pump activity, dependent on the tissue studied. 6'7 For exam- ple, in hepatocytes 29 and pancreatic acinar cells, 3° PKC stimulation enhanced Na/K-pump activity. However, in shark rectal gland, 31 in renal proximal convoluted tubule cells, 32 and in Xenopus Iaevis oocytes, 33 PKC stimulation decreased pump activity. The precise sites of phosphorylation of the Na/K pump by protein kinases have not been determined, but phosphopeptide analyses indicate that they ap- parently reside on serine or threonine residues or both on cytoplasmic loops of the ~ subunit. 31'34 Thus noncatalytic-site phosphorylation of the pump may be significant in short-term modulation of activity, and the balance between phosphorylation and de- phosphorylation, as directed by protein kinases and phosphatases, regulates pump activity. Our results indicate that H20 2 did not change phosphorylation of the ~1 subunit, as assessed by 32p labeling of immunoprecipitated ~1 subunit. Thus noncatalytic- site al-SUbunit phosphorylation is not likely the

mechanism of H202-induced increases in endothelial pump activity.

Inhibitors of PKC (calphostin, H7, and staurosporine) enhanced Na/K-pump activity in endothelial cells. These results suggest that there may be tonic inhibition of Na/K-pump activity by PKC in endothelial cells. Inhibitors of PKC are not specific and may have other effects on cells. In these experiments we used three PKC inhibitors that act by different mechanisms, and we depleted PKC by 24- hour incubation with PDBU. All of these maneuvers stimulated Na/K-pump activity. Furthermore, we determined that the inhibitors were at least partially effective in reducing PKC activity in whole endothelial cell lysates. In addition, we verified that 24-hour incubation with PDBU depleted PKC by assessing PKC activity in cytosol and membrane preparations. The mechanism of the effect of PKC inhibition on Na/K-pump activity is not known. Possible mechanisms include direct actions on pump subunits, in- direct effects of some unidentified intermediate(s), or an effect on the plasma-membrane environment of the Na/K pump.

Others reported that oxidants increase endothelial cell PKC activity. 35'36 Because PKC activation had been reported to enhance Na/K-pump activity in other tissues, 6'7 we assessed whether inhibition of

J Lab Clin Med Volume 130, Number 2

PKC prevented H202-induced increases in endothelial cell Na/K-pump activity. Depletion of PKC did not prevent HzOz-induced increases in Na/K-pump activity, indicating that H202 does not exert its effect by stimulation of PKC. However, activation of PKC by brief exposure to PDBU did prevent H2O 2- induced increases in Na/K-pump activity. Because activation of PKC by PDBU did not change Na/K- pump activity in control cells, these results suggest that some phosphorylated intermediate blocks the H 2 O 2 effect on pump activity.

In summary, our results indicate that the mechanism of H2Oz-induced increases in Na/K-pump activity is not through changes in membrane expression of the pump, changes in the eq subunit phosphorylation, or mediated by PKC activation. Other possible mechanisms of the H202 effect, not addressed by these studies, include increased pump cycling caused by changes in the lipid environment of the pump, changes in pump association with cytoskeletal filaments, or increased responsiveness to oxidant-induced increases in intracellular sodium. Further delineation of the mechanism of oxidant- induced increases in pump activity awaits further studies. The increase in Na/K-pump activity observed in response to PKC inhibition and depletion suggests that PKC may be an endogenous modula- tor of endothelial cell Na/K-pump activity.

The lack of polarized distribution of the Na/K pump ~1 subunit on main PAECs suggests that in those cells the pump primarily has a "housekeeping" role, rather than a role in vectorial transendothelial ion transport. However, increases in pump activity with oxidant-induced vascular injury may be an important protective mechanism against further endothelial damage in conditions such as adult respira- tory distress syndrome or sepsis.

We thank Nancy Huang, Nancy Parks, Lili Geng, and Rose- marie Smith for technical assistance. We thank Dr. Linda Nici of the Pulmonary Section and Dr. Judith Parmelee of the Clinical Neuroscience Department of Brown University School of Medi- cine for helpful discussions. We thank Dr. Michael Caplan of Yale University for providing monoclonal antibody to % subunit. We also thank Dr. Rick Rogers, Bruce Ekstein, and Jean Lal of the Biological Imaging Laboratory of the Harvard School of Public Health for performing the confocal microscopy.

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Documents

Studies on the mechanism of short-term regulation of pulmonary artery endothelial cell Na/K pump activity