r3H]Burnetanide Binding in Vascular Endothelial Cells

THEJOURNAL O F BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 34, Issue of December 5,pp. 20326-20330, 1989 Printed in U.S.A.

r3H]Burnetanide Binding in Vascular Endothelial Cells QUANTITATION OF Na-K-C1COTRANSPORTERS* (Received for publication, June 15, 1989)

Martha E. O’DonnellS From the Department of Cell Biology and Anatomy, University of Health Sciences, The Chicago Medical School, North Chicago, Illinois 60064

Vascularendothelial cells previouslyhave been pmol K+/g of protein/min. Na-K-C1 cotransport of aortic shown to possess a prominent Na-K-Cl cotransport endothelial cells is known to beregulated by a variety of system which mediates a K+ influx of approximately vasoactive hormones and neurotransmitters and their second 20 pmol/g of protein/min. Endothelial cell cotransport messengers. Thus, cotransport activity is stimulated by vahas also been shown to be regulated by a variety of sopressin, bradykinin, angiotensin11, or by elevation of intravasoactive agents and their second messengers, sug- cellular Ca2+ and is inhibited by norepinephrine, histamine, gesting that the transportsystem may have an impor- acetylcholine, rat atriopeptin 111, phorbol esters, and cyclic tant role in endothelial cell function. In the present nucleotides (3, 6). This observation suggests that the promistudy we investigated the possibility that thehigh level nent endothelial cell cotransporter could play a role in mediof cotransport in these cells is due to a large number of vasculature. More Na-K-C1 cotransporters in theplasma membrane. This ating theeffects of vasoactive agents on the was done by evaluating specific saturable binding of recently, evidence has been provided by this laboratory that Na-K-Cl cotransport may participate in regulation of endo[3H]bumetanide to cultured bovine aortic endothelial cells. We found a maximal r3H]bumetanide binding of thelial cell volume (7). These findings indicate that Na-K-C1 0.83 pmol/mg protein with a dissociation constant of cotransport may be of great importance to normal functioning 0.13 p ~ From . these data, the number of [3H]bumetan- of the endothelium. ide binding sites/endothelial cellwas determined to be In the present study, Na-K-Cl cotransport of endothelial approximately 230,000, and the turnover number for cells was further investigated by evaluating the number of cotransport activity was calculated to be 300 K+ ions/ cotransporters present per endothelialcell. The large magnisite/s. These findings indicatethat endothelial cells do tude of cotransport-mediated K’ influx in thesecells could be indeed exhibita large number of Na-K-Cl cotransport- the result of a large number of Na-K-Cl cotransporters presers/cell relative to other cell types. We also investi- ent in the plasma membrane, a high turnover rate for each gated the effectson r3H]bumetanide binding of agents cotransporter, or a combination of both. In previous studies, known to modulateNa-K-Cl cotransport activity. Sat- saturable binding of [3H]bumetanide has been used successurable binding of [3H]bumetanide was found to be re- fully to quantitate the number of Na-K-C1 cotransporters duced significantly by treatment of the cells with 8- presentinmembranepreparations from dog, bovine, and bromo-cyclic AMP, 8-bromo-cyclic GMP, phorbol es- rabbit kidney, winter flounder intestine, shark rectal gland, 111,all of which and rabbit parotid gland (15-20) as well as in intact cells, ters, norepinephrine,or rat atriopeptin have been shown to inhibit Na-K-Cl cotransport-me- including vascular smooth muscle cells, Ehrlich ascites tumor diated K+ influx. cells, and duck and ferret erythrocytes (21-25). Thus, in the present study, specific saturable [3H]bumetanide binding to intact bovine aortic endothelial cells was used to quantitate the numberof cotransporters present in the cells. In addition, Vascular endothelial cells possess a Na-K-C1 cotransport the ion dependence of [3H]bumetanide bindingin endothelial system that mediates a major portion of total K+ influx (3, cells was investigated. Finally, several agents known to mod6). The magnitude of cotransport in these cells is large relative ulate Na-K-Cl cotransport activity were evaluated for their to othercell types. That is, cultured bovine aortic endothelial effect on [3H]bumetanidebinding. cells exhibit a basal cotransport level of approximately 20 pmol of K+/g of protein/min (3). In contrast, studies of NaEXPERIMENTAL PROCEDURES K-Cl cotransport ina variety of other cell types, including rat Materials-Bumetanide was the gift of Hoffmann-La Roche. [3H] and rabbit aorticvascular smooth muscle cells (%lo), human Bumetanide (66 Ci/mmol) was generously provided by Dr. H. C. fibroblasts (11), NIH3T3 cells (12),Madin-Darkycanine Palfrey, Universityof Chicago. The synthesis and purification of [3H] kidney cells (13), and chick heart cells (14),have revealed bumetanide has been described by Forbush and Palfrey (1). Ouabain, basal cotransport rates ranging from approximately 3 to 10 norepinephrine,8-bromoadenosine 3’,5’-cyclic monophosphate (8-

* This work was supported by the American Heart Association of Metropolitan Chicago and by National Institutes of Health Biomedical Research Support Grant Program Grant BRSG-S07-RR0536624. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 4 To whom reprint requests should be addressed Dept. of Cell Biology and Anatomy, University of Health Sciences, The Chicago Medical School, 3333 Green Bay Rd., North Chicago, IL 60064.

Br-CAMP),’ 8-bromoguanosine 3’,5’-cyclic monophosphate (8-BrcGMP), andphorbol 12-myristate 13-acetate (PMA) were purchased from Sigma. Rat atriopeptin 111, vasopressin, and bradykinin were purchased from Peninsula Laboratories, Inc. (Belmont, CA). The abbreviations used are: 8-Br-cAMP, 8-bromoadenosine3’,5’cyclic monophosphate; 8-Br-cGMP, 8-bromoguanosine 3’,5’-cyclic monophosphate; PMA, phorbol 12-myristate 13-acetate; FBS, fetal bovine serum; MEM, minimum essential medium; Hepes, 4-(2-hydroxyethy1)-1-piperazineethanesulfonic acid; SDS, sodium dodecyl sulfate.

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[3H]BumetanideBinding in Endothelial Cells Cells-Cultured vascular endothelial cells used for these studies were bovine aortic endothelial cells, passaged from calf thoracic aorta as described by Gordon and Martin (2). Cells were grown in DulbecCO'S modified Eagle's medium (Hazelton/K. C., Lenexa, KS) containing 10% fetalbovine serum (FBS, HyClone Laboratories, Logan, UT) at 37 "C in a 95% air, 5%CO, atmosphere and were used between the 5th and 25th passages. For[3H]bumetanidebindingexperiments, cells were removed from stock culture dishes (100 mm) by trypsinization and subcultured onto multiwell plates (24-well, 16-mm, Costar, Cambridge, MA). Cells were used 3-5 days later. For transport studies, cells were subcultured onto 60-mm culture dishes and were used 3-5 days later as confluent monolayers. PHIBumetanide Binding Meas~rements-[~H]Bumetanidebindingmeasurements were performed on cells attached to multiwell plates. Prior to the start of the binding assay, wells were aspirated free of Dulbecco's growth medium (DMEM + 10% FBS) and rinsed once with 1 ml of FBS-free, Hepes-buffered minimumessential medium (MEM). Theassay was initiated by adding to each well 0.2 ml of Hepes-buffered MEM containing 0.1% FBS, various concentrations of [3H]bumetanide (at constantspecific activity), and either 40 or 0 /IM bumetanide(unlabeled). In some experiments,additional agents were present during the assay (i.e. 8-Br-cAMP, 8-Br-cGMP, PMA, norepinephrine, or rat atriopeptin 111). In all cases, the final volume of the assay medium in each wellwas 0.2 ml. Cells were incubated with the assay medium for 60 min at 22 "C. To terminate the assay, wells were aspirated free of medium and rapidly rinsed with three 1-ml volumes of ice-cold lysing buffer (20 mM Hepes + 1 mM CaC1,). Total rinse time was approximately 10s. After air drying, wells were extracted with 0.4 ml of 0.2 N NaOH and radioactivity of a 0.3-ml aliquotdetermined by liquid scintillationmethods. The effectiveness of the rinsing procedure and NaOH extraction has been verified previously (3). To evaluate proteincontent, several wells were rinsed with lysing buffer as described above, extracted with 1.0 ml of 0.2% sodium dodecyl sulfate (SDS), and theprotein content of a 0.3ml aliquot determined fluorometrically (4). Na-K-GI Cotransport Measurements-Na-K-C1 cotransport was measured in these studies as ouabain-insensitive bumetanide-sensitive K' influx. Details of this method have been published previously (3). 86Rbwas used as a tracerfor K+ because it has been demonstrated in previous studies that Rb' quantitatively substitutes for K' in this transport system (5). Prior tomeasurement of Na-K-C1 cotransport, endothelial cells on 60-mm culture dishes were equilibrated for 10 min with Hepes-buffered MEM in an air atmosphere at 37 "C on a gyratory water bath. For the cotransport experiments of the present study, thecells were then preincubated for 5 min at 37 "C in a gyratory water bath in Hepes-buffered MEM containing 145 mM Na+, 6 mM K', and various concentrations ofC1-. Also present in the preincubation medium was 1 mM ouabain and 0 or 10 /IMbumetanide. To assay Na-K-C1 cotransport, the medium was replaced with fresh medium of identical composition but containing 86Rb(1&i/ml) and the cells incubated for 5 min. The assay was terminated by rapidly aspirating theassay medium and rinsing the dishes in ice-cold isotonic MgC1,. After air drying the dishes, radioactivity was extracted with 1.0 ml of 0.2% SDS and the amountof radioactivity present in a 0.5ml aliquot of the extract determined by liquid scintillation methods. Protein content of the dishes was determined on a 0.3-ml aliquot of the SDS extractby fluorometric methods (4).=Rb uptake by bovine aortic endothelial cells has been demonstrated previously to remain linear for at least 10 min (3). Thus,=Rb uptake was calculated in the present study as theslope of the uptake uersus time plot, as described previously (5). The K+ influx data are expressed as micromoles of K+/g of protein/min.

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FIG. 1. [3H]Bumetanide binding in vascular endothelial cells. A , specific saturable [3H]bumetanide binding was evaluated in bovine aortic endothelial cells attached tomultiwell culture dishes in Hepes-buffered medium (containing 145 mM Na+, 6 mM K+, and 50 mM C1-/100 mM gluconate) as described under "Experimental Procedures." Specific binding of [3H]bumetanide was calculated as (total [3H]bumetanide bound) - ([3H]bumetanide bound in the presence of excess unlabeled bumetanide [40 wM]). Data are mean values & S.E. of triplicate determinations from arepresentativeexperiment. B, Scatchard analysis of data in A . Data are plotted as the ratio of specific bound to free [3H]bumetanide uersus specific bound [3H] bumetanide. Maximal binding (Elmax) was determined as the x axis intercept of this plot. The dissociation constant (Kd) was determined as the negative reciprocal of the slope of this plot. From these data, a Bmax value of 1.03 pmol/mg and a Kd value of 75 nM were determined. From seven separate experiments, mean Bmax and Kd values of 0.83 & 0.08 pmol/mg and 0.127 & 0.015 pM, respectively, were determined. Using this Emaxvalue, the number of [3H]bumetanide binding sites/ cell was calculated to be 230,000.

22 "C (data not shown).By Scatchard analysisof the binding data, the maximal binding (Elmax) was determined to be 1.0 pmol/mg protein for the representative experiment depicted in Fig. 1B. From seven separate experiments, the B,,, for RESULTS [3H]bumetanide bindingto bovine aortic endothelialcells was Fig. 1A demonstrates the presence of specific saturable [3H] determined to be 0.83 f 0.08 pmol/mg and the dissociation bumetanide binding in the endothelialcells. In these experi- constant (&) was found to be 0.13 f 0.02 p M . The Kd value ments,endothelial cells cultured on multiwell plates were is in agreement with the Ki value for bumetanide inhibition bovine aortic endothelialcells (0.25 incubated for 60 min at 22 "C with increasing concentrations of Na-K-Cl cotransport in , that the saturable [3H]bumetanide binding of [3H]bumetanidein the absence orpresence of an excess of p ~ ) suggesting unlabeled bumetanide(40 FM) to determine total and nonspe-does reflectbinding of bumetanide to the cotransporter. From of [3H]bumetanide binding sites prescific binding, respectively. Specific binding was determined these data, the number cells was calculated to be 230,00O/cell. as [totalbinding] minus [nonspecific binding]. An incubation ent in the endothelial time of 60 min was chosen because in preliminary studies The data shown in Fig. 1 are consistent with thepresence of specific binding of [3H]bumetanide to the endothelial cells only one class of bindingsites for [3H]bumetanide in the was found to reachequilibrium after approximately50 min at endothelial cells. However, the analysisused is notsufficiently

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rigorous to rule out the presence of more than one type of binding site in the cells. The effects of extracellular [Na+], [K’], and [Cl-] on 13H] bumetanide binding to endothelial cells are shown in Fig. 2. In this series of experiments, specific binding of 0.3 p~ [3H] bumetanide was determined in the presence of various con1-2 1

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FIG. 2. Effect of extracellular [Na+][K+], and[Cl-] on saturable binding of [3H]bumetanideto endothelial cells. Bovine aortic endothelial cells were prepared for binding studies as described under “Experimental Procedures.” Cells were incubated for 60 min at 22 “C with 0.3 p~ [3H]bumetanide in the presence or absence of 40 PM unlabeled bumetanide to determine specific binding, as described in Fig. l . T h e incubation medium contained 145 mM Na+, 6 mM K+, and 150 mMC1-, unless otherwise indicated. In each experiment, the concentration of only one of these ions was varied. [Cl-] was varied by isosomotic substitution with gluconate. [Na+] and [K+] were varied by isosmotic substitution with choline. Data are mean values +- S.E. of five separateexperiments (Na’ and K+) or four experiments (Cl-).

in Endothelial Cells centrations of Na+, K’, and C1-. The concentration of only one ion species was varied in each experiment. Concentrations of ion species when not varied were [Na’] = 145 mM, [K’] = 6 mM, and [Cl-] = 150 mM. [Cl-] was varied by isosmotic replacement with gluconate, and [Na’] and [K+]were varied by isosmotic replacement with choline. All three ion species were found to be required for [3H]bumetanide binding to the cells. That is, specific binding of 13H]bumetanideto theendothelial cells was abolished when any one of the ion species transported by the Na-K-Cl cotransporter was omitted from the binding medium. With increasing concentrations of Na+ and K’, specific [3H]bumetanide binding was found to increase and then to saturate. K1,>values from these experiments were determined to be 4 mM for Na’ and 3 mM for K’. Increasing the concentration ofC1- in the binding medium was found to increase specific [3H]bumetanidebinding up to approximately 50 mMC1-. At higher concentrations of Cl-, bumetanide binding decreased. Thus, C1- appears to have a biphasic effect on binding of 13H]bumetanideto theendothelial cells. For this reason, the binding assay medium used in the experiments depicted in Fig. 1 and Table I contained 50 mMC1-. The Kshvalue for the stimulatory effect of [Cl-] on bumetanide binding was determined to be approximately 10 mM. In order to calculate a turnover number for Na-K-Cl cotransport of the bovine aortic endothelial cells, it was necessary to determine the level of cotransport activity that occurs under the conditions employed in the [3H]bumetanidebinding studies. Fig. 3 demonstrates the effect of extracellular [Cl-] on Na-K-Clcotransportin the endothelial cells. Na-K-Cl cotransport was assessed as ouabain-insensitive bumetanidesensitive K’ influx as described under “Experimental Procedures.” [Cl-] was varied by isosmotically replacing C1- in the assay medium with gluconate. [Na+]and [K’] were held constant in the assay medium at 145 and 6 mM, respectively. The titration curve obtained for K+ influx uersus [Cl-] was found to be sigmoidal. A maximal level of Na-K-Cl cotransport was observed with a [Cl-] of approximately 100 mM. A t 50 mM [C1-],145 mM [Na’], and 6 mM[K’] ( i e . the concentrations employed in the binding studies), cotransport was found to be 15 pmol of K+/g of protein/min. Using these data and the value of 230,000 binding sites/endothelial cell, a turnover number of 300 K+/site/s was calculated. The effects on specific [3H]bumetanide binding of agents known to modulate endothelial cell Na-K-C1 cotransport activity were also investigated in the present study. The results TABLEI Effects of second messengers and vasoactive agents on PHI bumetanide binding in endothelial cells Bovine aortic endothelial cells were grown on multiwell dishes (24 well, 16 mm) and [3H]bumetanidebindingmeasured as described under “Experimental Procedures.” Specific binding of [3H]bumetanide was determined at 0.3 p~ [3H]bumetanide in the presence of various agents, as indicated. Bindingassays were performed in Hepesbuffered media (containing 145 mM Na+, 50 mM Cl-, and 6 mM K+), and specific binding was determined as described in Fig. 1. Data represent mean values f S.E. of triplicate determinations from the number of experiments shown in parentheses. Specific [3H]burnetanide binding

Condition

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Control 8-Br-CAMP (50 p M ) 8-Br-cGMP (50 p M ) Phorbol12-myristate13-acetate Norepinephrine (10 pM) atriopeptin Rat 111 (100nM)

(10nM)

100.0 65.7 k 5.8 (8) 68.0 f 3.2 (8) 66.2 t 7.2 (4) 66.8 t 4.6 (5) 73.9 -+ 7.9 (3)

[3H]BumetanideBinding in Endothelial Cells 25

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rcu, mM FIG. 3. Effect of extracellular [Cl-] on endothelial cell NaK-Cl cotransport. Bovine aortic endothelial cells were cultured and subcultured as described under “Experimental Procedures.” Na-K-CI cotransport was determined as ouabain-insensitive bumetanide-sensitive K+ influx (with =Rb as tracer). Cells were preincubated for 5 min in Hepes-buffered media containing 145 mM Na+, 6 mM K+, and various concentrations of C1- (substituted with gluconate) t 1 mM ouabain t 10 PM bumetanide. The medium was then replaced with identical freshmedium containing 1 pCi/ml =Rb and thecells assayed for 5 min. Values represent means +. S.E. of quadruplicate determinations from two separate experiments. A t 50 mM C1- ([Cl-] used in the [3H]bumetanide binding studies) Na-K-C1 cotransport-mediated K+ influx is 15.0 pmol of K+/g of protein/min.

of these experiments are shown in Table I. [3H]Bumetanide was present in the binding assay medium at 0.3 pM. Determination of specific 13H]bumetanide binding was done as described under “ExperimentalProcedures.” Addition of norepinephrine (10 p ~ or) rat atriopeptin 111 (100 nM) to the binding assay medium was found to significantly reduce specific binding of [3H]bumetanide below the control value. Elevation of intracellular cyclic AMP or cyclic GMP by treatment of the cells with the permeableanalogs, 8-bromo-cyclic ) caused a AMP (50 p M ) or 8-bromo-cyclic GMP (50 p ~ also significant reduction in specific [3H]bumetanide binding as didtreatment of the cells with phorbol12-myristate 13acetate (10 nM) to activate protein kinase C. These agents have all been previouslyreported to reduce activity of Na-KC1 cotransport. In contrast, treatmentof the cells during the binding assay with eithervasopressin (100 nM) or bradykinin (100 nM), agents previously shown tostimulate Na-K-C1 cotransport, was found to eitherincrease binding or be without effect, such that no statistically significanteffectwas observed (data not shown). DISCUSSION

In the present study, cultured bovine aorticendothelial cells were found to exhibitspecific saturable [3H]bumetanide binding with a B,,, value of 0.83 pmol/mg protein and a K d value of 0.13 kM. This K d value is ingood agreement with the K, value previously determined for bumetanide inhibition of Na-K-Cl cotransport in the endothelial cells (3) and therefore supports thehypothesis that specific binding of [3H]bumetanide to the cells does indeed represent binding to theNa-K-C1 cotransporter, as has been reported for other cell and tissue types (16,19,20,22). The number of binding sites determined to be presentperendothelial cell (230,000) indicates that these cells possess many copies of the cotransporter relative to other cell types. That is, other studies of [3H]bumetanide bindinginintact cells have shown thatavianandferret erythrocytes exhibit1,000 and 12,000 [3H]bumetanide binding sites/cell,respectively (22, 23). Ehrlich ascites tumor cells have been reported topossess 2 X lo6 sites/cell(24), although

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the cells usedin thatstudyarereportedtoexhibitboth bumetanide-sensitiveNa-ClcotransportandNa-K-ClCOtransport. Vascular smooth muscle cells appear to exhibit approximately 150,000 [3H]bumetanidebinding sites/cell (21). The ion dependence of [3H]bumetanide binding in bovine aortic endothelial cells is similar to that reported for intact duck erythrocytes and membrane preparations of dog kidney, rabbit parotid gland, and winter flounder intestine (16, 19, 20, 22). That is, all three transported ion species, Na+, K’, and C1-, are required for binding to occur in the endothelial cells. In addition, theeffect of [Cl-] on [3H]bumetanide binding was found tobe biphasic, withbinding increasingas [Cl-] is increased, up to approximately 50 mMC1-, and binding decreasing at higher concentrations of C1-. The optimal [Cl-] observed in the present studyfor [3H]bumetanide binding to bovine aortic endothelial cells is comparable with that reported for intact duck erythrocytes (22).An absolute dependence on thepresence of Na+, K’, and C1- for [3H]bumetanide binding to occur in the endothelial cells suggests that the specific binding of [3H]bumetanide represents binding to NaK-Cl cotransporters in these cells and not to Na-Cl or K-Cl cotransporters. An evaluation of the effect of [ C1-] on bumetanide-sensitive K’ influx in the endothelial cells revealed a sigmoidal relationship, as has been reported for Na-K-C1 cotransport of fibroblasts and ferret erythrocytes(11,25) when gluconate is the substituting anion. This finding is consistent with the presence of more than one transport site for C1- on the cotransporter and with the 1Naf:1K+:2C1- stoichiometry of NaK-Cl cotransport that has been demonstrated in other cell types (16). From these data, the magnitude of Na-K-C1 cotransport observed under the same ionic conditions as used in the binding assays( i e . 145 mM Na+, 6 mMK’, and 50 mM C1-) was found to be 15 pmol of K+/g of protein/min. This information, together with the B,,, valuefor specific [3H] bumetanide binding, was used to calculate a turnover number of 300 K+/site/s for Na-K-Cl cotransport in the endothelial cells. This number is ratherlow compared with that reported for avianerythrocytes at 41 “C (4000 K+/site/s(22))but higher thantheturnovernumber calculatedforvascular smooth muscle cells (70 K+/site/s (21)). A number of agents previously shown to modulate activity of endothelial cell Na-K-C1 cotransport were found in the present study to also alter [3H]bumetanide binding. Thus, 8Br-CAMP, 8-Br-cGMP, PMA, norepinephrine, andrat atriopeptin I11 were observed to reduce specific [3H]bumetanide binding by approximately 30%.These agents previously have been shown to inhibit Na-K-Cl cotransport-mediated K+ influx by the same degree (3). This finding provides further support for the hypothesis that specific [3H]bumetanide binding in the endothelial cells does indeed represent binding to the Na-K-C1 cotransporters. There are a t least two possible explanations for the reduction of [3H]bumetanidebinding that isobserved in the presence of these agents: 1)the number of Na-K-Cl cotransporters present in the plasma membrane could decrease; or 2) the cotransporter could be altered in a manner (e.g. via a phosphorylation) that changes its interactionwithbumetanide. Vasopressin andbradykinin, two agents previously shown to stimulate Na-K-Cl cotransport activity in endothelial cells (3), were not found to significantly alter I3H]bumetanide binding in these cells. This suggests that the signal transduction pathway by which vasopressin and bradykinin stimulate Na-K-Cl cotransport activitydoes not involve a covalent modification or conformational change

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in the cotransporter that is reflected by a change in [3H] bumetanide binding. In conclusion, the present study demonstrates thatlarge the magnitude of Na-K-C1 cotransport-mediated K+ influx observed in cultured bovine aortic endothelial cells appears to be dueto thepresence of many copies of the Na-K-Cl cotransporter in these cells. The high density of [3H]bumetanide binding sites in the endothelial cells provides further evidence of the potential importanceof Na-K-C1 cotransport in these cells. In addition, these findings suggest that the cultured aorticendothelial cells, a homogenous population of cells readily available in large quantities, may be useful in studies designed to isolate and purify the Na-K-Cl cotransporter. Acknowledgments-I wish to thankDr. H. C.Palfrey for providing [3H]bumetanide and for helpful discussions and Kim Steutermann for her excellent technical assistance. REFERENCES 1. Forbush, B., 111, and Palfrey, H. C. (1983) J. Biol. Chem. 258, 11787-11792 2. Gordon, J. L., and Martin, W. (1983)Br. J.Phurmacol. 79,531541 3. O’Donnell, M. E. (1989)Am. J. Physiol. 257,C36-C44 4. Avruch, J., and Wallach, D. F. H. (1971)Biochim. Biophys. Acta 33,234-237 5. Owen, N. E., and Prastein, M.L. (1985)J. Biol. Chem. 260, 1445-1451 6. Brock, T. A., Brugnara, C., Canessa, M., and Gimbrone, M. A.,

Jr. (1986)Am. J. Physiol. 250, C888-C895 7. O’Donnell, M. E. (1989)J. Cell Biol. 109,314a 8. O’Donnell, M. E., and Owen, N. E. (1986)Proc. Natl. Acad. Sci. U. S.A. 83,6132-6136 9. Owen, N. E. (1984)Biochem. Biophys. Res. Commun. 125,500508 10. Smith, J. B., and Smith, L. (1987)J. Membr. Biol. 99,51-63 11. Owen,N.E., and Prastein, M. L. (1985)J. Biol. Chem. 260, 1445-1451 12. Atlan, H., Snyder, D., and Panet, R. (1984) 81,181-188 13. McRoberts, J. A., Erlinger, S., Rindler, M. J., and Saier, M. H., Jr. (1982)J. Biol. Chem. 257,2260-2266 14. Frelin, C., Chassande, O., and Lazdunski, M. (1986)Biochem. Bwphys. Res. Commun. 134,326-331 15. Forbush, B., 111, and Palfrey, H. C. (1983) J. Bwl. Chem. 258, 11787-11792 16. O’Grady, S. M., Palfrey, H. C.,and Field, M. (1987)J. Membr. Biol. 96,ll-18 17. Griffiths, N. M., and Simmons, N. L. (1987)Q. J. Exp. Physiol. 72,313-329 18. Forbush, B., 111, and Haas, M. (1988)Biophys. J. 53,222 19. Forbush, B., 111, and Palfrey, H. C. (1982) Biophys. J. 37, 161a 20. Turner, R. J., and George, J. N. (1988)J. Membr. Bwl. 102,71-

77 21. O’Donnell, M. E., and Owen, N. E. (1988)Am. J. Physiol. 255, C169-C180 22. Haas, M., and Forbush, B., 111 (1986)J. Bwl. Chem. 261,84348441 23. Mercer, R. W., and Hoffman, J. F. (1985)Biophys. J. 47, 157a 24. Hoffman, E. K., Schiodt, M., and Dunham, P. (1986)Am. J . Physiol. 250, C688-C693 25. O’Grady, S. M., Palfrey, H. C., and Field, M. (1987)Am. J . Physiol. 253,C177-Cl92