Madin-Darby Canine Kidney Epithelial Cell Line

The Madin-Darby canine kidney (MDCK) cell line, derived from distal tubule/ collecting duct, expresses differentiated properties of renal tubule epithelium in culture. We studied the expression of adrenergic receptors in MDCK to examine the role of catecholamines in the regulation of renal function. Radioligand-binding studies demonstrated, on the basis of receptor affinities of subtype-selective adrenergic agonists and antagonists, that MDCK cells have both atand ~2-adrenergic receptors. To determine whether these receptor types were expressed by the same cell, we developed a number of clonal MDCK cell lines. The clonal lines had stable but unique morphologies reflecting heterogeneity in the parent cell line. Some clones expressed only/~2-adrenergic receptors and were nonmotile, whereas others expressed both ~tand /~2-receptors and demonstrated motility on the culture substrate at low cell densities. In one clone, aand/~-receptor expression was stable for more than 50 passages. Catecholamine agonists increased phosphatidylinositol turnover by activating a-adrenergic receptors and cellular cyclic adenosine monophosphate accumulation by activating/3-adrenergic receptors. Guanine nucleotide decreased the affinity of isoproterenol for the ~2-receptor but did not alter the affinity of epinephrine for the c~l-receptor. These results show that c~land/32-receptors can be expressed by a single renal tubular cell and that the two receptors behave as distinct entities in terms of cellular response and receptor regulation. Heterogeneity of adrenergic receptor expression in MDCK clones may reflect properties of different types of renal tubule cells. Catecholamines regulate a variety of cellular functions in the mammalian kidney, including tubule water and ion reabsorption, renin release, renal hemodynamics, and gluconeogenesis (20, 35). The influence of renal sympathetic nerves (which release catecholamines as neurotransmitters) on renal function has been discussed in several reviews (1, 6, 32). Catecholamines act on target cells by initially binding to cell surface receptors, and we and others (20, 41-43) have identified a~-, ~2-, B~-, and/~2-adrenergic receptors in membrane preparations of the kidney cortex by radioligand-binding techniques. However, the sites of action of adrenergic agents along the nephron have not been clearly defined. Determination of the location of catecholamine action within the kidney is particularly important to distinguish indirect adrenergic effects (resulting from changes in vascular tone) from direct effects on epithelial transport and metabolism. The kidney consists of segments that are anatomically and functionally distinguishable, yet each segment may contain several morphologically distinct cell types (8). To understand the regulation of renal function by catecholamines and sympathetic nerves, it therefore is important to study homogeneous populations of renal cells. Such populations may be found in the established renal cell lines, which have provided valuable information regarding kidney transport and metabolism (15). We chose the Madin-Darby canine kidney (MDCK) ~ cell line as a model system in which to examine the actions of catecholamines on renal tubule epithelium. The MDCK cell line was established in 1958 from normal dog kidney (12). MDCK cells retain differentiated properties of renal tubule ~Abbreviations used in this paper. MDCK, Madin-Darby canine kidney cells; and PtdIns, phosphatidylinositol; ICYP, iodocyanopindolol; IHEAT, iodo-2-[/3(4-hydroxyphenyl)ethylaminomethyl]tetralone. THE JOURNAL OF CELL BIOLOGY VOLUME 97 AUGUST 1983 405 415 © The Rockefeller University Press • 0021-9525/83/08/0405/1151.00 4 0 5 on N ovem er 7, 2017 jcb.rress.org D ow nladed fom epithelium, including transepithelial water and solute transport (28). These cells appear to be derived from distal tubule/ collecting duct because of their enzyme markers (38), morphologic features (47), and antigenic determinants (16). Cells of the distal nephron are likely target sites for the catecholamines. The basolateral surface of the distal tubule is directly innervated by renal sympathetic fibers, and the distal nephron is the most responsive segment to B-adrenergic agonists as measured by cAMP accumulation (7). The presence of both aand ~-adrenergic responses in MDCK cells has been reported. In these cells, a-adrenergic agonists stimulate arachidonic acid metabolism (23) and K + effiux (3). ~-Adrenergic agonists increase cyclic adenosine monophosphate (cAMP) levels (38, 39) and transepithelial CIsecretion (2). The adrenergic receptor subtype specificities of these responses have not been identified, although knowledge of the nature of these subtypes is important for understanding the mechanisms mediating target cell responses (11) and for identifying endogenous agonists and exogenous agonists and antagonists that will preferentially occupy the target cell receptors (1, 6, 20, 32, 35). Moreover, several investigators (22, 24, 37, 47) have presented evidence that MDCK is not a homogeneous cell line. We, therefore, addressed the following questions: (a) Are aand ~-adrenergic receptors present on renal tubule epithelium? (b) If so, which adrenergic subtype(s) are present? (c) Are aand ~-adrenergic receptors coexpressed by the same epithelial cell? (d) Are responses to aand ~-adrenergic agonists independently mediated? In this paper, we describe the characterization of aand ~adrenergic receptors in MDCK cells in terms of their ligand specificities, guanine nucleotide regulation, and intracellular second-messenger systems. We have developed a number of clonal MDCK cell lines with different morphologic features and different adrenergic receptor expression. We conclude that: (a) MDCK cells express amand ~2-adrenergic receptors; (b) MDCK is a heterogeneous cell line consisting of at least two stable cell types, one with both a~and Be-receptors and the other with only ~2-receptors; (c) in a clonal cell line derived from parent MDCK cells, aland ~2-agonists mediate discrete intracellular responses that are independently regulated. Preliminary reports of some of these findings have been presented in abstract form (29, 30). MATERIALS AND METHODS Materials: The following compounds were received as gifts from the sources indicated: (-)-epinephrine, (+)-epinephrine, and (-)-norepinephrine (+)-bitartrate salts (Stealing Winthrop Research Institute, Rennselear, NY); (-)propranolol HC1, (+)-propranolol HCI (Ayerst Laboratories, New York); phentolamine mesylate, clonidine (Geigy Pharmaceutical, Summit, NJ); cyanopindolol HCI (Sandoz Inc., Basel, Switzerland); prazosin (Pfizer Inc., Groton, CT); practolol (ICI Americas, Inc., Wilmington, DE); 2[~-(4-hydroxyphenyl)ethylaminomethyl]tetralone HCI (Beiersdorf AG, Hamburg, Federal Republic of Germany); and IPS 339 (Dr. Kenneth Minneman, Emory University, Atlanta, GA). The following radiochemicals were purchased from the sources indicated: carrier-free Na~25I (New England Nuclear, Boston, MA, Amersham Corp., Arlington Hts., IL); m vo-[2H]inositol, [3H]prazofin (Amersham); [3H]yohimbine (New England Nuclear). Formula 946 liquid scintillation cocktail was from New England Nuclear. Cell culture media and sera were from Grand Island Biological Co., Grand Island, NY. Plasticware for cell culture was manufactured by Falcon Labware, Div. of Becton, Dickinson & Co. (Oxnard, CA), Costar (Cambridge, MA), or Lux Scientific Corp. (Naperville, IL). All other reagents were from standard sources. Preparation of [12Sl]lodocyanopindolol ([12SlJICYP) and [12Sl]lodo_ 2_[l~(4_hydroxyphenyl)ethylaminomethyl]tetralone ([12Sl]IHEA 7-): Cyanopindoiol (CYP) was iodinated by minor modification of the method of Engel et al. (10). The reaction mixture contained 20 #g CYP, 406 THE JOURNAL OF CELL BIOLOGY VOLUME 97, 1983 10 #1 13.5 mM HCI, 20 #1 potassium phosphate buffer (0.3 M, pH 7.6), 2 mCi cartier-free Nan~I, and 20 #1 chloramine T in aqueous solution (0.34 mg/ml). The mixture was incubated for 5 min at room temperature, after which the reaction was stopped by the addition of 300 ~! of aqueous Na2S203 (6.3 mM). NaOH (10 #1 of I N) was then added, and the reaction mixture was extracted four times with 300 #1 0.01% phenol in ethylacetate. The combined washes were concentrated under N2 and then subjected to ascending paper chromatography at room temperature on 3-ram paper (22 × 24 cm) (Whatman Ltd., Clifton, NJ) with 0.01% phenol in 0.1 M ammonium formate (pH 8.5) as the solvent (running time ~4 h). The iodinated product migrated with an Rf of 0.09 and was extracted from the appropriate strip of the chromatogram with 0.01% phenol in methanol for ~24 h at -20°C. The [~25I]ICYP migrated as a single spot when subjected to thin-layer chromatography on silica gel uniplates (Analtech Inc., Newark, DE) with pyridine/glacial acetic acid/water (0.33:0.6:9.07, vol/vol/vol) as the solvent. [I251]ICYP was stable for at least 12 wk when stored as a 100 nM solution in 0.01% phenol in methanol. 2[fl-(4-HydroxylphenyDethylaminomethyl]tetralone (HEAT) was iodinated by a modification of the method of Engel and Hoyer (9), which is the same as that described above for [m'sI]ICYP except the reaction time was 1 min. When subjected to ascending paper chromatography, the product migrated with an Rf of 0.13. The [1251]IHEAT migrated as a single spot when subjected to thinlayer chromatography on silica gel with pyridine/glacial acetic acid/water (0.33:0.6:9.07, vol/vol/voi) as the solvent (Rf = 0.7). When methanol/chloroform (30:50, vol/vol) was used as solvent, the product ran with an Rf of 0.79, and trace radiochemical impurities with Rrs of 0.25 and 0.92 were detected. The [~25I]IHEAT was stable for at least 4 mo when stored at -20°C as a 100 nM solution in 0.01% phenol in methanol; the amounts of the radiochemical impurities did not increase during storage. Cell Culture: Low-passage MDCK cells (catalog No. CCL34, pat~age 53) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). ATCC-derived cells of a later passage number were obtained from Dr. Milton Saier, Department of Biology, University of California, San Diego. The cells were maintained in Dulbecco's modified Eagle's medium supplemerited with 7.5% heat-inaetivated horse serum, 2.5% heat-inactivated fetal calf serum, 15 mM HEPES, and 20/~g/ml gentamycin. Serum-free medium was prepared according to the formulation of Taub et al. (46). Confluent cells were subcultured once weekly with a trypsin-EDTA solution and were inoculated at dilution ratios between 1:4 and 1:10 into 250-ml culture flasks containing 20 ml of medium. The cells were maintained in a humidified 37"C incubator in an atmosphere of 90% air and 10% CO2. Clonal cell lines were obtained by limiting dilution (<0.5 cell/ml) of single-cell suspensions prepared from subconfluent cultures into 24-well plates (1 ml/well; 2 cm 2 surface area/well). Cional growth was confirmed by frequent microscope inspection of the wells. Cell lines were stored at -70°C af~cr detachment with trypsin-EDTA and resuspension in growth medium containing 10% dimethyl sulfoxide (vol/vol). Membrane Preparation: Cell membranes were prepared by a hypotonic lysis method from MDCK cells grown to confluence (3-6 d) in 150mm culture dishes. Each dish was washed twice with 5 ml of ice-cold lysis buffer (1 mM Tris-HCl, 2 mM MgC12, pH 7.5) and incubated with 10 ml of lysis buffer for 10 rnin at 4°C. The lysed cells were removed from the dishes by scraping with a Costar cell scraper. The dishes were washed once with 5 ml of lysis buffer, and the pooled lysate was centrifuged at 30,000g for 10 rain at 4°C. The pellet was washed two more times with lysis buffer and resuspended in icecold incubation buffer (145 mM NaCI, 20 mM Tris-HCI, 2 mM MgCI2, pH 7.5). This procedure resulted in uniform lysis of the cells. The resulting preparation consisted of cell membrane "ghosts" and all of the nuclei could be stained by the addition oftrypan blue. The cell concentration in the suspension was determined for each experiment by mixing 100 #1 of suspension and 100 #i of 0.4% trypan blue and then counting the intact, trypan blue-stained nuclei in a hemacytometer. Membrane protein was determined by the method of Lowry (25) using bovine serum albumin standards. The protein content was 0.146 + 0.03 my/10 ~ lysed cells and was ~ 15% higher for subconfluent cells than for confluent cells. Radioligand Binding Assays: Binding assays were performed.in duplicate or triplicate by incubating 0.125-0.2 ml of freshly prepared membrane suspension (0.5-3 × 10 ~ cells/tube)with 0.025 ml of [J25I]ICYP or [mI]IHEAT or 0.05 ml of [3H]prazusin and 0.025-0.1 ml of various drugs in a final volume of 0.25 ml (0.5 ml for [3H]prazusin) in polypropylene test tubes (16 × 105 ram, Walter Sarstedt, Inc., Princeton, NJ). Assays were initiated by the addition of membrane and were carried out in incubation buffer (145 mM NaC1, 20 mM Tris-HCl, 2 mM MgCI2, pH 7.5). For equilibrium binding studies, membranes were incubated for 60 rain at 37°C in a shaking water bath (~80 cycles/rain). The incubation was terminated by the addition of 10 ml of 37°C incubation buffer. Bound and free radioligand were then separated by rapid (<10 s) filtration over Whatman GF/C glass fiber filters (Whatman Ltd.) on a Millipore filtration manifold (Millipore Corp., Bedford, MA), and the filters were washed on N ovem er 7, 2017 jcb.rress.org D ow nladed fom with 10 ml of 37°C incubation buffer. Radioactivity retained on the filters was determined using a Searle gamma counter (Searle Analytic, Inc., Des Plaines, IL) at 63% efficiency (~2sI), or with 4.5 ml of scintillation cocktail in a Beckman scintillation counter (Beckman Instruments, Inc., Fuilerton, CA) at 30% efficiency (3H). Nonspecific binding was defined with the following agents: for [3HI prazosin and [3H]yohimbine, 10 pM phentolamine; for [~251]IHEAT, 0.5 or 1.0 /~M prazosin; and for [125I]ICYP, 1.0/~M (__.)propranolol. Specific binding was determined by subtraction of nonspecific binding from total binding. The specific binding was routinely the following percents of the total binding at concentrations of the radioligands near their dissociation constants: >60% for [3H]prazosin, >70% for [125I]IHEAT and >75% for [12~I]ICYP. Specific binding of the radioligands was linear with cell number. When catecholamines were used in binding experiments, ascorbic acid was included at a final concentration of 0.5 mg/ml to prevent drug oxidation. Replicate data points varied by <10%. Data Analysis: The dissociation constant (KD) and maximum number of binding sites ( B ~ ) were determined from Scatchard analysis (40) of saturation binding isotherms. The line of the Seatehard plot was fitted by linear regression analysis. Competitive binding curves were analyzed by a computer program (LIGAND) that performs iterative nonlinear regression (33). Phosphatidylinositol Assay: MDC[(-D cells were seeded in 35-mm culture dishes containing 3 rnl of medium find grown to confluence (2 d). Assays were performed with triplicate dishes in a humidified 37°C incubator with a 5% CO2 atmosphere. Experiments were initiated by rinsing each dish twice with 20 ml of 37°C Krebs-Henscleit buffer (KHB: 118 mM NaCi, 4.7 mM KCI, 3.0 mM CaCI2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 0.5 mM EDTA, 25 mM NaHCO3, and 10 mM glucose, pH 7.4) and then adding 750/~1 ofmyo[3H]inositol (6.6 Ci/mmol, ~4.5/~Ci/dish) in KHB. 60 min later, 7.5 pl of drug or control solution was added, and the incubation was continued for an additional 30 rain. Ascorbic acid (5 mg/ml) was included in the freshly prepared epinephrine stock solutions to prevent drug oxidation bafore addition to the cells (final concentration, 0.05 mg/ml). Incubations were terminated by removal of the incubation mixture by aspiration followed by three washes with 2 ml of ice-cold saline. Cold saline (0.5 ml) was then added, and the cells were removed from each dish by scraping. Each dish was washed two more times with 0.5 ml of cold saline, and the pooled washes were centrifuged for 30 s in a microfuge. The supernatant was removed, and 0.75 mi of ice-cold chloroform/methanol (1:2) was added to the pellet. The termination procedure was completed in <3 rain. Phosphatidylinositol (PtdIns) was extracted by the addition of 0.2 ml of 2 M KCI to each sample (final proportions of chloroform/methanol/2 M KCI, 10:5:4) followed by sonication using a Kontes cell disrupter in three 10-s bursts. A two-phase system was formed by the addition of 0.25 ml of chloroform and 0.25 ml of 2 M KCI followed by centrifugation at 1,600g for 20 min at 4°C. The aqueous upper phase and any interfacial material were removed, the organic lower phase was decanted, and the remaining tissue pellet was discarded. The lower phase was washed twice with 0.5 ml of chloroform/methanol/water (3:47:48) and dried under N2, and the tritium content was determined in 3.5 ml of OCS scintillation cocktail (Amersham)frriton X-100 (2:1). Control experiments showed that <0.05% of the free myo-[3H]inositoi contaminated the lower phase and that 100 + 1% of the total lower phase counts were recovered as PtdIns, as determined by lipid separation by two-dimensional thin-layer chromatography (first dimension: chloroform/methanol/water/18 M NI-I4OH, 130:70:8:0.5; second dimension: chloroform/acetone/methanol/acetic acid/water, 10:4:2:2:1) on 20 x 20-cm silica gel glass plates (Merck & Co., Rahway, NJ) (36). cAMP Assay: MDCK cells were grown to confluence (1-3 d; ~ 5 x 105 cells/well) in 24-v~11 culture dishes. The medium was removed by aspiration, and the cells were washed twice with 1 ml of Hanks' balanced salt solution. Incubation medium (0.4 ml) consisting of a freshly prepared solution of drugs in HBSS was then added, and the dishes were incubated at 37°C for 5 rain. At the conclusion of the incubation, 100 pl of 40% trichloroacetic acid was added to each well. After a 10-rain incubation at room temperature, the trichloroacetic acid extracts were removed from each well and transferred to 0.5 ml Dowex AG 1 x 8 columns. The columns were washed with 2.5 ml of water, and the cAMP was eluted with 4 ml water. Recovery of the cAMP was 90-95% as determined in control columns using [3H]cAMP. The cAMP ¢luate was dried with a Speedvac concentrator (Savant Instruments, Inc., Hicksville, NY). Cyclic AMP was measured by a competitive binding protein assay using aliquots of the dried sample fractions resuspended in sodium acetate buffer (18, 19).