JEZ 776 460 THE M.W.JOURNAL MUSCH ET OFAL. EXPERIMENTAL ZOOLOGY 277:460–463 (1997) RAPID COMMUNICATIONS Hypotonic Stress Induces Translocation of the Osmolyte Channel Protein pICln in Embryonic Skate (Raja eglanteria) Heart MARK W. MUSCH,1 CARL A. LUER,3 ERIN M. DAVIS-AMARAL,2 LEON GOLDSTEIN2* 1 Inflammatory Bowel Disease Center, Department of Medicine, The University of Chicago, Chicago, Illinois 60637 2 Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912 3 Mote Marine Laboratories, Sarasota, Florida 34236 AND ABSTRACT Volume expansion of cardiac cells from a wide variety of species stimulates the efflux of the β-amino acid taurine through an osmolyte channel. Previous studies have suggested that the osmolyte channel in epithelial cells is a swelling-activated anion channel (pICln). In skate heart, a 37-kDa protein is present which is recognized by a specific antibody to a protein characterized in MDCK cells as pICln. This protein is present predominantly in the cytosol (only 10% in the membrane fraction) of heart incubated under isotonic conditions. After transfer to hypotonic medium (one-half osmolarity), the distribution of this protein is markedly altered and significant amounts of the protein are found in the membrane fraction. After hypotonic exposure, the amount of the protein in the membrane fraction rises to 38 ± 11% (range 18–53, n=3). The translocation to the membrane fraction suggests that this protein may play a role in the taurine efflux in this tissue stimulated by hypotonic stress. J. Exp. Zool. 277:460–463, 1997. © 1997 Wiley-Liss, Inc. Under volume-expanded conditions, cardiac cells, like a wide variety of other cells, demonstrate an efflux of osmolytes to accomplish a regulatory volume decrease (RVD; for review see Strange et al., ’96). A number of solutes participate in the RVD including the β-amino acid taurine, polyols, methlamines, and electrolytes. Cardiac cells of the skate, like those of many species, accumulate millimolar concentrations of the nonmetabolized amino acid taurine, which is a major solute lost during the RVD from this tissue (Goldstein et al., ’93). In the chicken heart, volume-activated taurine efflux is thought to occur through a swelling-activated anion channel (Zhang et al., ’93) similar to channels found in MDCK cells (Banderali and Roy, ’92), lung cells (Kirk and Kirk, ’93), and C6 glioma cells (Jackson and Strange, ’93). Strange et al. (’96) have proposed that during RVD, osmolytes may be lost from epithelial cells via a pathway involving the protein pICln. Antibodies to this protein recognize a 37-Kda protein (pICl n) in cells from a wide variety of tissues and species (Krapivinsky et al., ’94) and cloning techniques have identified this protein in rabbit heart (Okada et al., ’95) and ciliary © 1997 WILEY-LISS, INC. epithelium (Anguita et al., ’95). This protein is found in the cytosol and is not predicted to be an integral membrane protein by any of the hydropathy algorithms used. Therefore, it is unclear how this protein may mediate the efflux of solutes across the membrane. A model has been proposed that upon such stimuli as hypotonic shock, the protein forms a dimer, perhaps in a beta-barrel configuration, and that this form inserts into the cell membrane and acts like a channel or regulates a preexisting anion channel (Buyse et al., ’96). Therefore, the aim of the present studies was to test this model and determine where this protein resides in the skate cardiac cytosol under isotonic conditions and if redistribution to the cell membrane occurs under hypotonic conditions. Contract Grant sponsor: NSF; Contract Grant number IBN9505567; Contract Grant sponsor: NIH; Contract Grant number DK-38510, DK-42086; Contract Grant sponsor: Walt Disney Company. *Correspondence to: Leon Goldstein, Ph.D., Division of Biology and Medicine, Brown University, Box G B311, Providence, RI 02912. Received 9 October 1996; Revision accepted 18 December 1996 VOLUME-STIMULATED CHANNEL TRANSLOCATION MATERIALS AND METHODS Distribution of pICln in embryonic skate heart Skate embryos for these studies were used 7– 11 weeks after oviposition with cardiac masses of 4–17 mg. Details of the surgical technique used to obtain hearts were described previously (Goldstein et al., ’93). Hearts were incubated in isoosmotic elasmobranch incubation medium (940 EIM; 940 mosmoles/1, composition in mmol/l: 300 NaCl, 5 CaCl2, 5.2 KCl, 2.7 MgSO4, 15 Tris pH 7.4, 370 urea) or one-half osmolarity (460 EIM, NaCl reduced to 100 and urea to 250 mM). Hearts were placed individually into beakers containing 30 mL of either 940 EIM or 460 EIM. Hearts were incubated for 1 h with constant, gentle oxygenation of the incubation medium. Following incubation, the hearts were frozen immediately on dry ice. Frozen hearts were placed in 200 µl buffer (composition in mmol/l: 10 Tris pH 7.4, 5 MgCl2, 1 phenylmethylsulfonyl fluoride, with 10 µg/ml leupeptin, aprotinin, pepstatin, and tosylphenylchloroketone). Hearts were minced finely with a razor blade and then homogenized by 30 strokes with a tight-fitting glass homogenizer. Poorly broken material was pelleted (500 g for 2 min at 4°C). The membrane fraction was resuspended in homogenization buffer and aliquots of both cytosol and membrane fractions removed for protein analysis using the bicinchononic acid procedure (Smith et al., ’85). One-half volume of 3X Laemmli stop solution (1% 2-mercaptoethanol, 0.5 M Tris pH 6.8, 0.5% w/v SDS, 50% v/v glycerol) was added to the remainder and samples were heated to 85°C for 10 min and frozen. For analysis of pICln 10 µg of protein was separated on 12.5% SDS-PAGE, and transferred to a polyvinylidene fluoride membrane (Immobilon) using Towbin’s buffer (25 mM Tris, 192 mM glycine, pH 8.8). Blots were blocked in 5% w/v nonfat dry milk in Hanks buffered saline (HBS) with 0.2% v/v Nonidet P40 (5% Blotto) for one hour. Blots were incubated with affinity-purified polyclonal anti-pICln antiserum (1 µg/ml) in HBS with 1% w/v bovine serum albumin, overnight at 4°C. For determination of separation of cytosol and membrane fractions, marker proteins were analyzed. Na-K-ATPase was selected as the membrane marker and hsp73/72 as the cytosol marker. Blots were then washed in 5% Blotto and 1% Blotto (milk reduced to 1% w/v) and then incubated in peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1:4,000) for 60 min in 1% Blotto. 461 Blots were then washed in 1% Blotto and then in HBS with Nonidet P40. pICln protein and the marker proteins were detected using an enhanced chemiluminescent system. Materials Antibody to pICln was a generous gift of Drs. D. E. Clapham and G. B. Krapivinsky (Mayo Institute, Rochester, MN). Immobilon membrane was obtained from Millipore (Medford, MA), peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse from Southern Biotechnology (Birmingham, AL), anti-Na-K-ATPase from Upstate Biotech (Lake Placid, NY), anti-hsp73/72 from Affinity Bioreagents (Boulder, CO), Nonidet P 40 from Amersham (Cleveland, OH), and chemiluminescent reagents from Pierce (Rockford, IL). All other reagents were of the highest grade available and obtained from Fisher (Itasca, IL) or Sigma Chemical (St. Louis, MO). RESULTS Identification of pICln in skate heart Western blots were used to determine whether pICln is found in skate heart and is similar in size to the protein in MDCK cells. A specific polyclonal antiserum recognizes a protein of similar molecular mass in both cell types (Fig. 1). The abundance of the protein is likely lower in cardiac myocytes since 10 µg of MDCK protein versus 200 µg of skate heart protein were required to obtain similar Western blots. Although intact heart was used in these experiments, cardiac myocytes compose a majority of the cells in the tissue. Additionally, as erythrocytes do not have pICln, these cells would not interfere with the analysis (DavisAmaral et al., ’97). Hypotonicity stimulates redistribution of pICln Under isotonic conditions, pICl n is predominantly in the cytosol of skate cardiac cells (Fig. 2). The amount of pICln in the cytosol and membrane fractions of skate hearts was analyzed densitometrically using NIH IMAGE 1.54 software. In two of three cases, less than 10% of pICln was in particulate fraction and in the other case, this value was 19.8%. When the hearts are incubated in one-half hypotonic medium, the distribution of pICln changes dramatically (Fig. 2). Large amounts of the protein redistribute so that substantial amounts of pICln are found in the particulate fraction. This redistribution was observed 462 M.W. MUSCH ET AL. Fig. 2. Distribution of pICln under isotonic and hypotonic conditions. Cell fractions were made as described in Materials and Methods and pICln in the fractions determined by Western blotting. One-twentieth of the total protein in each fraction was used to determine distribution of pICln. Chemiluminescent images shown represent distribution of pICln of two hearts under hypotonic conditions and two different hearts under isotonic conditions. M = membrane fraction. C = cytosol fraction. Fig. 1. Demonstration of pICln protein in skate heart. MDCK (10 µg protein) was used since pICln is readily identified in these cells and 200 µg protein of skate heart homogenate was used. Western blots were developed with an affinity-purified polyclonal antisera. Chemiluminescent image shown is representative of one of three different hearts. in every heart used. The means (± S.E., n=3) for particulate and cytosol distribution under isotonic conditions were 10 ± 4% and 90 ± 4%, respectively, and under hypotonic conditions these values were 38 ± 11% and 62 ± 10%. The values for the cytosolic percentage were significantly different at the 0.01 level by paired Student’s t test. To confirm that cytosol and membrane fractions were separated and that the distribution of marker proteins did not change with hypotonic stress, blots were analyzed using antibodies against Na-K-ATPase as a membrane marker and hsp73/72 as a cytosol marker. As shown in Figure 3, clear separation of hearts into cytosol and membrane fractions was accomplished since the marker proteins were found only in the correct fractions in all three experiments. Hypotonic stress did not alter the distribution in any experiment (n=3). Fig. 3. Distribution of membrane (Na-K-ATPase) and cytosol (hsp73/72) markers under isotonic and hypotonic conditions. Cell fractions were made as described in Materials and Methods and pICln in the fractions determined by Western blotting. One-twentieth of the total protein in each fraction was used to determine distribution of pICln and the marker proteins. Chemiluminescent image shown is representative of those in three separate experiments. DISCUSSION The regulation of cell volume within a narrow range is vitally important to marine species which VOLUME-STIMULATED CHANNEL TRANSLOCATION may encounter a wide range of environmental osmolarities. The maintenance of cell volume by all cells is an important function and all cells have developed multiple pathways to accomplish this action. Along with the efflux of electrolytes, numerous organic molecules are used to reduce cell volume and accomplish the regulatory volume decrease. These organic molecules are of a wide variety of chemical groups. However, they share certain characteristics: (1) they are all of a limited hydrated size, (2) they are all accumulated by cells, and (3) they are generally not used as central metabolites in the cell. The β-amino acid taurine is one of these important organic osmolytes; others include betaine, choline, and sorbitol. pICln is a protein that mediates a hypotonicstimulated Cl current and is pivotal in Cl efflux in hyptonically treated Xenopus oocytes (Paulmichl et al., ’92; Ackerman et al., ’94). This protein may be the one which is responsible for the functions attributed to a volume-sensitive organic anion channel (VSOAC) characterized in MDCK and C6 cells (Strange et al., ’96). pICln appears to be ubiquitous in tissues as diverse as brain, spleen, intestine, and cardiac muscle all have this protein. It also appears to be well conserved phylogenetically as a protein of the same molecular mass is recognized in elasmobranchs and mammals. In addition to the results reported here for cardiac cells, a recent preliminary report has shown that pICln translocates from cytosol to membrane fractions in other epithelial cells under hypotonic stress (Laich et al., ’96). It appears therefore that the phenomenon may be of general importance to volume regulation in animal cells. LITERATURE CITED Ackerman, M.J., K.D. Wickman, and D.E. Clapham (1994) Hypotonicity activates a native chloride current in Xenopus oocytes. J. Gen. Physiol., 103: 153–179. Anguita, J., M.L. Chalfant, M.M. 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