Receptor Regulation of the Volume-Sensitive Efflux of Taurine and Iodide from Human SH-SY5Y Neuroblastoma Cells: Differential Requirements for Ca and Protein Kinase C

The basal (swelling-induced) and receptor-stimulated effluxes of I and taurine have been monitored to determine whether these two osmolytes are released from human SH-SY5Y cells under hypotonic conditions via common or distinct mechanisms. Under basal conditions, both I (used as a tracer for Cl ) and taurine were released from the cells in a volumedependent manner. The addition of thrombin, mediated via the proteinase-activated receptor-1 (PAR-1) subtype, significantly enhanced the release of both I and taurine (3–6-fold) and also increased the threshold osmolarity for efflux of these osmolytes (“set-point”) from 200 to 290 mOsM. Inclusion of a variety of broad-spectrum anion channel blockers and of 4-[(2butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden5-yl)oxy]butanoic acid attenuated the release of both I and taurine under basal and receptor-stimulated conditions. Basal release of I and taurine was independent of Ca or the activity of protein kinase C (PKC). However, although PAR-1stimulated taurine efflux was attenuated by either a depletion of intracellular Ca or inhibition of PKC by chelerythrine, the enhanced release of I was independent of both parameters. Stimulated efflux of I after activation of muscarinic cholinergic receptors was also markedly less dependent on Ca availability and PKC activity than that observed for taurine release. These results indicate that, although the osmosensitive release of these two osmolytes from SH-SY5Y cells may occur via pharmacologically similar membrane channels, the receptor-mediated release of I and taurine is differentially regulated by PKC activity and Ca availability. Cell volume is constantly subject to change as a consequence of solute accumulation, oxidative metabolism, or fluctuations in the osmolarity of the extracellular fluid. To survive, cells need to regulate their volume within relatively narrow limits, and this homeostatic mechanism is of particular importance to the brain because of the restrictions of the skull. A common cause of brain swelling is hyponatremia, a condition that disproportionately affects the elderly, infants, marathon runners, and military personnel (Upadhyay et al., 2006). Hyponatremia is associated with a variety of neurological symptoms, such as disorientation, mental confusion, and seizures (Kimelberg, 2000; Pasantes-Morales et al., 2000, 2002). In response to hypotonic stress, cells swell with a magnitude proportional to the reduction in osmolarity. This is followed by a homeostatic mechanism termed regulatory volume decrease (RVD) that involves the extrusion of intracellular ions such as K , Cl , and a number of organic osmolytes, which together facilitate the loss of water to normalize cell volume (Pasantes-Morales et al., 2000). Inorganic ions constitute two-thirds of the osmolytes released during RVD, and the remainder are accounted for by “compatible” organic osmolytes such as polyols, methylamines, and amino acids. Of these, taurine, an amino acid present in eukaryotic cells at concentrations of up to 40 mM, is considered to be an ideal osmolyte because of its metabolic inertness and abundance (Huxtable, 1992; Lambert, 2004). It is proposed that extrusion of these osmolytes from the cell is mediated via a volume-sensitive organic osmolyte and anion channel (VSOAC), which is primarily permeable to Cl but impermeable to cations (for reviews, see Lang et al., The work was supported by National Institutes of Health Grants NS23831 (to S.K.F.) and F31 NS053020-01 (to T.A.C.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.115741. ABBREVIATIONS: RVD, regulatory volume decrease; VSOAC, volume-sensitive organic osmolyte and anion channel; DDF, 1,9-dideoxyforskolin; NPPB, 5-nitro-2-(3-phenylpropylamino) benzoic acid; DCPIB, 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid; mAChR, muscarinic cholinergic receptor; PAR, proteinase-activated receptor; PKC, protein kinase C; Y-27632, (R)-( )-trans-4-(1aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride; DIDS, 4,4 -diisothiocyanatostilbene-2,2 -disulfonic acid; DMEM, Dulbecco’s modified Eagle’s medium; Oxo-M, oxotremorine-M; Ca i, cytoplasmic calcium; ANOVA, analysis of variance. 0022-3565/07/3203-1068–1077$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 320, No. 3 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 115741/3179025 JPET 320:1068–1077, 2007 Printed in U.S.A. 1068 at A PE T Jornals on M ay 0, 2017 jpet.asjournals.org D ow nladed from 1998; Nilius and Droogmans, 2003). Evidence to support the involvement of a VSOAC in response to hypotonic stress comes from studies in which RVD, volume-sensitive Cl current, and organic osmolyte release can all be blocked by broad-spectrum anion channel inhibitors, such as DDF or NPPB, and by a highly selective agent, DCPIB (Decher et al., 2001; Abdullaev et al., 2006). Similarities in the pharmacological inhibition profile of swelling-activated efflux of organic osmolytes and Cl in response to anion channel blockers has led to the suggestion that a common pathway exists for the extrusion of both Cl and organic osmolytes (Banderali and Roy, 1992; Jackson and Strange, 1993; SanchezOlea et al., 1996; Abdullaev et al., 2006). However this possibility is at variance with results obtained from some nonneural tissues in which Cl and taurine effluxes were found to exhibit differences in kinetics of release, osmotic sensitivity, and/or the degree of inhibition by anion channel blockers, results which suggest the existence of separate volume-sensitive channels for Cl and organic osmolytes (Lambert and Hoffmann, 1994; Davis-Amaral et al., 1996; Shennan et al., 1996; Stutzin et al., 1999; Shennan and Thomson, 2000; Tomassen et al., 2004). When measured in vitro, the efflux of organic osmolytes is relatively insensitive to hypotonic stress, often requiring substantial ( 25%) reductions in osmolarity. However, recent studies from this and other laboratories have demonstrated that the volume-sensitive efflux of organic osmolytes from neural preparations can be enhanced after activation of cellsurface receptors. The latter include P2Y purinergic receptors in rat astrocytes (Mongin and Kimelberg, 2002, 2005), M3 muscarinic cholinergic receptors (mAChRs), lysophosphatidic and sphingosine 1-phosphate receptors in human SHSY5Y neuroblastoma cells (Loveday et al., 2003; Heacock et al., 2004, 2006), and proteinase-activated receptor (PAR)-1 in human 1321N1 astrocytoma and rat astrocytes (Cheema et al., 2005). In each case, Ca availability and PKC activity are required for the maximum release of organic osmolytes. The goals of the present study were 2-fold: first, to determine whether the release of I (used as a tracer for Cl ) from hypotonically stressed SH-SY5Y neuroblastoma cells was, like that of taurine, subject to receptor regulation and, second, to evaluate whether these two osmolytes are released from the cells via similar or distinct mechanisms. The results indicate that the activation of either PAR-1 or mAChRs elicits a significant increase in the osmosensitive release of both I and taurine and that the efflux of these osmolytes exhibits a similar, if not identical, inhibition profile in response to a variety of putative pharmacological inhibitors of VSOAC. However, the receptor-mediated efflux of I can be readily differentiated from that of taurine on the basis of its more limited dependence on Ca availability and, to a lesser extent, PKC activity. Thus, in SH-SY5Y cells, although both osmolytes may exit via a common (or pharmacologically similar) channel(s), distinct biochemical requirements exist for the receptor-stimulated release of I and taurine. Materials and Methods Materials. [1,2-H]Taurine (1.15 TBq/ml) and sodium iodide (I -labeled; 3885 MBq/ml) were obtained from GE Healthcare (Piscataway, NJ). Chelerythrine, thapsigargin, toxin B, Y-27632, and niflumic acid were obtained from Calbiochem (San Diego, CA). Thrombin, DIDS, NPPB, 1,9-dideoxyforskolin, and oxotremorine-M (Oxo-M) were purchased from Sigma-Aldrich (St. Louis, MO). DCPIB was obtained from Tocris Cookson Inc. (Ellisville, MO). Thrombin receptor-activating peptides, TFLLRN, TFRGAP, and GYPGKF, were purchased from Bachem California (Torrance, CA). Fura 2/acetoxymethyl ester was purchased from Invitrogen (Eugene, OR). Dulbecco’s modified Eagle’s medium (DMEM) and 50 penicillin/streptomycin were obtained from Invitrogen (Carlsbad, CA). Fetal calf serum was obtained from Cambrex Bio Science Walkersville, Inc. (Walkersville, MD). Tissue culture supplies were obtained from Corning Glassworks (Corning, NY), Starstedt (Newton, NC), and BD BioSciences (Franklin Lakes, NJ). Universol was obtained from Valeant Pharmaceuticals (Costa Mesa, CA). Cell Culture Conditions. Human SH-SY5Y neuroblastoma cells (passages 75–90) were grown in tissue culture flasks (75 cm/250 ml) in 20 ml of DMEM supplemented with 10% (v/v) fetal calf serum with 1% penicillin/streptomycin. The osmolarity of the medium was 330 to 340 mOsM. Cells were grown at 37°C in a humidified atmosphere containing 5% CO2. The medium was aspirated, and the cells were detached from the flask with a TrypLE Express (Cambrex Bio Science, Walkersville, MD) or sterile D1 solution (Heacock et al., 2004). Cells were then resuspended in DMEM-10% fetal calf serum with penicillin/streptomycin and subcultured into 35-mm, six-well culture plates for 5 to 6 days. Experiments were routinely conducted on cells that had reached 70 to 90% confluence. Measurement of Efflux of Taurine or I . Osmolyte efflux from SH-SY5Y neuroblastoma cells was monitored essentially as described previously (Heacock et al., 2004; Tomassen et al., 2004). In brief, cells were prelabeled overnight with 18.5 kBq/ml [H]taurine or 92.5 kBq/ml I at 37°C. After prelabeling, the cells were washed two or three times with 2 ml of isotonic buffer A (142 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl2, 3.6 mM NaHCO3, 1 mM MgCl2, 30 mM HEPES, pH 7.4, and 1 mg/ml D-glucose; 340 mOsM). Cells were then allowed to incubate in 2 ml of hypotonic buffer A (295–195 mOsM; rendered hypotonic by a reduction in NaCl concentration) in the absence or presence of thrombin or Oxo-M. In some experiments, buffer A was made hypertonic (370 mOsM) by the addition of NaCl. Osmolarities of buffer A were monitored by means of an Osmette precision osmometer (PS Precision Systems, Sudbury, MA). At the times indicated, aliquots of the extracellular medium (200 l for taurine and 1 ml for I ) were removed, and radioactivity was determined after the addition of 6 ml of Universol scintillation fluid. The reactions were terminated by rapid aspiration of the buffer, and cells were lysed by the addition of 2 ml of ice-cold 6% (w/v) trichloroacetic acid for taurine or 1 ml of 0.1 M NaOH for I . Efflux of taurine or I was calculated as a fractional release, i.e., the radioactivity released in the extracellular medium as a percentage of the total radioactivity present initially in the cells. The latter was calculated as the sum of radioactivity recovered in the extracellular medium and that remaining in the lysate at the end of the assay. For I efflux, radioactivity released at the zero time point was subtracted from the observed release of I . Throughout this study, “basal” release of taurine or I is defined as that which occurs at a specified osmolarity in the absence of agonists. Measurement of Phosphoinositide Turnover. To monitor phosphoinositide turnover, SH-SY5Y cells that had been prelabeled with 148 kBq/ml [H]inositol for 96 h were incubated in hypotonic buffer A (230 mOsM) that contained 5 mM LiCl. The accumulation of radiolabeled inositol phosphates present in the trichloroacetic acid cell lysates was determined as described previously (Thompson and Fisher, 1990). Measurement of Cytoplasmic Calcium Concentration. Cytoplasmic free calcium concentrations, [Ca ]i, were determined in suspensions of SH-SY5Y neuroblastoma cells after preloading cells with the Ca indicator, fura-2/acetoxymethyl ester (Molecular Probes), as described previously (Fisher et al., 1989; Cheema et al., 2005). The fluorometer used was a Shimadzu RF-5301PC spectrofluorometer (Shimadzu Scientific Instruments, Columbia, MD). Differential Regulation of I and Taurine Release 1069 at A PE T Jornals on M ay 0, 2017 jpet.asjournals.org D ow nladed from Data Analysis. Experiments were performed in triplicate and repeated at least three times. Values quoted are given as means S.E.M. for the number (n) of independent experiments indicated. A two-tailed Student’s t test (paired) was used to evaluate differences between two experimental groups (level of significance, p 0.05). One-way or repeated-measures analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test was used for statistical significance of differences between multiple groups (GraphPad Instat Software, Inc., San Diego, CA).