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NEUROPHARMACOLOGY

Activation of Muscarinic Cholinergic Receptors on Human SH-SY5Y Neuroblastoma Cells Enhances Both the Influx and Efflux of K⁺ under Conditions of Hypo-Osmolarity

Departments of Pharmacology (D.J.F., S.K.F.) and Neurosurgery and Molecular and Integrative Physiology (R.F.K.), and Molecular and Behavioral Neuroscience Institute (A.M.H., S.K.F.), University of Michigan, Ann Arbor, Michigan

Received December 19, 2007; accepted February 14, 2008.

	Abstract

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The ability of receptor activation to regulate osmosensitive K⁺ fluxes (monitored as ⁸⁶Rb⁺) in SH-SY5Y neuroblastoma has been examined. Incubation of SH-SY5Y cells in buffers rendered increasingly hypotonic by a reduction in NaCl concentration resulted in an enhanced basal efflux of Rb⁺ (threshold of release, 200 mOsM) but had no effect on Rb⁺ influx. Addition of the muscarinic cholinergic agonist, oxotremorine-M (Oxo-M), potently enhanced Rb⁺ efflux (EC₅₀ = 0.45 µM) and increased the threshold of release to 280 mOsM. Oxo-M elicited a similarly potent, but osmolarity-independent, enhancement of Rb⁺ influx (EC₅₀ = 1.35 µM). However, when incubated under hypotonic conditions in which osmolarity was varied by the addition of sucrose to a fixed concentration of NaCl, basal- and Oxo-M-stimulated Rb⁺ influx and efflux were demonstrated to be dependent upon osmolarity. Basal- and Oxo-M-stimulated Rb⁺ influx (but not Rb⁺ efflux) were inhibited by inclusion of ouabain or furosemide. Both Rb⁺ influx and efflux were inhibited by removal of intracellular Ca²⁺ and inhibition of protein kinase C activity. In addition to Oxo-M, agonists acting at other cell surface receptors previously implicated in organic osmolyte release enhanced both Rb⁺ efflux and influx under hypotonic conditions. Oxo-M had no effect on cellular K⁺ concentration in SH-SY5Y cells under physiologically relevant reductions in osmolarity (0–15%) unless K⁺ influx was blocked. Thus, although receptor activation enhances the osmosensitive efflux of K⁺, it also stimulates K⁺ influx, and the latter permits retention of K⁺ by the cells.

After hypo-osmotic stress, cells swell in proportion to the reduction in osmolarity and then normalize their volume in a recovery process known as regulatory volume decrease in which osmolytes (K⁺, Cl^–, and small organic molecules) are extruded, and cell volume is normalized via the exit of obligated water (McManus et al., 1995). Inorganic osmolytes, such as K⁺ and Cl^–, constitute the quantitatively major component of the osmolyte pool (60–70%), whereas organic osmolytes such as taurine, glutamate, and inositol comprise the remainder (Pasantes-Morales et al., 2002). In most (but not all) tissues, the extrusion of Cl^– and organic osmolytes seems to occur via a common, volume-sensitive organic osmolyte and anion channel, which is primarily permeable to Cl^– but impermeable to cations (Sánchez-Olea et al., 1996; Lang et al., 1998; Nilius and Droogmans, 2003; Abdullaev et al., 2006). Although less extensively studied, the efflux of K⁺ has been reported to occur via a variety of different K⁺ channels, including those gated by voltage or activated by stretch, swelling, or Ca²⁺ (Pasantes-Morales et al., 2006).

When monitored in vitro, the efflux of both inorganic and organic osmolytes is relatively insensitive to hypo-osmotic stress, often requiring reductions in osmolarity (>30%) that are not typically encountered in vivo. However, recent studies from this and other laboratories have demonstrated that the volume-sensitive efflux of osmolytes from neural tissues can be enhanced after the activation of certain G-protein-coupled receptors (GPCRs), including the P_2Y purinergic receptors (Mongin and Kimelberg, 2002, 2005), M3 muscarinic cholinergic receptors (mAChR; Loveday et al., 2003; Heacock et al., 2004), lysophospholipid receptors (Heacock et al., 2006), and the protease-activated-1 receptors (Cheema et al., 2005, 2007; Ramos-Mandujano et al., 2007). Receptor activation not only increases the extent of osmolyte release but also lowers the threshold osmolarity ("set-point") at which osmolytes are released. The latter observation raises the possibility that tonic agonist activation of cell surface receptors may permit neural cells to respond to more physiologically relevant reductions in osmolarity.

Although inorganic osmolytes are released from cultured neural cells to the same or greater extent than is observed for organic osmolytes under both basal (swelling-activated) and receptor-stimulated conditions (Abdullaev et al., 2006; Cheema et al., 2007), chronic hyponatremia results in a disproportionately greater percentage loss of organic osmolytes than of inorganic osmolytes from the brain (Lien et al., 1991; Videen et al., 1995; Pasantes-Morales et al., 2002; Massieu et al., 2004). One potential explanation for this observation is that, under hypo-osmotic conditions, the volume-dependent efflux of inorganic osmolytes is accompanied by a compensatory uptake phase, as previously proposed for K⁺ (Mongin et al., 1994, 1996). However, the issue of whether receptor activation can promote the uptake of osmolytes under hypoosmotic conditions has not, to the best of our knowledge, been previously investigated. To address this question, in the present study, we have examined the ability of mAChRs (and other GPCRs) to regulate K⁺ homeostasis in human SH-SY5Y neuroblastoma cells under conditions of hypo-osmotic stress. The results indicate that receptor activation facilitates both the efflux and influx of K⁺ in an osmosensitive manner. Under conditions of either isotonicity or limited reductions in osmolarity (15%), the efflux of K⁺ is effectively countered by an influx of K⁺, such that no net loss of cell K⁺ occurs. Only under more pronounced reductions in osmolarity (30%) does the rate of K⁺ efflux exceed that of influx and result in a net loss of K⁺. Thus, receptor activation serves to regulate both the release and uptake of osmolytes. A preliminary account of part of this work has appeared elsewhere (Foster et al., 2008).

	Materials and Methods

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Materials. Rubidium chloride (⁸⁶Rb⁺-labeled; 241 MBq/mg) was obtained from PerkinElmer Life and Analytical Sciences (Waltham, MA). 3-O-Methyl-D-[1-³H]glucose (148 GBq/mmol) was from GE Healthcare (Chalfont St. Giles, UK). Oxotremorine-M, sphingosine 1-phosphate, thrombin, bumetanide, DIOA, ouabain, furosemide, tetraethylammonium chloride, barium chloride dihydrate, atropine, and 3-O-methyl-D-glucose were purchased from Sigma-Aldrich (St. Louis, MO). Iberiotoxin, apamin, and glibenclamide were obtained from Tocris Bioscience, Inc. (Ellisville, MO). Chelerythrine, thapsigargin, and phloretin were obtained from Calbiochem (San Diego, CA). Lysophosphatidic acid was purchased from Avanti Polar Lipids (Alabaster, AL). Dulbecco's modified Eagle's medium (DMEM) and 50x penicillin/streptomycin were obtained from Invitrogen (Carlsbad, CA). Fetal calf serum was obtained from Lonza Walkersville, Inc. (Walkersville, MD). Tissue culture supplies were obtained from Corning Inc. (Corning, NY), Starstedt (Newton, NC), and BD Biosciences (San Jose, CA). Universol was obtained from Valeant Pharmaceuticals (Costa Mesa, CA).

Cell Culture Conditions. Human SH-SY5Y neuroblastoma cells (passages 70–89) were grown in tissue culture flasks (75 cm²/250 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 10% CO₂. The medium was aspirated, and cells were detached from the flask with a trypsin-versene mixture (Cambrex Bio Science). Cells were then resuspended in DMEM/10% fetal calf serum with penicillin/streptomycin and subcultured into 35-mm, six-well culture plates at a density of 250 to 300,000 cells/well for 4 to 5 days. Cells that had reached 70 to 90% confluence were routinely used.

Measurement of K⁺ Efflux. K⁺ efflux from SH-SY5Y neuroblastoma cells was determined using ⁸⁶Rb⁺ as a tracer for K⁺. In brief, cells were prelabeled overnight to isotopic equilibrium with 19 to 37 KBq/ml ⁸⁶Rb⁺ at 37°C. After prelabeling, the cells were washed 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 buffer A (370–200 mOsM; routinely rendered either hypertonic or hypotonic by an increase or decrease in NaCl concentration, respectively) in the absence or presence of agonists. In some experiments, osmolarities of the buffers were adjusted under conditions of a constant NaCl concentration (79 mM NaCl) by the addition of sucrose. Osmolarities of buffers were monitored by means of an Osmette precision osmometer (PS Precision Systems, Sudbury, MA). At the times indicated, aliquots of the extracellular medium (1 ml) 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 0.1 M NaOH. The rate of efflux of ⁸⁶Rb⁺ was calculated as a fractional release per minute, i.e., the radioactivity released per minute into 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 all measurements, radioactivity released at the zero time point was subtracted from the observed value. Throughout the study, "basal" release of ⁸⁶Rb⁺ is defined as that which occurs at a specified osmolarity in the absence of agonists.

Measurement of K⁺ Influx. K⁺ influx was determined using ⁸⁶Rb⁺ as a tracer for K⁺. SH-SY5Y neuroblastoma cells were washed twice with 2 ml of isotonic buffer A (340 mOsM) and then incubated in buffer A (370–200 mOsM routinely rendered either hypertonic or hypotonic, unless otherwise stated, by an increase or decrease in NaCl concentration, respectively) containing ⁸⁶Rb⁺ (28–56 KBq/ml) with or without agonist at 37°C. In some experiments, osmolarities of the buffers were adjusted under conditions of a constant NaCl concentration (79 mM NaCl) by the addition of sucrose. At the times indicated, the extracellular medium was aspirated, cells were washed three times with 2 ml of isotonic buffer A, and then the cells were lysed with 2 ml of 0.1 M NaOH. Aliquots of lysate (1 ml) were removed, and radioactivity was determined after the addition of 6 ml of Universol scintillation fluid. In all measurements, radioactivity accumulated at the zero time point was subtracted from the observed value. Protein contents of cell lysates were determined using a bicinchoninic acid protein assay reagent kit (Pierce Chemical, Rockford, IL). From the measurement of ⁸⁶Rb⁺ uptake, K⁺ influx was calculated as nanomoles per milligram of protein per minute with the assumption that ⁸⁶Rb⁺ transport into the cells reflects that of K⁺.

Intracellular Water Space. The intracellular water space was measured essentially as described previously (Novak et al., 1999). SH-SY5Y neuroblastoma cells were washed with 5 x 2 ml of buffer A without D-glucose and then incubated in buffer A with increasing extracellular concentrations of 3-O-[³H]methyl-D-glucose at 37°C until equilibrium had been achieved (50 min). Cells were then washed with 5 x 2 ml of ice-cold buffer A without glucose containing 0.1 mM phloretin and lysed with 2 ml of 0.1 M NaOH. Aliquots (1 ml) of lysate were removed, and radioactivity was determined after the addition of 6 ml of Universol scintillation fluid. Intracellular concentrations of 3-O-[³H]methyl-D-glucose were monitored at equilibrium, and a plot of this parameter versus the concentration of extracellular 3-O-[³H]methyl-D-glucose yields a line whose slope is the volume of intracellular water with respect to protein. Determination of water space by this method requires that 3-O-methyl-D-glucose not be metabolized or actively transported, and these assumptions were validated by the linearity of the plot and its extrapolation through the origin (Kletzien et al., 1975).

K⁺ Mass Measurements. SH-SY5Y cells were washed with 2 x 2 ml of isotonic buffer A. Cells were then incubated for 10 min in buffer A (340–230 mOsM rendered hypotonic by a reduction in NaCl concentration) at 37°C. The extracellular medium was then aspirated, cells were then washed with 2 ml of K⁺ free buffer A (142 mM NaCl, 2.2 mM CaCl₂, 3.6 mM NaHCO₃, 1 mM MgCl₂, 30 mM HEPES, pH 7.4, and 1 mg/ml D-glucose, 335 mOsM), and lysed in 2 ml of 0.1 M NaOH. Protein contents of cell lysates were determined using a bicinchoninic acid protein assay reagent kit (Pierce Chemical). Lysates from three separate 35-mm wells were combined (total volume, 6 ml) and centrifuged at 3000g for 30 min at 5°C. Supernatants were then adjusted to a final pH of between 5 and 11 with 4 N HCl. K⁺ values were obtained using a glass combination K⁺ electrode (Cole Parmer) and an Acorn Series Ion 6 meter (Oakton Instruments, Vernon Hills, IL).

Data Analysis. All experiments shown were performed in duplicate or 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 or unpaired) was used to evaluate differences between two experimental groups (level of significance, p < 0.05). Ordinary or repeated measures analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test was used for statistical significance of differences between multiple groups. EC₅₀ values were obtained using Prism 4.0a (GraphPad Software Inc., San Diego, CA).

	Results

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Agonist Activation of mAChRs on SH-SY5Y Neuroblastoma Cells Enhances Both ⁸⁶Rb⁺ Influx and ⁸⁶Rb⁺ Efflux. When SH-SY5Y cells were exposed to hypotonic buffer A (230 mOsM; 30% reduction in osmolarity), conditions previously determined to be optimal for the release of organic osmolytes, there was a time-dependent increase in both ⁸⁶Rb⁺ influx and ⁸⁶Rb⁺ efflux under basal conditions ("basal" is defined as influx or efflux monitored at a specified osmolarity in the absence of agonist). Inclusion of Oxo-M (100 µM) elicited a marked enhancement of ⁸⁶Rb⁺ influx over basal at 3 min and thereafter and resulted in a doubling of the rate of ⁸⁶Rb⁺ uptake (Fig. 1A). Inclusion of Oxo-M also significantly enhanced the efflux of ⁸⁶Rb⁺ in an approximately linear manner up to 10 min of incubation (rate constants for ⁸⁶Rb⁺ efflux under basal- and Oxo-M-stimulated conditions were 0.85 and 2.61%/min, respectively; Fig. 1B). In subsequent experiments, both basal- and agonist-stimulated ⁸⁶Rb⁺ influx and ⁸⁶Rb⁺ efflux were routinely monitored after either 5- or 10-min incubations. The addition of Oxo-M resulted in a stimulation of ⁸⁶Rb⁺ influx and ⁸⁶Rb⁺ efflux with EC₅₀ values of 0.45 and 1.37 µM, respectively, and with Hill coefficients close to unity (Fig. 2, A and B). The inclusion of 10 µM atropine completely blocked Oxo-M stimulation of both ⁸⁶Rb⁺ influx and ⁸⁶Rb⁺ efflux (data not shown).

View larger version (9K): [in this window] [in a new window]   Fig. 1. Kinetics of basal- and Oxo-M-stimulated 86Rb+ influx and 86Rb+ efflux. A, for measurement of 86Rb+ influx, SH-SY5Y cells were washed twice with 2 ml of isotonic buffer A before incubation in hypotonic buffer A (230 mOsM) that contained 0.5 µCi/ml 86Rb+, in the absence () or presence () of 100 µM Oxo-M. Reactions were terminated at the times indicated, and 86Rb+ uptake was monitored. Results are expressed as K+ (86Rb+) influx (nanomoles per milligram of protein per minute), assuming that 86Rb+ uptake reflects that of K+. Values shown are the means ± S.E.M. for three independent experiments, each performed in triplicate. Where error bars are absent, the S.E.M. fell within the symbol. Influx of 86Rb+ at 340 mOsM was also linear with time from 0 to 20 min. *, p < 0.05, different from basal (by unpaired Student's t test). B, for measurement of 86Rb+ efflux, SH-SY5Y cells that had been labeled to isotopic equilibrium in the presence of 86Rb+ were washed three times with 2 ml of isotonic buffer A before incubation in hypotonic buffer A (230 mOsM), in the absence () or presence () of 100 µM Oxo-M. Reactions were terminated at the times indicated, and 86Rb+ efflux was monitored. Results are expressed as rate of K+ (86Rb+) efflux (percentage of total cell radioactivity released per minute). Values shown are the means ± S.E.M. for three independent experiments, each performed in triplicate. Where error bars are absent, the S.E.M. fell within the symbol. Efflux of 86Rb+ was also linear for at least 10 min when monitored at 340 mOsM. *, p < 0.05, different from basal (by unpaired Student's t test).

View larger version (8K): [in this window] [in a new window]   Fig. 2. Dose-response relationships for Oxo-M-stimulated 86Rb+ influx and 86Rb+ efflux. 86Rb+ influx (A) and 86Rb+ efflux (B) were monitored in SH-SY5Y neuroblastoma cells incubated under hypotonic conditions (230 mOsM) in the presence or absence of Oxo-M at the concentrations indicated. Reactions were terminated after 10 min. Results are expressed as percentage of maximal agonist response (obtained at 1 mM Oxo-M) and are the means ± S.E.M. for three independent experiments, each performed in triplicate. Where error bars are absent, the S.E.M. fell within the symbol. The calculated EC50 value for 86Rb+ influx was 0.45 µM with a Hill coefficient of 0.80. Addition of Oxo-M stimulated the efflux of 86Rb+ with an EC50 of 1.37 µM and a Hill coefficient of 0.75.

Osmolarity Dependence of Basal- and Oxo-M-Stimulated ⁸⁶Rb⁺ Influx and ⁸⁶Rb⁺ Efflux. Because the degree of facilitation of osmolyte release observed after mAChR activation has previously been demonstrated to be dependent on the extent of hypo-osmotic stress (Loveday et al., 2003; Heacock et al., 2004), the ability of mAChR activation to regulate ⁸⁶Rb⁺ influx and ⁸⁶Rb⁺ efflux was monitored in SH-SY5Y cells under conditions of isotonicity (340 mOsM; defined by the osmolarity of the DMEM/fetal calf serum medium in which the cells were grown), mild-to-severe hypotonicity (310–200 mOsM), or mild hypertonicity (370 mOsM). Two experimental paradigms were used to evaluate the dependence of ⁸⁶Rb⁺ fluxes on osmolarity. In the first, buffers were rendered either hypertonic or hypotonic by increases or decreases, respectively, in the NaCl concentration (because Na⁺ and Cl^– are the primary osmolytes found in plasma and reductions in plasma osmolarity observed under pathological conditions, such as hyponatremia, principally reflect changes in the concentrations of these ions). Under these conditions, the magnitude of basal ⁸⁶Rb⁺ influx was constant at all osmolarities tested. The addition of Oxo-M resulted in an increase in ⁸⁶Rb⁺ influx of 75%, compared with basal, at all osmolarities (370–200 mOsM; Fig. 3A). In contrast, the basal efflux of ⁸⁶Rb⁺ was enhanced over that observed under isotonic conditions (340 mOsM) when osmolarity was reduced to 200 mOsM. Moreover, although the addition of Oxo-M resulted in a relatively small increase in ⁸⁶Rb⁺ efflux at both 340 and 370 mOsM, the extent of Oxo-M-stimulated ⁸⁶Rb⁺ efflux was significantly increased over isotonic at an osmolarity of 280 mOsM (a reduction in osmolarity of 18%) with a maximal enhancement observed at 230 mOsM (386% of basal; Fig. 3B). In the second experimental paradigm, osmolarities of buffers were adjusted under conditions of a constant NaCl concentration (79 mM NaCl) by the addition of sucrose. Under these conditions, the basal influx of ⁸⁶Rb⁺ was significantly enhanced over that observed under isotonic conditions when the osmolarity was reduced to 230 or 200 mOsM (134 and 159% of that at 340 mOsM, respectively). The extent of Oxo-M-stimulated ⁸⁶Rb⁺ influx was also dependent upon osmolarity, and, although an increased influx was monitored under isotonic conditions, significantly greater increases were observed at 230 and 200 mOsM than at 340 mOsM (Fig. 4A). The magnitudes of both basal- and Oxo-M-stimulated ⁸⁶Rb⁺ efflux were also found to be dependent upon the osmolarity of the buffer under conditions of a fixed concentration of NaCl, and the values obtained for ⁸⁶Rb⁺ efflux were quantitatively similar for the two experimental paradigms (Fig. 4B).

View larger version (16K): [in this window] [in a new window]   Fig. 3. Basal- and Oxo-M-stimulated 86Rb+ influx and 86Rb+ efflux as a function of osmolarity. 86Rb+ influx (A) and 86Rb+ efflux (B) were monitored in SH-SY5Y neuroblastoma cells in buffers rendered hypertonic or hypotonic by an increase or decrease in NaCl concentration, respectively, in the absence (open bars) or presence (closed bars) of 100 µM Oxo-M. Reactions were terminated after 5 min. Results are expressed as either K+ (86Rb+) influx (nanomoles per milligram of protein per minute) or as rate of K+ (86Rb+) efflux (percentage of total cell radioactivity released per minute). Values shown are the means ± S.E.M. for three independent experiments, each performed in triplicate. *, p < 0.01, Different from basal release monitored under isotonic (340 mOsM) conditions (by repeated measures ANOVA followed by Dunnett's multiple comparison test). #, p < 0.01, different from Oxo-M treatment under isotonic (340 mOsM) conditions (by repeated measures ANOVA followed by Dunnett's multiple comparison test). At all osmolarities, Oxo-M addition significantly increased both 86Rb+ influx and 86Rb+ efflux when compared with uptake or release monitored under basal conditions (p < 0.05, by paired Student's t test).

View larger version (12K): [in this window] [in a new window]   Fig. 4. Basal- and Oxo-M-stimulated 86Rb+ influx and 86Rb+ efflux are volume-dependent. 86Rb+ influx (A) and 86Rb+ efflux (B) were monitored in SH-SY5Y neuroblastoma cells in buffers of the osmolarity indicated in the absence (open bars) or presence (closed bars) of 100 µM Oxo-M. Osmolarities of the buffers were adjusted under conditions of a constant NaCl concentration (79 mM NaCl) by the addition of sucrose. Reactions were terminated after 5 min of incubation. Results are expressed as either K+ (86Rb+) influx (nanomoles per milligram of protein per minute) or as rate of K+ (86Rb+) efflux (percentage of total cell radioactivity released per minute). Results shown are the means ± S.E.M. for three or four independent experiments, each performed in triplicate. *, p < 0.05, different from basal influx or efflux monitored under isotonic (340 mOsM) conditions (by repeated measures ANOVA followed by Dunnett's multiple comparison test). #, p < 0.05, different from Oxo-M treatment under isotonic (340 mOsM) conditions (by repeated measures ANOVA followed by Dunnett's multiple comparison test). At all osmolarities, Oxo-M addition significantly increased both 86Rb+ influx and 86Rb+ efflux when compared with basal conditions (p < 0.05, by paired Student's t test).

⁸⁶Rb⁺ Influx Is Mediated Primarily via Na⁺/K⁺-ATPase and the Na⁺-K⁺-2Cl^– Cotransporter Transporter under Both Basal and Oxo-M-Stimulated Conditions. K⁺ transport mechanisms, including Na⁺/K⁺ ATPase and the Na⁺-K⁺-2Cl^– cotransporter (NKCC) and the K⁺-Cl^– cotransporter (KCC), have previously been implicated in cell volume regulation. To determine the role, if any, played by these transporters in ⁸⁶Rb⁺ influx in SH-SY5Y neuroblastoma cells, both basal- and Oxo-M-stimulated ⁸⁶Rb⁺ influx were monitored in the absence or presence of pharmacological inhibitors at concentrations similar to those previously used (Yabaluri and Medzihradsky, 1997; Ernest et al., 2005). Inclusion of 800 µM concentrations of either bumetanide or furosemide, inhibitors of the NKCC, attenuated both basal- and Oxo-M stimulated ⁸⁶Rb⁺ influx by 50%. Because furosemide inhibits both NKCC and KCC, we also evaluated the ability of DIOA, a KCC inhibitor, to attenuate ⁸⁶Rb⁺ influx. Inclusion of a 40 µM concentration of DIOA had no effect on basal ⁸⁶Rb⁺ influx but resulted in a 20% inhibition of the Oxo-M-mediated component. Inclusion of 30 µM ouabain, a selective inhibitor of the Na⁺/K⁺-ATPase, resulted in a significant inhibition (40%) of both basal- and Oxo-M-stimulated ⁸⁶Rb⁺ influx. When both ouabain and furosemide were present, basal- and Oxo-M stimulated ⁸⁶Rb⁺ influx were essentially abolished (94 and 97% reductions, respectively; Fig. 5A). In contrast, neither the inclusion of bumetanide nor furosemide had any significant effect on basal ⁸⁶Rb⁺ efflux (Fig. 5B). Furthermore, the inclusion of the NKCC inhibitors resulted in either no effect (bumetanide) or a modest inhibition (19%, furosemide) when Oxo-M-stimulated ⁸⁶Rb⁺ efflux was monitored. Inclusion of DIOA also resulted in a small inhibition of Oxo-M-mediated ⁸⁶Rb⁺ efflux. Addition of ouabain had no effect on the magnitude of either basal- or Oxo-M-stimulated ⁸⁶Rb⁺ efflux, but when coadministered with furosemide, an inhibition of Oxo-M-stimulated ⁸⁶Rb⁺ efflux (20%) was again observed (Fig. 5B). Taken collectively, these results suggest that ⁸⁶Rb⁺ influx and ⁸⁶Rb⁺ efflux are mediated by distinct mechanisms.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Effect of ouabain, DIOA, furosemide, or bumetanide on basal- and Oxo-M-stimulated 86Rb+ influx and 86Rb+ efflux. 86Rb+ influx (A) and 86Rb+ efflux (B) were monitored in SH-SY5Y neuroblastoma cells under hypotonic conditions (230 mOsM) in the absence (open bars) or presence (closed bars) of 100 µM Oxo-M. In some experiments, bumetanide (800 µM), furosemide (800 µM), DIOA (40 µM) and ouabain (30 µM) were also present. Reactions were terminated after 10 min. Results are expressed as either K+ (86Rb+) influx (nanomoles per milligram of protein per minute) or as rate of K+ (86Rb+) efflux (percentage of total cell radioactivity released per minute). Values shown are the means ± S.E.M. for three to nine independent experiments, each performed in triplicate. *, p < 0.05, different from control basal influx (by one-way ANOVA followed by Dunnett's multiple comparison test). #, p < 0.05, different from control plus Oxo-M (by one-way ANOVA followed by Dunnett's multiple comparison test).

Basal- and Oxo-M-Stimulated ⁸⁶Rb⁺ Influx and ⁸⁶Rb⁺ Efflux: Dependence on Ca²⁺ Availability and PKC Activity. We have previously demonstrated that the mAChR-stimulated osmosensitive release of the organic osmolyte, taurine, from SH-SY5Y cells is more dependent upon Ca²⁺ availability and PKC activity than that of the inorganic osmolyte, Cl^–, suggesting that the release of these osmolytes may be differentially regulated (Heacock et al., 2006; Cheema et al., 2007). For this reason, in the present study, the roles played by Ca²⁺ availability and PKC activity in ⁸⁶Rb⁺ influx and ⁸⁶Rb⁺ efflux were evaluated. Although removal of extracellular Ca²⁺ had no effect on either basal- or Oxo-M-stimulated ⁸⁶Rb⁺ influx, the additional depletion of intracellular Ca²⁺ stores with 1 µM thapsigargin resulted in an increase in basal ⁸⁶Rb⁺ influx, whereas the ability of Oxo-M to enhance ⁸⁶Rb⁺ influx over the basal value was attenuated by approximately 35% under these conditions. Inclusion of 10 µM chelerythrine, a PKC inhibitor, had no effect on basal ⁸⁶Rb⁺ influx but significantly inhibited (50–60%) the Oxo-M-stimulated component, both in the presence or absence of Ca²⁺/thapsigargin (Fig. 6A). Removal of extracellular Ca²⁺ resulted in an increase in basal efflux of ⁸⁶Rb⁺, whereas the ability of Oxo-M to increase efflux over the basal value was unchanged relative to control incubations. Depletion of intracellular Ca²⁺ stores with thapsigargin resulted in a further increase in the basal efflux of ⁸⁶Rb⁺ but significantly attenuated (25%) the Oxo-M-mediated increase in ⁸⁶Rb⁺ efflux. As observed for ⁸⁶Rb⁺ influx, inclusion of chelerythrine had no effect on basal ⁸⁶Rb⁺ efflux but significantly inhibited (60%) Oxo-M-mediated ⁸⁶Rb⁺ efflux. Under conditions in which intracellular stores of Ca²⁺ were depleted and PKC activity inhibited, the ability of Oxo-M to stimulate ⁸⁶Rb⁺ efflux was severely attenuated (86% inhibition; Fig. 6B).

View larger version (15K): [in this window] [in a new window]   Fig. 6. The role of extra- and intracellular Ca2+ and PKC in basal and Oxo-M-stimulated 86Rb+ influx and 86Rb+ efflux. 86Rb+ influx (A) and efflux (B) were monitored in SH-SY5Y neuroblastoma cells under hypotonic conditions (230 mOsM). Cells were incubated in the absence (–Ca2+; Ca2+ was omitted from buffer, and 50 µM EGTA was added) or presence of extracellular Ca2+. For some experiments, cells were pretreated for 15 min in isotonic buffer A in the presence of 1 µM thapsigargin (Thp) to deplete intracellular pools of Ca2+. For evaluation of the involvement of PKC, cells were pretreated with 10 µM chelerythrine (Chel) in isotonic buffer A for 15 min. Reactions were allowed to proceed for 5 min in the absence (open bars) or presence (closed bars) of 100 µM Oxo-M. Results are expressed as either K+ (86Rb+) influx (nanomoles per milligram of protein per minute) or as rate of K+ (86Rb+) efflux (percentage of total cell radioactivity released per minute). Results shown are the means ± S.E.M. for four to six independent experiments, each performed in triplicate. *, p < 0.05, different from control basal influx or efflux (by repeated measures ANOVA followed by Dunnett's multiple comparison test). #, p < 0.05, different from control plus Oxo-M (by repeated measures ANOVA followed by Dunnett's multiple comparison test).

Activation of Multiple GPCRs Can Elicit Both ⁸⁶Rb⁺ Influx and ⁸⁶Rb⁺ Efflux under Hypotonic Conditions. In addition to the mAChR, activation of several other GPCRs has been shown to increase the efflux of osmolytes from SH-SY5Y cells under hypotonic conditions (Heacock et al., 2006; Cheema et al., 2007). These include the protease-activated receptor (PAR), which can be activated by thrombin and lysophospholipid receptors that can be selectively activated by either sphingosine-1-phosphate (S1P) or lysophosphatidic acid (LPA). To investigate whether activation of these receptors could also mediate changes in ⁸⁶Rb⁺ fluxes, thrombin (1.25 nM), S1P (5 µM), or LPA (10 µM) were added to SH-SY5Y cells under hypotonic conditions (230 mOsM), and ⁸⁶Rb⁺ influx and ⁸⁶Rb⁺ efflux were monitored after a 10-min incubation. Addition of each of the three agonists resulted in a significant increase in both ⁸⁶Rb⁺ influx and ⁸⁶Rb⁺ efflux, with a rank order of efficacy for both fluxes being thrombin = S1P > Oxo-M > LPA (Fig. 7, A and B).

View larger version (16K): [in this window] [in a new window]   Fig. 7. Activation of multiple GPCRs can induce both volume-dependent 86Rb+ influx and 86Rb+ efflux under hypotonic conditions. 86Rb+ influx (A) and 86Rb+ efflux (B) were monitored in SH-SY5Y neuroblastoma cells under hypotonic conditions (230 mOsM) in the presence or absence of 100 µM Oxo-M, 1.25 nM thrombin, 10 µM LPA, or 5 µM S1P. Reactions were terminated after 10 min. Results are expressed as either K+(86Rb+) influx (nanomoles per milligram of protein per minute) or as rate of K+ (86Rb+) efflux (percentage of total cell radioactivity released per minute). Values shown are the means ± S.E.M. for four independent experiments, each performed in triplicate. *, p < 0.01, different from basal (by repeated measures ANOVA followed by Dunnett's multiple comparison test).

	Discussion

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Previous studies of osmolyte loss from neural cells after their exposure to a hypo-osmotic medium have focused almost exclusively on measurement of efflux. However, the net loss of an osmolyte from a cell reflects both its release and uptake. The principal finding to emanate from the present study is that, under hypo-osmotic conditions, the activation of mAChRs in SH-SY5Y neuroblastoma cells can enhance the osmosensitive efflux and influx of ⁸⁶Rb⁺. Furthermore, these two fluxes exhibit similar characteristics in terms of kinetics, agonist concentration dependence, and requirements for Ca²⁺ availability and PKC activity. Because SH-SY5Y cells exhibit a relatively homogeneous population of M3 mAChRs (>80% of total; Wall et al., 1991; Slowiejko et al., 1994), it is possible that this mAChR subtype mediates the increase in ⁸⁶Rb⁺ fluxes, as has been previously demonstrated for the osmosensitive release of taurine (Heacock et al., 2004). In addition to the mAChR, activation of several other GPCRs that have been demonstrated previously to regulate the osmosensitive release of taurine from SH-SY5Y cells, i.e., PAR-1, S1P, and LPA receptors (Heacock et al., 2006; Cheema et al., 2007), also promoted both the efflux and influx of ⁸⁶Rb⁺ under hypo-osmotic conditions (Fig. 7). Furthermore, activation of PAR-1 and S1P receptors also facilitated both the influx and efflux of ⁸⁶Rb⁺ in primary cultures of rat astrocytes (data not shown). Taken collectively, these results suggest that the ability of GPCRs to regulate both the influx and efflux of ⁸⁶Rb⁺ under hypo-osmotic conditions may be a general property of neural cells. Although Oxo-M-stimulated efflux and influx of ⁸⁶Rb⁺ share similar characteristics, the two fluxes are mediated via distinct mechanisms. Under hypo-osmotic conditions, both basal- and Oxo-M-mediated ⁸⁶Rb⁺ influx are abolished after administration of ouabain and furosemide, a result that indicates that ⁸⁶Rb⁺ influx is mediated principally through the NKCC and Na⁺/K⁺-ATPase (Fig. 5A). These findings are consistent with previous studies in which the basal influx of ⁸⁶Rb⁺ in astrocytes and C6 glioma cells, monitored under iso-osmotic or hypo-osmotic conditions, was also inhibited by ouabain and furosemide (Kimelberg and Frangakis, 1985; Mongin et al., 1994, 1996). KCC may also play a minor role in ⁸⁶Rb⁺ influx in SH-SY5Y cells, as previously suggested for C6 glioma (Gagnon et al., 2007). In contrast to the results obtained for ⁸⁶Rb⁺ influx, none of the agents tested had any effect on basal ⁸⁶Rb⁺ efflux, and only the addition of either furosemide or DIOA resulted in a small inhibition (<20%) of the Oxo-M-mediated component, a result consistent with a limited involvement of KCC in ⁸⁶Rb⁺ efflux (Fig. 5B). At least part of the ⁸⁶Rb⁺ efflux seems to be mediated by K⁺ channels because inclusion of either TEA or Ba²⁺ attenuated both basal- and Oxo-M-stimulated efflux. However, the identity of the specific K⁺ channel(s) involved remains to be determined.

The magnitude of basal- and Oxo-M-stimulated efflux of taurine and ¹²⁵I^– (used as a tracer for Cl^–) from SH-SY5Y cells is dependent upon the degree of osmotic stress, when monitored under conditions in which the buffers are rendered increasingly hypotonic by a reduction in NaCl concentration (Heacock et al., 2004; Cheema et al., 2007). However, under the same conditions, basal- and Oxo-M-stimulated ⁸⁶Rb⁺ influx seemed to be independent of osmolarity (Fig. 3A). Although this experimental paradigm mimics the changes encountered under physiological conditions, it also involves alterations in three experimental variables, i.e., osmolarity and the concentrations of Na⁺ and Cl^– ions. When monitored under conditions in which NaCl concentration was held constant, and osmolarity varied by means of the addition of sucrose, it was evident that the magnitude of both basal- and Oxo-M-stimulated ⁸⁶Rb⁺ influx was dependent upon osmolarity (Fig. 4A). Both basal- and Oxo-M-stimulated efflux of ⁸⁶Rb⁺ was found to be dependent upon the degree of osmolarity, regardless of which experimental paradigm was used (Figs. 3B and 4B). Thus, we conclude that although ⁸⁶Rb⁺ efflux occurs via an osmolarity-sensitive, but NaCl-independent, mechanism, ⁸⁶Rb⁺ influx is mediated by a mechanism that is dependent on both osmolarity and NaCl, consistent with the involvement of NKCC and Na⁺/K⁺-ATPase. Although both the influx and efflux of ⁸⁶Rb⁺ in SH-SY5Y cells are osmosensitive, the efflux component is more dependent on changes in osmolarity, as is evident from the observation that although the Oxo-M-mediated component of ⁸⁶Rb⁺ influx doubles when osmolarity is reduced from 340 to 200 mOsM, the corresponding increase for ⁸⁶Rb⁺ efflux is 6- to 7-fold (Fig. 4, A and B).

The osmosensitive efflux of taurine and ¹²⁵I^– from SH-SY5Y cells after activation of mAChRs (but not that monitored under basal conditions) is differentially regulated, with the efflux of ¹²⁵I^– exhibiting less dependence on Ca²⁺ availability and PKC activity than that observed for taurine (Cheema et a., 2007). Thus, although removal of extracellular Ca²⁺ attenuates mAChR-stimulated taurine efflux by >60%, and depletion of intracellular Ca²⁺ abolishes the response, ¹²⁵I^– efflux is unaffected by removal of extracellular Ca²⁺ and only minimally reduced by depletion of intracellular Ca²⁺ (30%). Likewise, mAChR-stimulated taurine efflux is more susceptible to inhibition of PKC than is that of ¹²⁵I^– release (Heacock et al., 2006; Cheema et al., 2007). In the current study, the Ca²⁺ requirements observed for ⁸⁶Rb⁺ influx and efflux resembled more closely those previously obtained for ¹²⁵I^– release than for taurine efflux. Thus, removal of extracellular Ca²⁺ had no effect on the magnitude of either mAChR-stimulated ⁸⁶Rb⁺ influx or efflux, and only under conditions in which the intracellular pool of Ca²⁺ was depleted was the Oxo-M-mediated component reduced by 25 to 35% (Fig. 6, A and B). In contrast, both the basal influx and efflux of ⁸⁶Rb⁺ were increased by removal of Ca²⁺. An increase in ⁸⁶Rb⁺ efflux under Ca²⁺-depleted conditions has also been observed for astrocytes, although the mechanism remains unclear (Quesada et al., 1999). Oxo-M-stimulated ⁸⁶Rb⁺ influx and efflux were also dependent on PKC activity and could be attenuated by 50% after preincubation of the cells with chelerythrine. From this series of experiments, two conclusions can be drawn. First, the differential Ca²⁺ requirements observed for basal- and mAChR-stimulated ⁸⁶Rb⁺ release provide additional support for the proposal that distinct mechanisms underlie the swelling-activated and receptor-mediated components of osmolyte release (Mongin and Kimelberg, 2005; Heacock et al., 2006). Second, because both Oxo-M-mediated efflux of ⁸⁶Rb⁺ and ¹²⁵I^– exhibit requirements for Ca²⁺ and PKC activity that are distinct from those necessary for taurine release, these results indicate that the receptor-mediated release of inorganic (⁸⁶Rb⁺ and ¹²⁵I^–) and organic (taurine) osmolytes occurs via distinct mechanisms.

Although the use of the radiotracer, ⁸⁶Rb⁺, provides a convenient means whereby the characteristics of K⁺ influx and efflux pathways are readily evaluated, this approach does not permit a quantitative assessment of the relative contributions made by each pathway to K⁺ content of cells. To address this issue, we monitored changes in the concentration of K⁺ in SH-SY5Y cells under hypo-osmotic conditions using a K⁺-specific electrode. The results indicated that the addition of Oxo-M had no effect on the K⁺ content of SH-SY5Y cells when exposed to either iso-osmolarity or a mild reduction in osmolarity (290 mOsM) unless K⁺ influx was concurrently prevented by inclusion of ouabain and furosemide (Table 1). This result suggests that under normal conditions, the agonist stimulation of K⁺ efflux is countered by an equivalent stimulation of K⁺ influx. When monitored under more hypo-osmotic conditions (230 mOsM), Oxo-M addition results in a 10% reduction of K⁺ content, and this loss is further accentuated when K⁺ influx is prevented (23% reduction). The results obtained from measurement of K⁺ content are consistent with those derived from radiolabeling studies, which indicate that although both K⁺ influx and efflux are osmosensitive, it is the efflux pathway that is most strongly regulated by a reduction in osmolarity (Fig. 4). Thus, although an increase in K⁺ efflux is offset by a comparable increase in K⁺ influx under conditions of limited reductions in osmolarity, when cells are incubated under more hypoosmotic conditions, the efflux of K⁺ predominates, and a net loss of K⁺ occurs.

Previous studies have indicated that, under conditions of acute or chronic hyponatremia, the brain selectively retains its inorganic osmolytes (Melton et al., 1987; Pasantes-Morales et al., 2002; Massieu et al., 2004). In the present study, we have demonstrated that activation of GPCRs not only enhances the efflux of K⁺ but also its influx. Under conditions of limited reductions in osmolarity, such as those most likely to occur under pathological conditions, the basal uptake of K⁺, which is mediated via the NKCC and Na⁺/K⁺-ATPase, is markedly facilitated by receptor activation, and this permits K⁺ to be more effectively retained by the cells. These results raise the possibility that, under hyponatremic conditions, tonic receptor activity in the CNS may serve not only to enhance the efflux of osmolytes but also to maintain relatively high intracellular concentrations of K⁺.

	Footnotes

 
This study was supported by National Institutes of Health Grants NS23831 (to S.K.F.), NS 034709 (to R.F.K.), and GM007767 (to D.J.F.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.135475.

ABBREVIATIONS: CNS, central nervous system; GPCR, G-protein-coupled receptor; mAChR, muscarinic cholinergic receptor; DIOA, R-(+)-[(2-n-butyl-6,7-dichlooro-2-cyclopentenyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid; DMEM, Dulbecco's modified Eagle's medium; ANOVA, analysis of variance; Oxo-M, oxotremorine-M; NKCC, Na⁺-K⁺-2Cl^– cotransporter; KCC, K⁺-Cl^– cotransporter; PKC, protein kinase C; PAR, protease-activated receptor; S1P, sphingosine-1-phosphate; LPA, lysophosphatidic acid.

Address correspondence to: Dr. Stephen K. Fisher, Molecular and Behavioral Neuroscience Institute, University of Michigan, 5039 Biomedical Science Research Building, Ann Arbor, MI 48109-2200. E-mail: skfisher{at}umich.edu

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