QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.109496v1 319/2/963    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Heacock, A. M. Articles by Fisher, S. K. Search for Related Content PubMed PubMed Citation Articles by Heacock, A. M. Articles by Fisher, S. K.

CELLULAR AND MOLECULAR

Prostanoid Receptors Regulate the Volume-Sensitive Efflux of Osmolytes from Murine Fibroblasts via a Cyclic AMP-Dependent Mechanism

Molecular and Behavioral Neuroscience Institute (A.M.H., S.K.F.) and Department of Pharmacology (D.J.F., S.K.F.), University of Michigan, Ann Arbor, Michigan

Received June 19, 2006; accepted August 24, 2006.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
The ability of prostanoid receptors to regulate the volume-dependent efflux of the organic osmolyte taurine from murine fibroblasts (L cells) via a cAMP-dependent mechanism has been examined. Incubation of L cells under hypoosmotic conditions resulted in a time-dependent efflux of taurine, the threshold of release occurring at 250 mOsM. Addition of prostaglandin E₁ (PGE₁) potently (EC₅₀ = 2.5 nM) enhanced the volume-dependent efflux of taurine at all time points examined and increased the threshold for osmolyte release to 290 mOsM. Maximal PGE₁ stimulation (250–300% of basal) of taurine release was observed at 250 mOsM. Of the PGE analogs tested, only the EP₂-selective agonist butaprost (9-oxo-11,16S-dihydroxy-17-cyclobutyl-prost-13E-en-1-oic acid) was able to enhance taurine efflux. Inclusion of 1,9-dideoxyfoskolin, 5-nitro-2-(3-phenylpropylamino) benzoic acid, or 4-[(2-butyl-6,7-dicloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]-butanoic acid blocked the ability of PGE₁ to enhance taurine release, indicating the mediation of a volume-sensitive organic osmolyte and anion channel. The ability of PGE₁ to increase osmolyte release from L cells was mimicked by the addition of agents that inhibit cAMP breakdown, directly activate adenylyl cyclase, or are cell-permeant analogs of cAMP. Taurine release elicited by either PGE₁ or 8-(4-chlorophenylthio)-cAMP was attenuated by >70% in L cells that had been stably transfected with a mutant regulatory subunit of cAMP-dependent protein kinase (PKA). PGE₁ stimulation of taurine efflux was not attenuated by either depletion of intracellular calcium or inhibition of protein kinase C. The results indicate that activation of prostanoid receptors on murine fibroblasts enhances osmolyte release via a cAMP and PKA-dependent mechanism.

When measured in vitro, the efflux of organic osmolytes is relatively insensitive to hypoosmotic stress, often requiring substantial (nonphysiological) reductions in osmolarity before a significant efflux of osmolytes occurs. This observation, along with previous reports that swelling-induced osmolyte release can be enhanced by Ca²⁺ ionophores, phorbol esters, or agents known to elevate cAMP (Strange et al., 1993; Novak et al., 2000; Moran et al., 2001), raises the possibility that, in vivo, the activity of VSOAC may be regulated by the activity of G protein-coupled receptors (GPCRs). In this context, we and others have recently identified a number of Ca²⁺-mobilizing GPCRs that, when activated, enhance the volume-sensitive efflux of osmolytes from both neural and non-neural cells: P2Y purinergic receptors in rat primary astrocytes (Mongin and Kimelberg, 2002, 2005); H₁ histamine receptors in HeLa cells (Falktoft and Lambert, 2004); m₃ muscarinic cholinergic (mAChR), lysophosphatidic acid, and sphingosine 1-phosphate receptors in human SH-SY5Y neuroblastoma (Loveday et al., 2003; Heacock et al., 2004, 2006); and the protease-activated receptor-1 in myoblasts and human 1321N1 astrocytoma (Manopoulos et al., 1997; Cheema et al., 2005). Receptor activation has been demonstrated to facilitate the ability of the cells to release osmolytes under conditions of very limited reductions in osmolarity (5–10%) via a mechanism that seems to involve intracellular Ca²⁺ and protein kinase C (PKC) activity.

A major signal transduction pathway used by a large number of GPCRs is the activation of adenylyl cyclase with the concomitant formation of cAMP. In this context, the addition of forskolin, a direct activator of adenylyl cyclase, has been reported to increase osmolyte release in some, but not all, tissues (Strange et al., 1993; Manopoulos et al., 1997; Moran et al., 2001). Electrophysiological recordings indicate that cAMP can also increase I_Cl,swell, although inhibitory effects of the cyclic nucleotide have also been reported (Carpenter and Peers, 1997; Du and Sorota, 1997; Nagasaki et al., 2000; Shimizu et al., 2000). Although these results indicate a potential role for cAMP in osmoregulation, the ability of endogenously expressed adenylyl cyclase-linked receptors to regulate osmolyte efflux has not been systematically examined. In the present study, we have evaluated the ability of prostanoid receptors present in murine L fibroblasts to regulate osmolyte efflux under conditions of hypoosmotic stress. These cells are known to possess prostanoid receptors that robustly couple to adenylyl cyclase and PKA activation (Maganiello and Vaughn, 1972; Uhler and Abou-Chebl, 1992). The results indicate that activation of prostanoid receptors (principally of the EP₂ subtype) facilitates a volume-dependent increase in osmolyte release that is mediated via a VSOAC. The stimulatory effect of PGE₁ on taurine efflux can be mimicked by agents that elevate intracellular cAMP and that are attenuated in an L cell line that exhibits reduced PKA activity. Moreover, in contrast to the responses elicited by agonist occupancy of Ca²⁺-mobilizing receptors, osmolyte efflux triggered by prostanoid receptor activation is independent of both intracellular Ca²⁺ and PKC.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Materials. [1,2-³H]Taurine (1.1 TBq/mmol) was obtained from GE Healthcare (Piscataway, NJ). [-³²P]ATP (111 TBq/mmol) was from PerkinElmer Life and Analytical Sciences (Boston, MA). NPPB, sphingosine 1-phosphate, forskolin, thrombin, 8-CPT-cAMP, 8-bromo-cAMP, pepstatin A, phenylmethylsulfonyl fluoride, 1,10-phenanthroline, dithiothreitol, cAMP, ATP, and Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) were purchased from Sigma-Aldrich (St. Louis, MO). Prostaglandin E₁ was from BIOMOL Research Laboratories (Plymouth Meeting, PA). DDF, Gö6983 and IBMX were obtained from Calbiochem (San Diego, CA). DCPIB was purchased from Tocris Cookson Inc. (Ellisville, MO). Guanidinethyl sulfonate was obtained from Toronto Chemicals (Toronto, ON, Canada). Fura-2/acetoxymethyl ester (Fura-2/AM) was from Invitrogen (Eugene, OR). Butaprost (9-oxo-11,16S-dihydroxy-17-cyclobutyl-prost-13E-en-1-oic acid, free acid), 17-phenyl trinor PGE₂ (9-oxo-11,15S-dihydroxy-17-phenyl-18,19,20-trinor-5Z,13E-dien-1-oic acid), and sulprostone [N-(methylsulfonyl)-9-oxo-11,15R-dihydroxy-16-phenoxy-17, 18,19,20-tetranor-prosta-5Z,13E-dien-1-amide] were obtained from Cayman Chemical (Ann Arbor, MI). Dulbecco's modified Eagle's medium (DMEM), genetecin (G418), 50x penicillin/streptomycin, and horse serum were obtained from Invitrogen. Universol was obtained from ICN (Aurora, OH).

Cell Culture Conditions. Murine Ltk^– fibroblasts (L cells; passage numbers 5–19) and RAB-10 cells (an L cell-derived cell line that exhibits reduced PKA activity; passage numbers 5–11; Uhler and Abou-Chebl, 1992) were grown in tissue culture flasks (75-cm²/250 ml) in 20 ml of DMEM supplemented with 10% (v/v) horse serum and 1% penicillin/streptomycin. For the RAB-10 cells, 750 µg/ml G418 was included. 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 detached from the flask with a tryspin-versene mixture (Cambrex Bio Science, Walkersville, MD). Cells were then resuspended in DMEM/10% horse 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 95 to 100% confluence were routinely used.

Measurement of Taurine Efflux. Osmolyte efflux from L cells and RAB-10 cells was monitored essentially as described previously for SH-SY5Y neuroblastoma (Heacock et al., 2004, 2006). In brief, L cells were prelabeled overnight with 18.5 KBq/ml [³H]taurine at 37°C. Under these conditions, approximately 90 to 95% of the added radiolabel was taken up into the cells. Uptake of radiolabel into L cells was time-dependent (t_1/2 of 3 h) and temperature-sensitive (inhibited >98% by lowering the temperature to 4°C) and was inhibited >70% by inclusion of 500 µM guanidinethyl sulfonate, an inhibitor of the taurine uptake transporter (Lambert, 2004). After prelabeling, the cells were washed two 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, and 30 mM HEPES, pH 7.4, 1 mg/ml D-glucose; 335 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 PGE₁. 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 times indicated, aliquots (200 µl) of the extracellular medium were removed, and radioactivity was determined after the addition of 5 ml of Universol scintillation fluid (MP Biomedicals, Solon, OH). 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. Taurine efflux was calculated as a fractional release, i.e., the radioactivity released in the extracellular media 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 (Novak et al., 1999). "Basal" release of taurine is defined as that which occurs at a specified osmolarity in the absence of agonists or cyclic AMP analogs.

Measurement of PKA Activity. PKA activity was determined essentially as described by Uhler and McKnight (1987). L cells or RAB-10 cells were harvested by detaching the cells from the flasks with a rubber policeman and then centrifuged at 5000g for 5 min. Cell pellets were then resuspended in homogenization buffer (10 mM NaPO₄ buffer, pH 7.0, 1 mM EDTA, 1 mM dithiothreitol, 1 mM iodoacetic acid, 0.1 mM phenylmethylsufonyl fluoride, 1 mM 1,10-phenanthroline, 1 mM pepstatin A, and 250 mM sucrose), sonicated (six times for 1 s each), and protein concentrations was adjusted to 1 mg/ml. Assays (50 µl final volume) were conducted for 5 min at 30°C and contained (final concentrations) 100 µM ATP (250 dpm/pmol), 5 mM magnesium acetate, 15 µM Kemptide, 250 µM IBMX, 5 mM dithiothreitol, 2.5 mM NaF, and 10 mM Tris-HCl, pH 7.4. When included in the assay, the concentration of cAMP was 5 µM. The phosphorylation of Kemptide was determined by spotting 25 µlofthe incubation mixture on Whatman PE81 phosphocellulose filter papers (2 x 2 cm; Whatman, Maidstone, UK) and washing them four times with 200 ml of 10 mM orthophosphoric acid. After a final wash in 95% ethanol, individual filters were allowed to dry at room temperature, and radioactivity was determined after the addition of 5 ml of Universol scintillation fluid.

Measurement of Cytoplasmic Calcium Concentrations. Cytoplasmic free calcium concentrations, [Ca²⁺]_i, were determined in suspensions of L cells after preloading cells with the Ca²⁺ indicator Fura-2/AM, as described previously (Fisher et al., 1989). The fluorometer used was a Shimadzu RF-5301PC spectrofluorometer (Shimadzu Scientific Instruments, Columbia, MD).

Data Analysis. Experiments were performed in triplicate and repeated at least three times. Except where stated otherwise, 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). 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. EC₅₀ values were obtained using Prism 4.0a (GraphPad Software Inc., San Diego, CA).

	Results

Top Abstract Materials and Methods Results Discussion References

 
Osmosensitive Efflux of Taurine from L Cells Is Enhanced by the Addition of PGE₁. When L cells that had been prelabeled with [³H]taurine were exposed to hypotonic buffer (250 mOsM), there was a time-dependent efflux of the radiolabeled amino acid, the initial rate of which (<5 min) exceeded that observed in more prolonged incubations (Fig. 1). Inclusion of 20 µM PGE₁ significantly enhanced the rate of release of taurine at all time points examined and increased the magnitude of response to approximately 250 to 300% of basal (basal release being that monitored in the absence of an agonist). As a result of these observations, both basal and agonist-stimulated taurine effluxes were routinely monitored after 10 min of incubation in subsequent experiments. During the course of the present study, some inter-experimental variability in the magnitude of the basal release of taurine (5.2 ± 2.6% of total, mean ± S.D.; n = 65) and in the extent of PGE₁-stimulated taurine efflux (274 ± 74% of control, mean ± S.D.; n = 65) was observed. The addition of PGE₁ resulted in a concentration-dependent stimulation of taurine efflux with an EC₅₀ value of 2.5 ± 0.4 nM (n = 3) and a Hill coefficient close to unity (1.1 ± 0.2; n = 3; Fig. 2). To determine the subtype of prostanoid receptor coupled to osmolyte efflux, L cells were incubated in the presence of 1 µM concentrations of PGE₁, butaprost (EP₂-selective), 17-phenyl-trinor PGE₂ (EP₁- and EP₃-selective), or sulprostone (EP₃-selective). Of these analogs, only butaprost elicited a significant increase in taurine release (Fig. 3).

View larger version (10K): [in this window] [in a new window]   Fig. 1. Kinetics of basal and PGE1-stimulated taurine efflux from L cells. L cells that had been prelabeled overnight in the presence of [3H]taurine were washed twice with 2 ml of isotonic buffer A before incubation in 250 mOsM buffer A in the presence or absence of 20 µM PGE1. Reactions were terminated at the times indicated, and taurine efflux was measured. Results are expressed as taurine efflux (percentage of total soluble radioactivity) and (where error bars are shown) are the means ± S.E.M. for three independent experiments, each performed in triplicate. Where errors bars are absent, the result from a single experiment is shown.

View larger version (10K): [in this window] [in a new window]   Fig. 2. Dose-response relationships for PGE1-stimulated taurine efflux. Cells that had been prelabeled with [3H]taurine were washed with isotonic buffer A and then incubated in 250 mOsM buffer in the presence of PGE1 at the concentrations indicated. Reactions were terminated after 10 min, and taurine efflux was monitored. Results are expressed as taurine efflux (percentage of total soluble radioactivity), and are the means ± S.E.M. for triplicate replicates obtained from a single experiment, representative of three conducted. Where an error bar is not shown, the S.E.M. fell within the symbol. In the experiment shown, the calculated EC50 value for stimulated taurine efflux was 2.0 nM, and the Hill coefficient was 0.8.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Butaprost, an EP2-selective agonist, mimics the ability of PGE1 to stimulate taurine release. Cells that had been prelabeled with [3H]taurine were washed in isotonic buffer A and then incubated in 250 mOsM buffer in the presence or absence of 1 µM concentrations of PGE1, butaprost, 17-phenyl trinor PGE2 (17-PT-PGE2), or sulprostone. Reactions were terminated after 10 min, and taurine efflux was monitored. Results are expressed as taurine efflux (percentage of total soluble radioactivity) and are the means ± S.E.M. for five independent experiments, each performed in triplicate. *, p < 0.01, different from basal (by paired Student's t test).

The Ability of PGE₁ to Enhance the Volume-Sensitive Efflux of Taurine from L Cells Is Dependent on Osmolarity. The ability of PGE₁ to enhance the release of taurine at different osmolarities was examined. Both basal and PGE₁-stimulated release of taurine were monitored under conditions of isotonicity (335 mOsM; defined by the osmolarity of the DMEM/horse serum medium in which the cells were grown), mild-severe hypotonicity (295–190 mOsM), or mild hypertonicity (370 mOsM). In the series of experiments conducted, the basal release of taurine (i.e., that monitored in the absence of an agonist) was not significantly enhanced until the osmolarity of the buffer had been reduced to 250 mOsM. In contrast, the addition of PGE₁ resulted in a significant increase in taurine efflux under mildly hypotonic conditions (290 mOsM; Fig. 4). Moreover, as the osmolarity of the buffer was reduced, the ability of PGE₁ to enhance taurine efflux over the basal component was further increased. The maximal enhancement of taurine efflux was observed at an osmolarity of 250 mOsM (350% of basal), but not under either isotonic or mildly hypertonic conditions (Fig. 4). As a result of these findings, an osmolarity of 250 mOsM was chosen for all subsequent experiments.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Basal and PGE1-stimulated release of taurine as a function of osmolarity. Cells prelabeled with [3H]taurine were first washed in isotonic buffer A and then incubated for 10 min in buffer A at the osmolarities indicated in the absence (open bars) or presence of 20 µM PGE1 (closed bars). Reactions were terminated after 10 min, and taurine efflux was monitored. Results are expressed as taurine efflux (percentage of total soluble radioactivity) and are the means ± S.E.M. for four independent experiments, each performed in triplicate. *, p < 0.05, different from taurine release observed in cells incubated in isotonic buffer A (335 mOsM) (by one-way ANOVA followed by Dunnett's multiple comparison test). **, p < 0.01, different from basal release (by paired Student's t test).

PGE₁-Mediated Efflux of Taurine from L Cells Is Mediated via a VSOAC. Previously, we demonstrated that osmolyte release triggered by the activation of mAChRs, protease-activated receptors, or lysophospholipid receptors is mediated via a VSOAC (Heacock et al., 2004, 2006; Cheema et al., 2005). To determine whether taurine release elicited by the activation of prostaglandin receptors also occurred via the same channel(s), basal and PGE₁-stimulated taurine effluxes were monitored in the presence of three putative blockers of VSOAC, namely, NPPB, DDF, and DCPIB. Each of these agents (at concentrations of 100 µM for DDF and NPPB or 30 µM for DCPIB) resulted in a significant inhibition of both basal and PGE₁-stimulated taurine release (45–62% and 74–90%, respectively; Fig. 5).

View larger version (11K): [in this window] [in a new window]   Fig. 5. Inhibition of basal and PGE1-stimulated taurine release by anion channel blockers. Cells that had been prelabeled overnight with [3H]taurine were washed in isotonic buffer A and then incubated in hypotonic buffer A (250 mOsM) with either 100 µM NPPB (A), 100 µM DDF (B), or 30 µM DCPIB (C) in the absence or presence of 20 µM PGE1. Reactions were terminated after 10 min, and efflux of taurine was monitored. Results are expressed as taurine efflux (percentage of total soluble radioactivity) and are the means ± S.E.M. of six to eight independent experiments, each performed in triplicate. *, p < 0.05, different from control basal, and **, p < 0.03, different from PGE1-stimulated efflux under control conditions (by paired Student's t test).

View larger version (32K): [in this window] [in a new window]   Fig. 6. Agents that increase cAMP concentrations in cells mimic the ability of PGE1 to enhance taurine release. Cells that had been prelabeled overnight with [3H]taurine were washed in isotonic buffer A and then incubated in hypotonic buffer A (250 mOsM) with either 1 mM IBMX, 20 µM PGE1, or both (A) or 50 µM forskolin (FSK), 1 mM 8-CPT-cAMP, or 1 mM 8-bromo cAMP (B). Reactions were terminated after 10 min, and efflux of taurine was monitored. Results are expressed as taurine efflux (percentage of total soluble radioactivity) and are the means ± S.E.M. of four to six independent experiments, each performed in triplicate. *, p < 0.05 and **, p < 0.01, different from control basal (by paired Student's t test).

View larger version (18K): [in this window] [in a new window]   Fig. 7. The ability of PGE1 or 8-CPT-cAMP to stimulate taurine efflux is attenuated in RAB-10 cells that exhibit a reduced PKA activity. A, L cells or RAB-10 cells that had been prelabeled overnight with [3H]taurine were washed in isotonic buffer A and then incubated in hypotonic buffer A (250 mOsM) with either 20 µM PGE1, 1 mM 8-CPT-cAMP, 1.25 nM thrombin, or 10 µM sphingosine 1-phosphate (S1P). Reactions were terminated after 10 min, and efflux of taurine was monitored. Results are expressed as taurine efflux (percentage of total soluble radioactivity) and are the means ± S.E.M. for five independent experiments, each performed in triplicate. *, p < 0.03 and **, p < 0.006, different from control basal (by paired Student's t test). B, PKA activity was measured in extracts of L cells or RAB-10 cells in the absence or presence of 10 µM cAMP. Results shown are means ± S.E.M. for seven independent experiments, each performed in triplicate. *, p < 0.001, different from L cells (by paired Student's t test).

PGE₁ Stimulation of Taurine Efflux Is Independent of Intracellular Ca²⁺ and PKC Activity. To date, a common characteristic of those agonists that have been demonstrated to promote the efflux of osmolytes from cells is their ability to elicit increases in [Ca²⁺]_i. Likewise, the addition of PGE₁ to Fura-2-loaded L cells also resulted in a 2- to 3-fold rise in [Ca²⁺]_i. Thrombin addition also elicited an increase in [Ca²⁺]_i (Fig. 8). The rise in [Ca²⁺]_i triggered by the addition of PGE₁ seems to be independent of cAMP formation, since neither the addition of forskolin nor the addition of 8-CPT-cAMP had any significant effect on [Ca²⁺]_i. Furthermore, no significant increase in [Ca²⁺]_i was observed in the presence of butaprost (data not shown). The agonist-induced increases in [Ca²⁺]_i evoked by PGE₁ and thrombin were both markedly attenuated when extracellular Ca²⁺ was omitted (>75%), and they were abolished following depletion of the intracellular pool of Ca²⁺ with thapsigargin (Fig. 8B).

View larger version (10K): [in this window] [in a new window]   Fig. 8. Addition of PGE1 or thrombin elicits an increase in cytoplasmic free calcium in L cells. A, Fura-2/AM-loaded cells were first resuspended in 250 mOsM buffer A and then either 20 µM PGE1 or 1.25 nM thrombin was added. Changes in [Ca2+]i were monitored after the addition of the agonists at 120 s (indicated by the arrow). Traces shown are representative of 15 to 17 experiments. B, for each agonist, the maximal increase in [Ca2+]i was monitored under control conditions (2.2 mM Ca2+; open bars), in the absence of extracellular Ca2+ (closed bars), or following pretreatment of the cells for 5 min with 1 µM thapsigargin in the absence of extracellular Ca2+(hatched bars). Results shown are means ± S.E.M. for 15 to 17 experiments (+Ca2+) or four experiments (–Ca2+, –Ca2+/thapsigargin). *, p < 0.05 and **, p < 0.001, different from control (by paired Student's t test).

The ability of PGE₁ to stimulate Ca²⁺ mobilization in L cells was unexpected and prompted us to examine the role, if any, played by Ca²⁺ in basal, PGE₁-, or thrombin-stimulated taurine efflux. Removal of extracellular Ca²⁺ reduced the swelling-induced (basal) release of taurine and that elicited by the addition of either PGE₁ or thrombin to the same extent, i.e., approximately 30 to 35%. However, when expressed relative to their controls, PGE₁-stimulated taurine efflux was only minimally reduced by the omission of Ca²⁺ (203 ± 13 and 183 ± 8% of control for PGE₁ in the presence and absence of extracellular Ca²⁺, respectively; n = 10; Fig. 9A). Likewise, thrombin-stimulated taurine efflux was also unaffected by the removal of extracellular Ca²⁺ (276 ± 28 and 289 ± 47% of control, in the presence and absence of Ca²⁺, respectively; n = 6; Fig. 9B). To examine the role of intracellular Ca²⁺, cells were first preincubated for 5 min in the presence of 1 µM thapsigargin (in the absence of extracellular Ca²⁺) to discharge the intracellular Ca²⁺ pools, and then they were challenged with either PGE₁ or thrombin. Under these conditions, PGE₁-stimulated taurine efflux was not significantly reduced (177 ± 6 and 183 ± 8% of control, in the presence and absence of thapsigargin, respectively). Basal release of taurine was also unaffected by depletion of intracellular Ca²⁺. In contrast, thrombin-stimulated taurine efflux was diminished by 65% following depletion of intracellular Ca²⁺ (167 ± 9 and 289 ± 47% of control, in the presence and absence of thapsigargin, respectively; p < 0.01; Fig. 9B).

View larger version (17K): [in this window] [in a new window]   Fig. 9. Role of extracellular and intracellular calcium in PGE1- or thrombin-stimulated taurine efflux. Cells that had been prelabeled overnight with [3H]taurine were washed in isotonic buffer A and then incubated for 5 min in hypotonic buffer A (250 mOsM) in the presence (open bars) or absence (closed bars) of extracellular Ca2+ (Ca2+ was omitted from the buffer and 50 µM EGTA added) before the addition of either 20 µM PGE1 (A) or 1.25 nM thrombin (B), as indicated. In some experiments, cells were preincubated for 5 min in Ca2+-free hypotonic buffer A (Ca2+ omitted and 50 µM EGTA added) in the presence (stippled bars) of 1 µM thapsigargin (TG) before the addition of agonist. Incubations were then allowed to proceed for an additional 10 min, and then reactions were terminated. Results are expressed as taurine efflux (percentage of total soluble radioactivity) and are the means ± S.E.M. for 10 independent experiments for PGE1 or six independent experiments for thrombin, each performed in triplicate. *, p < 0.05, different from Ca2+-containing control incubations (by one-way ANOVA followed by Dunnett's multiple comparison test); **, p < 0.01, different from Ca2+-free incubations (by one-way ANOVA followed by Dunnett's multiple comparison test).

To examine the involvement of PKC in PGE₁- and thrombin-stimulated taurine efflux, L cells were preincubated in isotonic buffer A for 15 min with 10 µM chelerythrine before agonist challenge under hypotonic conditions. Chelerythrine had no inhibitory effect on basal, PGE₁- or thrombin-stimulated taurine efflux, and preincubation of L cells with the PKC inhibitor slightly enhanced all three parameters (Fig. 10). When calculated relative to their respective controls, the addition of PGE₁ increased taurine efflux to 306 ± 36 and 320 ± 44% of control in the absence or presence of chelerythrine, respectively, whereas the corresponding values for thrombin-stimulated taurine efflux were 428 ± 125 and 430 ± 136% of control, respectively (n = 5; Fig. 10). In addition, chelerythrine had no effect on mAChR-stimulated taurine release from L cells that had been stably transfected with the m₃ mAChR (data not shown). The ability of Gö6983, a highly potent cell-permeant PKC inhibitor, to inhibit PGE₁-stimulated taurine efflux was also examined. When cells were pretreated for 15 min with 1 µM Gö6983 and then challenged with PGE₁, agonist-stimulated release of taurine was not significantly reduced (278 ± 12 and 284 ± 27% of basal in the absence and presence of Gö6983, respectively; n = 6). Preincubation of L cells with 1 µM bisindolylmaleimide, another broad-spectrum PKC inhibitor, also had no inhibitory effect on basal, PGE₁- or thrombin-stimulated taurine efflux (data not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 10. Inhibition of PKC does not attenuate either PGE1- or thrombin-stimulated taurine efflux. Cells were pretreated in the absence or presence of 10 µM chelerythrine in isotonic buffer A for 15 min before incubation of cells in hypotonic buffer A (250 mOsM) in the absence (control; open bars) or presence of chelerythrine (closed bars) in the presence or absence of PGE1 or thrombin, as indicated. Reactions were terminated after 10 min, and taurine efflux was monitored. Results are expressed as taurine efflux (percentage of total soluble radioactivity) and are the means ± S.E.M for five independent experiments, each performed in triplicate. *, p < 0.01, different from control release (by paired Student's t test).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Although the role of organic osmolyte release in volume regulation following hypoosmotic stress has been extensively studied, only recently has the possibility that this process is subject to neurohumoral control been systematically examined. To date, of the several GPCRs that have been demonstrated to facilitate the volume-dependent release of osmolytes from cells, all seem to share a common characteristic in that receptor activation triggers a mobilization of intracellular Ca²⁺, although the precise role of Ca²⁺ in osmolyte release remains to be determined. Furthermore, maximal receptor-stimulated osmolyte release also seems to require PKC activity (Mongin and Kimelberg, 2002, 2005; Falktoft and Lambert, 2004; Heacock et al., 2004, 2006; Cheema et al., 2005). In the present study, we demonstrate that a GPCR that couples to adenylyl cyclase activation is also able to facilitate osmolyte release via a mechanism that is distinct from that described previously. Evidence to support the conclusion that prostanoid receptors present on mouse fibroblasts regulate osmolyte efflux via a cAMP-dependent mechanism is based on three series of experimental observations. First, the ability of PGE₁ to enhance taurine efflux could be mimicked by the addition of agents that elevate intracellular cAMP concentrations via either the inhibition of cAMP breakdown or direct activation of adenylyl cyclase, or, alternatively, by acting directly as cell-permeant analogs of cAMP (Fig. 6). Moreover, when these agents were included in incubations that contained PGE₁, no further increase in osmolyte release was observed, a result consistent with a common mechanism of action. Second, of the PGE analogs tested, only butaprost, an analog that selectively activates the prostanoid EP₂ receptor subtype that couples to activation of adenylyl cyclase, could facilitate osmolyte release. Third, the ability of either PGE₁ or 8-CPT-cAMP (but not that of either thrombin or sphingosine 1-phosphate) to stimulate taurine efflux was significantly attenuated in RAB-10 cells, which exhibit a lower activity of PKA than L cells. Since PKA is the major downstream cellular target for cAMP action, this result is a further indication that the cyclic nucleotide is the mediator of osmosensitive increases in taurine efflux. The conclusion that prostanoid receptor-mediated changes in cAMP can regulate taurine release is consistent with a previous study in which isoproterenol, a -adrenergic agonist, was observed to increase osmolyte efflux from glial cells (Moran et al., 2001). Taken collectively, the results indicate that receptor-mediated increases in cAMP are potentially linked to the process of osmoregulation in cells.

One complication in the interpretation of our results is that, in addition to its previously documented ability to increase the concentration of intracellular cAMP in L cells (Maganiello and Vaughn, 1972; Uhler and Abou-Chebl, 1992), PGE₁ was also observed to elicit an increase in [Ca²⁺]_i (Fig. 8). However, two lines of evidence suggest that the rise in [Ca²⁺]_i and increases in cAMP concentration are distinct events in L cells. First, the addition of forskolin, 8-CPT-cAMP, or the EP₂-selective agonist butaprost (all of which elicit robust increases in taurine efflux) failed to mimic the ability of PGE₁ to increase [Ca²⁺]_i. Second, the PGE₁-mediated increase in osmolyte release was essentially independent of both extra- and intracellular Ca²⁺ (when calculated on a –fold stimulation basis), even though the agonist-mediated increase in Ca²⁺ was either substantially inhibited or abolished under these conditions (Fig. 8B). In contrast, although taurine release stimulated by thrombin addition was also independent of extracellular Ca²⁺, depletion of intracellular Ca²⁺ with thapsigargin strongly attenuated the response, a result consistent with our previous findings in astrocytoma cells (Cheema et al., 2005). The most parsimonious interpretation of these results is that L cells possess two populations of prostanoid receptors, one population that couples to the activation of adenylyl cyclase, PKA activation, and osmolyte release and a second population of receptors that is linked to an increase in Ca²⁺ mobilization. It seems that the latter population of receptors does not make a significant contribution to osmolyte release in L cells. In this context, it should be noted that distinct differences in the susceptibility of GPCR-stimulated osmolyte release to depletion of intracellular Ca²⁺ have been observed. Thus, whereas taurine release elicited by the addition of either lysophosphatidic acid or sphingosine 1-phosphate is reduced by 30 to 40%, the responses to ATP and muscarinic agonists are essentially abolished (Mongin and Kimelberg, 2005; Heacock et al., 2006).

PGE₁ stimulation of taurine release also seems to be independent of PKC, as determined from the inability of chelerythrine, Gö6983, or bisindolylmaleimide to significantly inhibit either basal or PGE₁-induced osmolyte release. The observation that inhibition of PKC also did not attenuate either thrombin- or mAChR-stimulated taurine release from L cells was unexpected and at variance with previous studies in which PKC activity was found to be necessary for the maximal release of osmolytes in response to either of these receptors (Cheema et al., 2005; Heacock et al., 2006). One interpretation of the present findings is that PKC activity may not invariably be a prerequisite for agonist-stimulated osmolyte release, even for Ca²⁺-mobilizing receptors.

Although taurine release elicited by prostanoid receptor stimulation seems to differ from that exhibited by previously studied receptors in terms of its apparent lack of Ca²⁺ and PKC dependence, two features common to all receptors can be identified. The first feature is that, similar to osmolyte release induced by Ca²⁺-mobilizing agonists, a VSOAC seems to mediate osmolyte efflux as indicated by the ability of nonselective anion channel inhibitors, such as DDF and NPPB, to block PGE₁-stimulated taurine efflux. This conclusion is strengthened by the observation that DCPIB, a highly selective inhibitor of VSOAC (Decher et al., 2001), also significantly inhibits PGE₁-stimulated taurine efflux (Fig. 5). A second characteristic shared by both the prostanoid receptor and those receptors primarily linked to Ca²⁺ mobilization is a reduction in the osmotic threshold for osmolyte release following receptor activation. Thus, in the absence of PGE₁ addition, the ability of L cells to significantly respond to hypoosmotic stress is restricted to a relatively large reduction in osmolarity (>33%), whereas in the presence of the agonist, osmolyte release occurs when the osmolarity is reduced by <15% (Fig. 4). The present results are consistent with data previously obtained for osmolyte release following agonist activation of other GPCRs such as the P2Y purinergic, m3 mAChR, protease-activated receptor-1, sphingosine 1-phosphate, and lysophosphatidic acid receptors (Mongin and Kimelberg, 2002, 2005; Heacock et al., 2004, 2006; Cheema et al., 2005) and also that elicited following activation of the epidermal growth factor receptor (Franco et al., 2004). These results indicate that, regardless of the underlying mechanism(s) of activation, receptors coupled to osmolyte efflux share a common property in their ability to facilitate osmolyte release from cells in response to relatively small changes in osmolarity, such as those that are likely to be encountered in vivo.

In summary, the results in the present study provide evidence that prostanoid receptors coupled to an increase in cAMP concentration can facilitate osmolyte release from L cell fibroblasts in a volume-dependent manner by a mechanism that is distinct from that described previously for Ca²⁺-mobilizing receptors. These results raise the possibility that cells may use multiple cell signaling mechanisms to regulate their volume in the face of hypoosmotic challenge.

	Acknowledgements

 
We thank Dr. Michael Uhler (University of Michigan, Ann Arbor, MI) for providing the L cells and RAB-10 cells and for constructive input into various aspects of this study.

	Footnotes

 
This work was supported by National Institutes of Health Grant NS23831 (to S.K.F.).

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

doi:10.1124/jpet.106.109496.

ABBREVIATIONS: VSOAC, volume-sensitive organic osmolyte and anion channel; I_Cl,swell, outwardly rectifying Cl^– current activated by hypotonicity; 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; GPCR, G protein-coupled receptor; EP, prostaglandin receptor; mAChR, muscarinic cholinergic receptor; PKC, protein kinase C; PKA, cAMP-dependent protein kinase; 8-CPT-cAMP, 8-(4-chlorophenylthio)-cAMP; Gö6983, 2-[1-(3-dimethyl-aminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide; PG, prostaglandin; IBMX, 3-isobutyl-1-methylxanthine; AM, acetoxymethyl ester; DMEM, Dulbecco's modified Eagle's medium; ANOVA, analysis of variance.

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

	References

Top Abstract Materials and Methods Results Discussion References

 

Abdullaev IF, Rudkouskaya A, Schools GP, Kimelberg HK, and Mongin AA (2006) Pharmacological comparison of swelling-activated excitatory amino acid release and Cl^– currents in cultured rat astrocytes. J Physiol (Lond) 572: 677–689.[Abstract/Free Full Text]

Carpenter E and Peers C (1997) Swelling-and cAMP-activated Cl^– currents in isolated rat carotid body type l cells. J Physiol (Lond) 503: 497–511.[Abstract/Free Full Text]

Cheema TA, Ward CE, and Fisher SK (2005) Subnanomolar concentrations of thrombin enhance the volume-sensitive efflux of taurine from human 1321N1 astrocytoma cells. J Pharmacol Exp Ther 315: 755–763.[Abstract/Free Full Text]

Decher N, Lang HJ, Nilius B, Bruggemann A, Busch AE, and Steinmeyer K (2001) DCPIB is a novel selective blocker of I_Cl,swell and prevents swelling-induced shortening of guinea-pig atrial action potential duration. Br J Pharmacol 134: 1467–1479.[CrossRef][Medline]

Du XY and Sorota S (1997) Modulation of dog atrial swelling-induced chloride current by cAMP protein kinase A-dependent and –independent pathways. J. Physiol (Lond) 500: 111–122.[Abstract/Free Full Text]

Falktoft B and Lambert IH (2004) Ca²⁺-mediated potentiation of the swelling-induced taurine efflux from HeLa cells: on the role of calmodulin and novel protein kinase C isoforms. J Membr Biol 201: 59–75.[CrossRef][Medline]

Fisher SK, Domask LM, and Roland RM (1989) Muscarinic receptor regulation of cytoplasmic Ca²⁺ concentrations in human SK-N-SH neuroblastoma cells: Ca²⁺ requirements for phospholipase C activation. Mol Pharmacol 35: 195–204.[Abstract]

Franco R, Lezama R, Ordaz B, and Pasantes-Morales H (2004) Epidermal growth factor receptor is activated by hypoosmolarity and is an early signal modulating osmolyte efflux pathways in Swiss 3T3 fibroblasts. Pflueg Arch Eur J Physiol 447: 830–839.[CrossRef][Medline]

Heacock AM, Dodd MS, and Fisher SK (2006) Regulation of volume-sensitive osmolyte efflux from human SH-SY5Y neuroblastoma cells following activation of lysophospholipid receptors. J Pharmacol Exp Ther 317: 685–693.[Abstract/Free Full Text]

Heacock AM, Kerley D, Gurda GT, VanTroostenberghe AT, and Fisher SK (2004) Potentiation of the osmosensitive release of taurine and D-aspartate from SH-SY5Y neuroblastoma cells after activation of M₃ muscarinic cholinergic receptors. J Pharmacol Exp Ther 311: 1097–1104.[Abstract/Free Full Text]

Kimelberg HK (2000) Cell volume in the CNS: regulation and implications for nervous system function and pathology. Neuroscientist 6: 14–25.[Abstract/Free Full Text]

Lang F, GL. Busch, Ritter M, Volkl H, Waldegger S, Gulbins E, and Haussinger D (1998) Functional significance of cell volume regulatory mechanisms. Physiol Rev 78: 247–306.[Abstract/Free Full Text]

Lambert IH (2004) Regulation of the cellular content of the organic osmolyte taurine in mammalian cells. Neurochem Res 29: 27–63.[CrossRef][Medline]

Loveday D, Heacock AM, and Fisher SK (2003) Activation of muscarinic cholinergic receptors enhances the volume-sensitive efflux of myo-inositol from SH-SY5Y neuroblastoma cells. J Neurochem 87: 476–486.[CrossRef][Medline]

Maganiello V and Vaughn M (1972) Prostaglandin E₁ effects on adenosine 3':5'-cyclic monophosphate concentration and phosphodiesterase activity in fibroblasts. Proc Natl Acad Sci USA 69: 269–273.[Abstract/Free Full Text]

Manopoulos VG, Voets T, Declerq PE, and Nilius B (1997) Swelling-activated efflux of taurine and other organic osmolytes in endothelial cells. Am J Physiol 273: C214–C222.[Medline]

McManus ML, Churchwell KB, and Strange K (1995) Regulation of cell volume in health and disease. N Engl J Med 333: 1260–1266.[Free Full Text]

Mongin AA and Kimelberg HK (2002) ATP potently modulates anion channel-mediated excitatory amino acid release from cultured astrocytes. Am J Physiol 283: C569–C578.

Mongin AA and Kimelberg HK (2005) ATP regulates anion channel-mediated organic osmolyte release from cultured rat astrocytes via multiple Ca²⁺-sensitive mechanisms. Am J Physiol 288: C204–C213.

Moran J, Morales-Mulia M and Pasantes-Morales H (2001) Reduction of phospholemman expression decreases osmosensitive taurine efflux in astrocytes. Biochim Biophys Acta 1538: 313–320.[Medline]

Nagasaki M, Ye L, Duan D, Horowitz B, and Hume JR (2000) Intracellular cyclic AMP inhibits native and recombinant volume-regulated chloride channels from mammalian heart. J Physiol (Lond) 523: 705–717.[Abstract/Free Full Text]

Nilius B and Droogmans G (2003) Amazing chloride channels: an overview. Acta Physiol Scand 177: 119–147.[CrossRef][Medline]

Nilius B, Eggermont J, Voets T, Buyse G, Manolopoulos V, and Droogmans G (1997) Properties of volume-regulated anion channels in mammalian cells. Prog Biophys Mol Biol 68: 69–119.[CrossRef][Medline]

Novak JE, Agranoff BW, and Fisher SK (2000) Regulation of myo-inositol homeostasis in differentiated human NT2-N neurons. Neurochem Res 25: 561–566.[CrossRef][Medline]

Novak JE, Turner RS, Agranoff BW, and Fisher SK (1999) Differentiated human NT2-neurons possess a high intracellular content of myo-inositol. J Neurochem 72: 1431–1440.[CrossRef][Medline]

Pasantes-Morales H, Cardin V, and Tuz K (2000) Signaling events during swelling and regulatory volume decrease. Neurochem Res 25: 1301–1314.[CrossRef][Medline]

Pasantes-Morales H, Franco R, Ordaz B, and Ochoa LD (2002) Mechanisms counteracting swelling in brain cells during hyponatremia. Arch Med Res 33: 237–244.[CrossRef][Medline]

Shimizu T, Morishima S, and Okada Y (2000) Ca²⁺-sensing receptor mediated regulation of volume-sensitive Cl^– channels in human epithelial cells. J Physiol (Lond) 528: 457–472.[Abstract/Free Full Text]

Strange K, Morrison R, Shrode L, and Putnam R (1993) Mechanism and regulation of swelling-activated inositol efflux in brain glial cells. Am J Physiol 265: C244–C256.[Medline]

Stutzin A and Hoffman EK (2006) Swelling-activated ion channel functional regulation in cell-swelling, proliferation and apoptosis. Acta Physiol 187: 27–42.[CrossRef]

Uhler MD and Abou-Chebl A (1992) Cellular concentrations of protein kinase A modulate prostaglandin and cAMP induction of alkaline phosphatase. J Biol Chem 267: 8658–8665.[Abstract/Free Full Text]

Uhler MD and McKnight GS (1987) Expression of cDNAs for two isoforms of the catalytic subunit of cAMP-dependent protein kinase. J Biol Chem 262: 15202–15207.[Abstract/Free Full Text]

This article has been cited by other articles:

				T. A. Cheema and S. K. Fisher Cholesterol Regulates Volume-Sensitive Osmolyte Efflux from Human SH-SY5Y Neuroblastoma Cells following Receptor Activation J. Pharmacol. Exp. Ther., February 1, 2008; 324(2): 648 - 657. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.109496v1 319/2/963    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Heacock, A. M. Articles by Fisher, S. K. Search for Related Content PubMed PubMed Citation Articles by Heacock, A. M. Articles by Fisher, S. K.