Osmotic swelling-provoked release of organic osmolytes in human intestinal epithelial cells

Sebastian F. B. Tomassen, Durk Fekkes,2 Hugo R. de Jonge,1 and Ben C. Tilly1

Departments of 1Biochemistry and 2Psychiatry, Erasmus University Medical Center, 3000 DR Rotterdam, The Netherlands

Submitted 27 October 2003 ; accepted in final form 10 February 2004


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Human Intestine 407 cells respond to osmotic cell swelling by the activation of Cl- and K+-selective ionic channels, as well as by stimulating an organic osmolyte release pathway readily permeable to taurine and phosphocholine. Unlike the activation of volume-regulated anion channels (VRAC), activation of the organic osmolyte release pathway shows a lag time of ~30–60 s, and its activity persists for at least 8–12 min. In contrast to VRAC activation, stimulation of organic osmolyte release did not require protein tyrosine phosphorylation, active p21rho, or phosphatidylinositol 3-kinase activity and was insensitive to Cl channel blockers. Treatment of the cells with putative organic anion transporter inhibitors reduced the release of taurine only partially or was found to be ineffective. The efflux was blocked by a subclass of organic cation transporter (OCT) inhibitors (cyanine-863 and decynium-22) but not by other OCT inhibitors (cimetidine, quinine, and verapamil). Brief treatment of the cells with phorbol esters potentiated the cell swelling-induced taurine efflux, whereas addition of the protein kinase C (PKC) inhibitor GF109203X largely inhibited the response, suggesting that PKC is involved. Increasing the level of intracellular Ca2+ by using A-23187- or Ca2+-mobilizing hormones, however, did not affect the magnitude of the response. Taken together, the results indicate that the hypotonicity-induced efflux of organic osmolytes is independent of VRAC and involves a PKC-dependent step.

regulatory volume decrease; taurine; volume-regulated anion channels; chloride channel; protein kinase C


BECAUSE OF THE WATER PERMEABILITY of the plasma membrane, perturbations of the osmotic equilibrium between the cell and its surrounding medium result in an immediate change in cell volume. To avoid the potential deleterious consequences of cell shrinkage or swelling, most eukaryotic cells have developed compensatory mechanisms that involve the activation of ion channels and/or transporters in the plasma membrane (for review, see Refs. 29, 31, 48, 49). Hyposmotic stimulation of human intestine 407 epithelial cells leads to a rapid, transient increase in cell volume accompanied by the activation of distinct K+- and Cl-selective ion channels [i.e., regulatory volume decrease (RVD)] (55). The cell swelling-induced activation of the anion channels involved was found to require protein tyrosine phosphorylation and to depend on active p21rho (43, 53). Similar results were obtained with several other cell models, including vascular endothelial (5, 44) and neuronal cells (7, 8), as well as plant cells (5).

In a number of tissues, the release of small molecules such as taurine, betaine, and sorbitol contributes significantly to RVD. In excitable cells, these metabolites were found to be the major osmolytes released (41, 45). Taurine especially has been implicated as an osmolyte involved in volume regulation (1, 10, 19).

As is the case for volume-regulated anion channels (VRAC), the molecular identity of the organic osmolyte release pathway has not been elucidated yet (for review, see Ref. 42). In a number of cell types, the efflux of organic osmolytes and anion conductance was regulated similarly, suggesting that a single release pathway is involved [i.e., volume-sensitive organic osmolyte and anion channel (VSOAC)] (16, 2224, 40, 47). In other cells, however, distinct pathways and/or transporters have been proposed (40, 46). Indeed, in hippocampal slice preparations, at least two pathways for taurine release have been identified that differ in their kinetics of activation/inactivation and in their sensitivity for inhibitors (11).

Using intestine 407 cells, we investigated the hypotonicity-provoked release pathway for organic osmolytes and compared its properties with those of VRAC activation. This cell line was particularly suitable for this study because no plasma membrane Cl conductances other than VRAC, such as CFTR and voltage-sensitive or Ca2+-activated anion channels, are expressed (18, 55). In this article, we report that osmotic swelling of intestine 407 cells results in massive release of taurine after a distinct lag period. The results indicate that the efflux of taurine is regulated independently of VRAC and uses a signaling pathway involving protein kinase C (PKC).


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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Materials. Radioisotopes (125I, [3H]taurine, and [3H]choline) were purchased from Amersham Netherlands (s'Hertogenbosch, The Netherlands). A-23187, GF109203X, and LY-294002 were obtained from Molecular Probes (Eugene, OR), Biomol (Plymouth Meeting, PA), and Upstate Biotechnology (Lake Placid, NY), respectively. All other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Cell culture. Intestine 407 cells were routinely grown as a monolayer in DMEM supplemented with 25 mM HEPES, 10% FCS, 1% nonessential amino acids, 40 mg/l penicillin, and 90 mg/l streptomycin in a humidified atmosphere of 95% O2 and 5% CO2 at 37°C. Before the experiments, cells were serum-starved overnight.

Radioisotope efflux assays. Confluent monolayers of intestine 407 cells were loaded with 5 µCi/ml 125I, 0.1 µCi/ml [3H]choline, or 0.1 µCi/ml [3H]taurine for 2 h in modified Meyler solution (108 mM NaCl, 4.7 mM KCl, 1.3 mM CaCl2, 1 mM MgCl2, 20 mM NaHCO3, 0.8 mM Na2HPO4, 0.4 mM NaH2PO4, 20 mM HEPES, and 10 mM glucose, pH 7.40) and washed three times with isotonic buffer (66 mM NaCl, 4.7 mM KCl, 1.3 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 123 mM mannitol, and 20 mM HEPES, pH 7.4). Hypotonic buffers were prepared by adjusting the concentration of mannitol. Full time courses were prepared by replacing the medium at 1- to 2-min intervals. For inhibitor studies, a single fraction of 8 min was collected. Radioactivity in the medium was determined by gamma (125I) or beta (3H) radiation counting and expressed as fractional efflux per minute as previously described (56).

The most abundant 3H-labeled osmolyte released after hyposmotic stimulation of [3H]choline-loaded cells was found to be phosphocholine (~87% of 3H radioactivity), as determined by thin-layer chromatography (Silica Gel 60 plates; Merck, Darmstadt, Germany) using a methanol-1.2% NaCl-13.3 M NH4OH (10:10:1 vol/vol) solvent system (52). In addition to phosphocholine, low levels of [3H]choline and [3H]glycerophosphocholine (~12 and 1% of 3H radioactivity, respectively) were found. In contrast to the release of [3H]phosphocholine, which was increased dramatically after hyposmotic stimulation (isotonic: 184 ± 95 dpm, hypotonic: 1,778 ± 219 dpm), the release of [3H]choline and [3H]glycerophosphocholine (isotonic: 147 ± 22 and 14 ± 2 dpm, respectively; hypotonic: 253 ± 46 and 27 ± 3 dpm, respectively) was only moderately affected by hypotonicity.

Amino acid analysis. Medium and lysate fractions of 250 µl were collected corresponding to 1- to 2-min stimuli. Taurine and {beta}-alanine contents of cell lysates and incubation medium were analyzed by reverse-phase HPLC after pre-column derivatization with o-phthaldialdehyde as previously described (9).


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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Osmotic cell swelling-induced release of organic osmolytes. Hyposmotic stimulation of intestine 407 cells not only triggers the activation of K+- and Cl-selective ionic channels but also activates an organic osmolyte release pathway, evidenced by the efflux of [3H]taurine from isotope-loaded cells (Figs. 1 and 2) and by quantitating the taurine content of the incubation medium (Fig. 1). The release of taurine started after a lag period of ~30–60 s and lasted for at least 8–12 min; thereafter a slightly elevated efflux remained (Fig. 1). During this period, the cellular content of taurine is reduced by 70% (Table 1). In contrast, the 125I efflux displayed no apparent lag period (Fig. 1). Compared with the activation of volume-sensitive anion channels, the release of organic osmolytes developed considerably more slowly and lasted for a longer period (Fig. 1). The organic osmolyte release pathway is not selective exclusively for taurine but also can conduct phosphocholine with similar kinetics, as determined using [3H]choline-loaded cells (Fig. 1), as well as {beta}-alanine, a precursor of taurine (Table 1).



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Fig. 1. Comparison of osmotic swelling-induced anion and taurine efflux. Cultures of intestine 407 cells were loaded with 125I (A), [3H]taurine, or [3H]choline (B), and fractional isotope efflux (%/min) induced by osmotic cell swelling (30% hypotonicity) was determined as described in MATERIALS AND METHODS. C: release of taurine into the incubation medium (µmol/106 cells). Data are expressed as means ± SD for n = 3. Arrows indicate shift to a hypotonic medium.

 


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Fig. 2. Iodide and taurine efflux from intestine 407 cells in response to a hypotonic challenge. [3H]taurine ({bullet}) and 125I ({circ}) efflux from intestine 407 cells was determined as described in MATERIALS AND METHODS. Data are presented as the increase in osmolyte efflux (peak value) relative to the unstimulated control (means ± SD for n = 3).

 

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Table 1. Hypotonicity-induced release of taurine and {beta}-alanine into the medium

 
To determine the threshold for activation, cultures of cells were treated with buffers of different osmolarity. As shown in Fig. 2, the release of taurine required a reduction in osmolarity of the external medium to <=225 mosmol/l, whereas saturation of the response was observed at an extracellular osmolarity of 100 mosmol/l. In contrast, hypotonicity-induced 125I efflux was already observed at 290 mosmol/l (i.e., a 10% reduction in osmolarity) and was maximal at 190–225 mosmol/l (Fig. 2). Taken together, the results indicate that the hypotonicity-provoked efflux of organic osmolytes acts as a relatively slow volume-correcting mechanism that starts only when the Cl conductance has reached its maximum.

Organic release pathway does not involve VRAC. To date, the molecular nature of the organic anion release pathway has not been elucidated. In a number of cell models, the release of both Cl and organic osmolytes seems to be mediated by the same channel protein; in other models, however, the release of Cl and the release of taurine are apparently distinct processes. In an attempt to further discriminate between the release pathways for Cl and taurine in intestine 407 cells, we also studied the effects of pharmacological inhibitors on anion conductance and the release of taurine.

Treating the cells with the purinoceptor antagonist and VRAC inhibitor suramin completely prevented the hypotonicity-induced release of taurine (Fig. 3), suggesting that the same channel and/or transporter is involved. In contrast, however, DIDS and millimolar concentrations of extracellular ATP, which efficiently inhibit VRAC activation (57), were unable to block the taurine efflux (Fig. 3).



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Fig. 3. Effects of volume-regulated anion channel inhibitors on the release of taurine and phosphocholine. [3H]taurine (hatched bars)-, [3H]choline (open bars)-, and 125I-loaded (closed bars) cells were exposed to a hypotonic medium, and fractional isotope efflux was determined as described in MATERIALS AND METHODS. Cultures were treated with suramin (100 µM, 5 min), ATP (5 mM, 5 min), wortmannin (10 nM, 1 h), LY-294002 (60 µM, 1 h), genistein (200 µM, 1 h), Na2VO3 (100 µM, 5 min), Clostridium botulinum C3 exoenzyme (C3-exo; 50 µg/ml, overnight incubation), and DIDS (50 µM, 1 h). Data are expressed as the percent change in osmolyte efflux relative to the hypotonic control (mean ± SD for n = 3). *P < 0.05, significant difference from control.

 
Previously, we (55) and others (32) demonstrated that activation of a tyrosine kinase, or inhibition of a phosphotyrosine phosphatase, is a prerequisite for the activation of volume-sensitive anion channels. To investigate putative involvement of protein tyrosine phosphorylation in the regulation of the cell swelling-induced taurine release, we treated cultures of intestine 407 cells with the tyrosine kinase inhibitor genistein and with vanadate, an inhibitor of phosphotyrosine phosphatases. Unlike the activation of VRAC, the release of taurine and phosphocholine was neither increased by vanadate nor reduced after genistein treatment (Fig. 3). In contrast, a small but significant increase of the efflux of taurine was observed in genistein-treated cells (Fig. 3). These results suggest a pronounced difference in regulation between volume-sensitive Cl channels and organic osmolyte release pathways. To further substantiate this notion, phosphatidylinositol 3-kinase (PtdIns 3-kinase) and p21rho, both of which are critically involved in VRAC activation, were inhibited using respectively wortmannin or LY-294001 and Clostridium botulinum C3 exoenzyme. Again, wortmannin and LY-294001, as well as C3 exoenzyme treatment, did not affect the cell swelling-induced taurine efflux (Fig. 3). Taken together, these results clearly indicate that the volume-sensitive taurine efflux in intestine 407 cells involves a different transport protein or channel and is regulated by a pathway independent of tyrosine kinases, PtdIns 3-kinase, and p21rho.

Role of organic anion or cation transporters. Movement of taurine and other organic osmolytes across the plasma membrane is mediated by at least four different transport proteins (OATs 1–3 and Taut; Refs. 15, 27, 51). To investigate whether these transporters contribute to the swelling-induced release of taurine and phosphocholine, cells were treated with established inhibitors of each of these transport systems. As shown in Table 2, inhibitors of the OAT family of transporters, such as probenecid, methothrexate, fenobarbital, and tunicamycin, which lead to an intracellular retention of OATs, did not inhibit or only partially inhibited (20–40% reduction) the hypotonicity-induced taurine efflux (20). In contrast, decynium-22, cyanine-863, and quinidine, inhibitors of organic cation transporters (OCTs; Refs. 28, 59), strongly diminished the efflux of both taurine and phosphocholine (Table 2). However, several OCT inhibitors (i.e., cimetidine, quinine, and verapamil) were found to be ineffective (Table 2). These combined pharmacological data argue against the concept of a single, "classical" organic osmolyte transporter (OAT or OCT) accounting for the cell swelling-induced release pathway.


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Table 2. [3H]taurine efflux from cultures treated with OCT and OAT inhibitors

 
PKC activation regulates the release of taurine. Among the cell models studied, considerable differences are observed in the regulation of taurine transport. Signaling pathways including PtdIns 3-kinase, calmodulin-dependent kinase, tyrosine kinases, and PKC have been reported. As described above, the release of taurine from hypotonicity-provoked intestine 407 cells did not require tyrosine kinase or PtdIns 3-kinase activity, nor did it depend on functional p21rho. Loading cultures with BAPTA-AM or treating them with extracellular EGTA hardly affected the magnitude of the response (EGTA: 102 ± 8%, BAPTA-AM: 116 ± 13% of untreated control). In addition, raising the intracellular concentration of calcium ([Ca2+]i) by adding the Ca2+ ionophore A-23187 or by stimulating the cells with bradykinin, histamine, or EGF did not potentiate the response to a submaximal hypotonic medium (fractional efflux: 126 ± 8, 111 ± 9, 80 ± 22, and 76 ± 16% of untreated control, respectively).

In the presence of the specific PKC inhibitor GF109203X, marked inhibition of the cell swelling-induced taurine efflux was observed, and vice versa, activation of PKC by brief treatment of the cells with the phorbol ester PMA increased the response approximately twofold (Fig. 4). The potentiation by PMA was fully reversed in the presence of GF109203X, indicating that PKC activation is involved. Notably, PMA did not increase basal taurine efflux (basal: 8 ± 2%, PMA: 2 ± 4% of hypotonic control). Prolonged treatment of the cultures with PMA in an attempt to downregulate PKC resulted in reduced basal efflux but only slightly affected the hypotonicity-induced efflux. Notably, the volume-sensitive anion efflux was not affected by PKC activation or inactivation (54). Taken together, the results suggest the involvement of a Ca2+-insensitive PKC isoenzyme in the regulation of the cell swelling-induced release of organic osmolytes.



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Fig. 4. Protein kinase C is involved in the regulation of osmotically induced taurine release. [3H]taurine-loaded cultures of intestine 407 cells were treated with the phorbol ester PMA (200 nM, 25 min or 18 h) and/or GF109203X (1 µM, 15 min) as indicated. Data are expressed as taurine release relative to the untreated isotonic control (means ± SD, n = 3).

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Most animal cells regulate their volume to avoid irreversible cellular damage or even cell death due to imbalances between intra- and extracellular osmolarity. We report a release pathway for organic osmolytes in intestine 407 epithelial cells, distinct from the compensatory anion efflux reported previously (53, 55, 57) and regulated by PKC. Hyposmotic release of organic osmolytes such as taurine, betaine, and inositol has been observed in many different cell models (2, 3, 12, 17, 25, 45, 46). Unlike chloride ions, which can have additional effects on enzyme activity, membrane potential, and, in muscle and nerve cells, the generation of action potentials, these organic osmolytes do not affect other cellular functions, making them especially suitable for volume regulation (2, 13). Stimulation of intestine 407 cells with mild hypotonic solutions (70–90% tonicity) was found to promote anion efflux in the absence of effects on taurine efflux, whereas the efflux of taurine and phosphocholine was observed only after a more severe hyposmotic challenge (<70% tonicity; cf. Fig. 1). These results suggest that, in this cell type, activation of Cl conductance is the first line of defense against osmotic cell swelling and that slower, more prolonged release of organic osmolytes serves as a last way out to prevent further cell swelling.

To date, at least two different mechanisms have been reported to be responsible for the release of organic osmolytes: a polyspecific one transporting both taurine and GABA and one selective for amino acids (25). The latter involves VSOAC (4), a pathway facilitating the release of both Cl and organic osmolytes (25, 26) and VSOC, a transporter of organic osmolytes only (11). The molecular identity of these pathways remains to be established. Our experiments clearly showed a difference in time course as well as in the threshold for activation between the release of 125I and [3H]taurine, suggesting that different pathways are involved. A differential sensitivity to DIDS of the Cl and taurine release from HeLa cells has been reported, also pointing to the existence of separate pathways (50). In contrast, the efflux of taurine was inhibited by chloride channel blockers, including DIDS and millimolar concentrations of ATP, in calf pulmonary endothelial (CPAE) cells (34). In intestine 407 cells, however, DIDS treatment did not affect hypotonicity-induced taurine efflux (Fig. 3). Recently, two different release pathways for taurine—a "fast" one and a "slow" one—have been observed in hippocampal brain slices, with the slow one being sensitive to DIDS (11). It is therefore likely that only the fast release pathway is present in intestine 407 cells.

In an attempt to identify the transporters involved, cells were treated with a variety of OAT and OCT inhibitors. None of the OAT inhibitors tested were found to inhibit the release of taurine dramatically; however, a moderate (20–40%) reduction in efflux was observed with some of the inhibitors tested. In contrast, taurine release was inhibited strongly by several OCT inhibitors but not by others. Because the taurine efflux was abolished almost completely after quinidine treatment, whereas the related and potent OCT inhibitor quinine (27) was ineffective, we think that a transport system other than the OCT is involved. This notion is supported by our observation that cimetidine and verapamil, inhibitors of all three OCT subtypes (58), did not reduce the hypotonicity-induced osmolyte efflux. However, we cannot completely rule out the possibility that a subset of transporters with different sensitivities for the inhibitors used may contribute to the observed organic osmolyte efflux.

In several selected cell models, the hypotonicity-provoked taurine release was found to be sensitive to inhibitors of protein tyrosine phosphorylation as well as to wortmannin, an inhibitor of PtdIns 3-kinase (11, 37). In Intestine 407 cells, however, in clear contrast to the activation of VRAC (53, 55), the release of taurine was found to be independent of tyrosine kinases and PtdIns 3-kinase and did not involve p21rho. A similar low sensitivity to genistein has been reported for isolated rat supraoptic astrocytes (6). Taken together, these results indicate not only that the release of taurine does not involve VRAC in intestine 407 cells but also that distinct signaling pathways are involved in the regulation of Cl and taurine efflux (54).

An increase in intracellular free Ca2+ has been reported to be involved in the activation of taurine efflux for several cell types, including erythroleukemia cells (21), cultured rat astrocytes (33), and rat cerebral cortex (30). In astrocytes and cerebral granule cells, however, the hypotonicity-provoked taurine efflux was independent of [Ca2+]i or required basal Ca2+ levels (36, 38, 40). With the use of intestine 407 cells, modulation of [Ca2+]i by BAPTA-AM loading, extracellular EGTA, Ca2+-mobilizing hormones, the Ca2+ channel blocker verapamil, or the Ca2+ ionophore A-23187 did not affect the volume-sensitive taurine efflux. However, we cannot exclude the possibility that a minimal basal [Ca2+]i is required, as observed previously for astrocytes (36).

Brief treatment of intestine 407 cells with the phorbol ester PMA potentiated the cell swelling-induced taurine efflux, whereas addition of the PKC inhibitor GF109203X largely inhibited the response, suggesting a major role for PKC as an activator of this process. The PKC family of serine kinases consists of a large group of isoenzymes playing crucial roles in cellular signaling and involved in a variety of biological processes (for review, see Ref. 35). The group can be divided into subgroups on behalf of their behavior and their protein sequence: the classical ({alpha}, {beta}, {gamma}), novel ({delta}, {epsilon}, {eta}, {theta}), and atypical ({iota}, {xi}) PKCs and the PKC-related kinases (PRK1, PRK2, PRK3). Whereas activation of PKC-{alpha}, -{beta}, and -{gamma} requires binding of phosphatidylserine, both classical and novel PKCs are activated by the phorbol ester PMA. Because binding of phosphatidylserine occurs in a Ca2+-dependent manner, the permissive role of Ca2+ (36) in the activation of hyposmotically triggered taurine efflux might be explained by the activation of these isoforms of PKC. In CPAE cells, however, the release of organic osmolytes was found to be independent of PKC (34).

To summarize, the results of this study indicate that in intestine 407 cells, the hypotonicity-induced efflux of organic osmolytes is independent of VRAC and involves an activation pathway that includes PKC. These observations are not generally applicable, however, because in several other model systems, a close relationship between Cl conductance and release of organic osmolytes has been established (16, 2224, 40, 47). Therefore, a definite answer to the question of whether the release pathways are the same or different awaits the molecular identification of the transporters involved.


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This study was supported by the Netherlands Organization for Scientific Research (Stichting Aard- en Levenswetenschappen).


    ACKNOWLEDGMENTS
 
We thank Marieke van der Heide-Mulder and Ans Voskuilen-Kooijman (Department of Psychiatry, Erasmus University Medical Center) for determining the taurine and {beta}-alanine content of the cells and incubation medium.


    FOOTNOTES
 

Address for reprint requests and other correspondence: B. C. Tilly, Dept. of Biochemistry, Erasmus Univ. Medical Center, PO Box 1738, 3000 DR Rotterdam, The Netherlands (E-mail: b.tilly{at}erasmusmc.nl).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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