Previous data indicate that adenosine 3′,5′-cyclic monophosphate activates the epithelial basolateral Na+-K+-Cl−cotransporter in microfilament-dependent fashion in part by direct action but also in response to apical Cl− loss (due to cell shrinkage or decreased intracellular Cl−). To further address the actin dependence of Na+-K+-Cl−cotransport, human epithelial T84 monolayers were exposed to anisotonicity, and isotopic flux analysis was performed. Na+-K+-Cl−cotransport was activated by hypertonicity induced by added mannitol but not added NaCl. Cotransport was also markedly activated by hypotonic stress, a response that appeared to be due in part to reduction of extracellular Cl− concentration and also to activation of K+ and Cl− efflux pathways. Stabilization of actin with phalloidin blunted cotransporter activation by hypotonicity and abolished hypotonic activation of K+ and Cl− efflux. However, phalloidin did not prevent activation of cotransport by hypertonicity or isosmotic reduction of extracellular Cl−. Conversely, hypertonic but not hypotonic activation was attenuated by the microfilament disassembler cytochalasin D. The results emphasize the complex interrelationship among intracellular Cl− activity, cell volume, and the actin cytoskeleton in the regulation of epithelial Cl− transport.
the bumetanide-inhibitableNa+-K+-(2)Cl−cotransporter participates in the homeostatic control of transmembrane ion gradients and cell volume in diverse cell types (10, 41). In addition, Na+-K+-Cl−cotransport is an integral component of the salt-transporting apparatus of many secretory and absorptive epithelia. Ion transfer by Na+-K+-Cl−cotransporters generally obeys chemical potential. However, acute regulation of cotransport is influenced not only by thermodynamic driving force but also by a number of additional factors, including Na+-K+-Cl−cotransporter protein phosphorylation and, possibly, the function of associated transmembrane regulatory proteins (6, 10, 41). In many epithelia, Cl− secretion can be elicited by agents acting via adenosine 3′,5′-cyclic monophosphate (cAMP). Activation of apical Cl− channels is generally viewed as the primary regulatory event of cAMP-elicited Cl− secretion. However, basolateral Na+-K+-Cl−cotransport must also increase to maintain cell electrolyte composition, and therefore active salt secretion demands coordinated control of apical Cl− exit and basolateral Cl− entry. The factors responsible for “cross talk” between apical and basolateral transport events are incompletely defined but have been proposed to include changes in cell volume, intracellular Cl− activity ([Cl−]i), and the actin cytoskeleton (3, 10, 12, 26, 31, 40, 41).
Hypertonic cell shrinkage activates Na+-K+-Cl−cotransport and mediates a compensatory regulatory volume increase (RVI) in many epithelial and nonepithelial cells (36). Linkage of apical and basolateral transport events during cAMP-elicited epithelial salt secretion could involve signaling cascades similar to those evoked during hypertonic shrinkage (40). In other words, cAMP activation of apical Cl− channels carries an obligatory cell water loss, and the resultant cell shrinkage could then secondarily trigger basolateral Na+-K+-Cl−cotransport. However, regulation of Na+-K+-Cl−cotransport during epithelial secretion appears to be more complex. For example, in HT-29 intestinal (33) and canine tracheal epithelial cells (11-13), cAMP has been shown to activate cotransport directly, that is, in the absence of cAMP-dependent salt efflux and cell shrinkage. Moreover, some cells have been shown to possess cAMP- and shrinkage-independent pathways for cotransporter activation; in tracheal epithelial cells, cotransporter protein phosphorylation can be increased by manipulations that simply produce a fall in [Cl−]i(12). Thus, in response to cAMP stimulation, epithelial Na+-K+-Cl−cotransport may increase as a response to cell shrinkage and/or decreased [Cl−]i(both secondary to the activation of apical Cl− channels) or as a direct downstream effect of elevated intracellular cAMP.
We previously showed that stimulation of Na+-K+-Cl−cotransport in T84 human intestinal epithelial cells under a number of circumstances appears to involve dynamic reorganization of the microfilamentous cytoskeleton and may be attenuated by the F-actin stabilizer phalloidin. Thus activation of cotransport by cAMP, 5′-AMP, and guanosine 3′,5′-cyclic monophosphate is blunted in cells loaded with phalloidin (31, 32, 34, 46), and in each of these cases, activation of transepithelial Cl− secretion is proportionately decreased. We also recently showed that F-actin disassembly using cytochalasin D activates T84 cell Na+-K+-Cl−cotransport in the absence of secretagogue (29). The motivation of the present study was to further address the actin dependence of regulation of Na+-K+-Cl−cotransport. Specifically, we wished to determine whether hypertonic cell shrinkage activates the cotransporter in T84 cells and whether such activation is attenuated by actin stabilization. In so doing, we unexpectedly found that Na+-K+-Cl−cotransport was activated not only by extracellular hypertonicity but also by hypotonicity. Additionally, hypertonic and hypotonic activation were found to display strikingly different sensitivity to chemical manipulation of the actin cytoskeleton by phalloidin and cytochalasin D.
Cell culture and buffers.
T84 human intestinal epithelial cells obtained from American Type Culture Collection and Dr. K. Barrett (University of California, San Diego) were maintained in culture as previously described (7, 31). For experiments, cells were seeded onto 1-cm2 collagen-coated permeable supports (Costar, Cambridge, MA) and used after confluence and stable transepithelial electrical resistances were achieved, ∼7–14 days after they were plated. A variety of buffered electrolyte solutions were used for these experiments to examine the effects of changes in extracellular tonicity and/or ionic composition. These buffers are modifications of aN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid-phosphate-buffered Ringer solution used in previous studies (29,31-33) and are described in detail in Table1. All solutions were prewarmed to 37°C, and experiments were carried out at 37°C, with temperature control by thermal probe-coupled heat lamps.
Isotopic flux studies.
The functional activity of the basolateral Na+-K+-Cl−cotransporter of confluent T84 monolayers was determined by radioisotopic methods (bumetanide-sensitive86Rb uptake), as previously described (29, 31-33). Briefly, monolayers grown on permeable supports were equilibrated inN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid-phosphate-buffered Ringer solution for 20 min. At the beginning of the experimental period, the apical buffer was removed, and monolayers were then washed by three rapid dips into a beaker containing 100–200 ml of the appropriate experimental buffer. Inserts were then placed in 12-well plates containing 1 ml of the appropriate experimental solution with or without 20 μM bumetanide. The identical buffer (1 ml, without bumetanide) was added to the apical side of the insert. To initiate uptakes, inserts were transferred to new wells containing the identical basolateral solution with the addition of 1–1.5 μCi/ml 86Rb. Uptake has previously been shown to be linear for up to 5 min, and for the experiments reported here a 3-min uptake period was routinely used. Uptakes were terminated by rapidly washing the monolayers by 10 rapid dips in ice-cold stop solution containing 100 mM MgCl2 and 10 mM tris(hydroxymethyl)aminomethane ⋅ HCl, pH 7.4. Filters were then cut out from the plastic support and placed in scintillation vials containing 3 ml of Aquasol. Radioactivity was counted using a Packard Liquid Scintillation counter. Representative monolayers were used for protein determination by bicinchoninic acid assay (Pierce Chemical). Uptakes were expressed as nanomoles of K+ per milligram of protein per minute. Activation of Cl−and K+ efflux pathways in response to osmotic challenge was measured by a modification of a method previously described by Venglarik et al. (50), using125I and86Rb as tracers, respectively, and previously reported techniques (31, 33). Efflux rate constants were expressed per minute, as in previous studies.
Short-circuit current (I sc), which represents electrogenic Cl−secretion in the T84 model, was measured in monolayers grown on 0.33-cm2 permeable supports using a dual voltage-current clamp and Ag-AgCl and calomel electrodes interfaced via “chopstick” KCl-agar bridges, as previously described (29, 31, 32).
Phalloidin (Sigma Chemical, St. Louis, MO) was dissolved in methanol as a stock solution. For experiments, aliquots of the methanol stock solution were dried under N2 and dissolved in media to a final concentration of 3–10 μM. Monolayers were loaded with phalloidin by overnight incubation, as previously described (29, 31, 32). Adequacy of phalloidin loading was assessed by measurement of inhibition of forskolin-stimulatedI sc responses compared with unloaded control monolayers. The absence of monolayer toxicity was confirmed by the preservation of transepithelial resistance and theI sc response to carbachol, as previously described (31).
Fluorescent staining of the F-actin cytoskeleton was performed as described previously (15, 29, 32). Briefly, monolayers grown on glass coverslips were rinsed in phosphate-buffered saline, fixed in 3.7% formaldehyde, and permeabilized in ice-cold acetone. Monolayers were then dried and stained with rhodamine-phalloidin, and coverslips were mounted upside down on glass slides in phosphate-buffered saline-glycerol-p-phenylendiamine. Cells were examined using a Zeiss IM-35 inverted microscope equipped for epifluorescence and photographed with Kodak Tri-X film (1,000 ASA). Morphological analysis consisted of blinded review of slides by one investigator without knowledge of the treatment groups.
Materials and statistical analysis.
Radionuclides were obtained from Dupont NEN (Boston, MA). All chemicals were obtained from Sigma Chemical, with the exception of forskolin, which was from Calbiochem (La Jolla, CA), and rhodamine-phalloidin, which was from Molecular Probes (Eugene, OR). Statistical analysis was by Student’s t-test for paired variates and by analysis of variance, where appropriate, withP < 0.05 considered statistically significant.
Response to hypertonicity.
The basal rate of Na+-K+-Cl−cotransporter activity measured by bumetanide-sensitive86Rb (K+) uptake across the basolateral membrane of confluent T84 monolayers was 4.63 ± 0.68 nmol K+ ⋅ mg protein−1 ⋅ min−1for 16 separate experiments each performed on duplicate or triplicate monolayers. We expected to demonstrate that hypertonic shock would activate Na+-K+-Cl−cotransport in these cells. However, exposure of monolayers to hypertonic NaCl buffer for 10 min only marginally affected Na+-K+-Cl−cotransport to a level that did not reach statistical significance (Fig. 1). We found that a more substantial cotransporter response to hypertonicity could be uncovered if the same degree of extracellular hypertonicity was imposed using hypertonic mannitol buffer rather than hypertonic NaCl (Fig. 1). The degree of activation of cotransport by hypertonic mannitol was considerably lower than the response to a typical secretory agonist such as forskolin (41.0 ± 9.6 nmol K+ ⋅ mg protein−1 ⋅ min−1,n = 3, 10 μM forskolin stimulation for 10 min). Hypertonicity did not increase transepithelialI sc (data not shown).
Although the response to hypertonicity under high salt conditions (hypertonic NaCl) was considerably less than under standard conditions, we did not find evidence that the response to hypertonicity could be meaningfully enhanced by a further reduction in extracellular Cl− concentration. For example, Na+-K+-Cl−cotransport after a 20-min equilibration in isotonic gluconate buffer (76.5 mM Cl−) was 8.92 ± 1.6 nmol K+ ⋅ mg protein−1 ⋅ min−1, and this increased to 16.9 ± 3.0 nmol K+ ⋅ mg protein−1 ⋅ min−1after an increase in osmolarity to 445 mosM using 135 mM added mannitol, a value comparable to that shown in Fig. 1.
Response to hypotonicity.
Hypotonic cell swelling evokes a regulatory volume decrease (RVD) in most cells that usually involves inhibition of Na+-K+-Cl−cotransport and activation of volume-sensitive K+ and Cl− channels (36). Shrinkage-elicited activation of cotransport in some cell systems has been shown to be enhanced by prior cell swelling (18). Because the T84 cotransporter response to hypertonicity seemed somewhat meager, we examined the effect of isotonic cell shrinkage on T84 cell cotransport. To do this, we initially exposed cells to hypotonic buffer, then returned the cells to isotonic buffer, a so-called RVI-after-RVD maneuver (18). To our surprise, we found that cotransport was dramatically activated during the initial exposure to a nominally 175 mosM hypotonic buffer. As shown in Fig. 2, the rate of bumetanide-sensitive K+ uptake in response to this hypotonic buffer was at least as great in magnitude as the response to hypertonic mannitol buffer. Subsequent return to isosmotic conditions after hypotonic shock in fact did not substantially increase Na+-K+-Cl−cotransport beyond the already-stimulated activity (not shown). The increase in cotransport evoked by hypotonicity was maximal ∼10 min after exposure (Fig. 2 B).
This unexpected activation of the Na+-K+-Cl−cotransporter by hypotonicity could be due to the acute reduction in extracellular tonicity and/or reduction in extracellular NaCl concentration. To examine these possibilities, first, the effect of an isosmotic reduction in extracellular NaCl concentration was studied. Monolayers were bathed in isotonic buffer or in buffer in which 67.5 mM NaCl was replaced with equimolar Na-gluconate (to decrease Cl− concentration only),N-methylglucamine (NMG)-Cl (to decrease Na+ concentration only), or mannitol (to decrease Cl−and Na+ concentrations). As shown in Fig.3 A, cotransporter activity markedly increased when Cl− concentration was reduced (isotonic gluconate and mannitol buffer) but not when Na+ concentration alone was decreased (isotonic NMG buffer). These data must be interpreted with caution, inasmuch as it would be expected that isosmotic replacement of a permeant ion (Cl−and/or Na+) with an impermeant ion species induces a degree of cell shrinkage (28). However, it seems unlikely that cell shrinkage could entirely account for the activation response to these ion substitutions; if this were the case, one would have expected to see substantial activation of cotransport by isotonic NMG-Cl buffer. Nevertheless, a contribution from cell shrinkage cannot be excluded; we did observe that isosmotic substitution of Cl− with failed to activate cotransport. Because permeation of anion transport pathways more closely approximates Cl−, little or no cell shrinkage would be expected (11, 12). Unfortunately, and further confounding the analysis, has been shown to display unusual inhibitory behavior with respect to the outward-facing and, possibly, the inward-facing conformation of the cotransporter compared with larger, more inert anion substituents (11,16, 51). Hypotonic activation exceeded activation by isotonic gluconate or isotonic mannitol buffer (P < 0.02). In fact, the degree of activation by hypotonicity exceeds the degree of activation by isosmotic ion substitution of Cl− (Na-gluconate) throughout a range of extracellular Cl− concentrations, as indicated in Fig. 3 B. This suggests that the activation response to extracellular hypotonicity is in part reflective of the reduction in extracellular Cl− concentration but that the hypotonic stimulus itself confers an additional component of activation on the cotransporter.
We next wished to determine whether hypotonic shock in the absence of a change in extracellular Cl−concentration affects Na+-K+-Cl−cotransport. To this end, a series of experiments was performed to explore the effect of hypotonicity at constant (but reduced) extracellular NaCl concentration. Monolayers were preequilibrated for 30 min in isotonic mannitol buffer in which 67.5 mM NaCl was replaced by 135 mM mannitol. Monolayers were then transferred to hypotonic buffer with the identical NaCl concentration (67.5 mM) but without mannitol. As shown in Fig. 4, hypotonicity persisted in activating Na+-K+-Cl−cotransport, although the response was considerably less than that elicited by hypotonicity with reduced extracellular Cl− concentration (Fig. 3). Others have reported that hypotonic stress activates transepithelial Cl− secretion in T84 monolayers, possibly by a Ca2+-dependent signaling pathway (35). However, in our hands, no such stimulation could be elicited in monolayers exposed to hypotonic buffer (n = 6). Despite the absence of a hypotonicity-elicitedI sc, hypotonicity did activate K+ and Cl− efflux pathways. Specifically, the rate constant of efflux of86Rb from preloaded monolayers substantially increased on exposure to hypotonic buffer (Fig.5 A). This increase in 86Rb efflux was confined to the basolateral aspect of the monolayers and also occurred in monolayers pretreated with 20 μM bumetanide. This observation suggests, first, that enhanced efflux of86Rb likely reflects the activation of volume-activated K+channels that are basolaterally restricted and, second, that the increase in cotransporter activity indeed represents enhanced net inward flux of K+ (and presumably the cotransported Na+ and Cl−) through this pathway. Activation of 125I efflux into the basolateral but not apical buffer was also evident (Fig.5 B), suggesting that osmosensitive Cl− channels are likely to be present predominantly in the basolateral domain.
Influence of the actin cytoskeleton.
We previously showed that activation of Na+-K+-Cl−cotransport by cyclic nucleotide-dependent agonists in T84 monolayers is markedly attenuated in monolayers loaded with the F-actin stabilizer phalloidin (31, 32). Activation of Cl− secretion by cyclic nucleotide agonists has been shown to be paralleled by a rearrangement of F-actin in the basal pole of T84 monolayers, an event that is also blunted in phalloidin-loaded cells (31, 32). Hypotonicity, but not hypertonicity, was associated with a rearrangement of basal F-actin that resembled that seen in our earlier reports for secretagogue-induced rearrangements (Fig.6) (31, 32) and previously described in other cell systems (20, 53). Isosmotic reduction of extracellular Cl− concentration using gluconate buffer, however, did not induce a significant rearrangement of microfilament architecture (Fig. 6).
We then examined whether phalloidin would also affect osmotic activation of cotransport. As shown in Fig.7, phalloidin reduced the increase in bumetanide-sensitive 86Rb uptake induced by hypotonic buffer by ∼40%. In contrast, hypertonic activation of cotransport was not inhibited by phalloidin; in fact, it was enhanced. Phalloidin marginally reduced the activation of Na+-K+-Cl−cotransport induced by isosmotic reduction in extracellular Cl− concentration, but this did not reach statistical significance (8.1 ± 1.0 vs. 6.4 ± 0.32 nmol K+ ⋅ mg protein−1 ⋅ min−1for control vs. phalloidin treatment after 10 min of exposure to isotonic gluconate buffer, each n = 6,P = NS). Hypotonic stimulation of bumetanide-insensitive Cl−(125I) and K+(86Rb) efflux apparently required F-actin remodeling, inasmuch as both responses were completely blocked in phalloidin-loaded cells (Table 2). In contrast to results with phalloidin, the F-actin disassembler cytochalasin D, which was recently reported to enhance bumetanide-sensitive 86Rb uptake in T84 cells (29), did not affect hypotonic activation of cotransport; however, the ability of hypertonicity to activate cotransport was diminished or abolished (Fig. 8).
Recent molecular studies have identified two major isoforms of the Na+-K+-Cl−cotransporter (8, 27, 41, 42, 52). The more widely distributed NKCC1 (or BSC2) represents the major Cl− loading pathway in secretory epithelia and mediates cotransport in a wide range of nonepithelial cell types. NKCC2 (or BSC1) is restricted to the kidney and mediates apical salt uptake in absorptive renal epithelia. A number of reports indicate that alterations in Na+-K+-Cl−cotransporter function may substantially modulate transepithelial Cl− secretory capacity (14,30, 31, 46); it is becoming apparent that NKCC1 is not simply a passive element responding to apical membrane events but that it may serve as an important independent regulatory site. In this study we establish that osmotic stress, both hypertonic and hypotonic, activates inward Na+-K+-Cl−cotransport across the basolateral membrane of polarized T84 human intestinal epithelial cells without eliciting a corresponding increase in transepithelial Cl−secretion. Clearly, cotransporter activation under these conditions is dissociated from events at the apical membrane, confirming that transport function of NKCC1 is not invariably linked to the secretory state of the epithelium. Moreover, such data serve to illustrate that, in epithelial cells, NKCC1 has the capacity to be regulated independently of apical Cl−channels, although such regulation is not sufficient for activation of transepithelial salt secretion. Similar conclusions have been drawn on the basis of results obtained in airway epithelia (22) and shark rectal gland (24).
Hypertonic cell shrinkage and cAMP lead to enhanced serine/threonine phosphorylation of NKCC1 in models such as the shark rectal gland, an event associated with an increase in the number of binding sites for [3H]benzmetanide in parallel with increased cotransport of ions (14, 24, 25, 30). The specific kinases and/or phosphatases that directly affect the state of NKCC1 phosphorylation in response to these stimuli remain undefined. Although cAMP increases cotransport, NKCC1 phosphorylation, and [3H]benzmetanide binding, no region of the cloned shark NKCC1 isoform conforms to consensus sequences for cAMP-dependent protein kinase (PKA) (41, 42,52). Human NKCC1 contains one such sequence, although whether this site plays a role in cAMP-dependent regulation is not established. Direct evidence for a putative kinase downstream from PKA has been lacking. Activation of Cl− secretion by cAMP results in cell shrinkage due to apical Cl− loss. Theoretically, a shrinkage-activated kinase [such as the “V-kinase” postulated to regulate transport events in erythrocytes (19, 23)] could participate in shrinkage- and cAMP-dependent activation of Na+-K+-Cl−cotransport. Indeed, Lytle (23) recently demonstrated that cAMP and hypertonicity phosphorylate erythrocyte NKCC1 at common sites. Klein and O’Neill (21) reported that myosin light chain kinase (MLCK) is a shrinkage-activated kinase in endothelial cells and that the MLCK inhibitor ML-7 inhibits shrinkage activation of cotransport. However, MLCK is unlikely to be directly involved in shrinkage- and cAMP-dependent activation of NKCC1, since inhibition by ML-7 did not decrease hypertonic phosphorylation of NKCC1; moreover, PKA decreases MLCK activity in a number of cells (38).
A decrease in [Cl−]isecondary to activation of cAMP-dependent apical channels may also account for enhanced Na+-K+-Cl−cotransport. Recent data from a number of cell types suggest the possibility that, beyond its role in setting thermodynamic driving force, [Cl−]imay modulate cotransport activity through direct kinetic inhibition of NKCC1 itself or of a key regulatory protein (2, 3, 11, 12, 41). This concept was first put forth by Breitwieser et al. (2) for hypertonic regulation of cotransport in squid giant axon. Lytle and Forbush (24,25) demonstrated an increase in cotransport activity, NKCC1 phosphorylation, and [3H]bumetanide binding in response to Cl−-free media in shark rectal gland. Haas et al. (11, 13) demonstrated activation of cotransport and enhanced bumetanide binding in dog tracheal epithelial cells in response to apical Cl− efflux elicited by the cAMP-independent agonist UTP. More recently, Haas et al. (12) reported [Cl−]i-dependent phosphorylation of NKCC1 in nystatin-permeabilized airway epithelial cells. How a change in [Cl−]ikinetically modifies cotransport and/or NKCC1 phosphorylation is a matter of speculation, but the presence of a [Cl−]i-sensitive regulatory element has now been postulated not only in the case of NKCC1 but also for Na+/H+exchange and a nonselective cation channel in rat salivary acinar cells (45) and for Na+/H+exchange in rat distal colonic crypts (44). Low [Cl−]imay create a “permissive” environment for phosphorylation-dependent regulation of such pathways (2). Treharne et al. (49) reported two novel protein kinases in airway epithelia that were progressively inhibited as Cl− concentration increased above 50 mM and suggested that such anion-sensitive kinase activity may explain the [Cl−]i-dependent regulation of various ion transporters. In our experiments, [Cl−]i(to the extent that it was affected by changes in extracellular Cl− concentration) appeared to exert some influence over hypertonic activation of cotransport. That is, at the same level of extracellular hypertonicity (445 mosM), inward bumetanide-sensitive fluxes were twice as large in buffer containing added mannitol as in buffer containing added NaCl. However, hypertonic stimulation of cotransport was not significantly enhanced under conditions of reduced (76 mM) extracellular Cl− concentration.
In this study, Na+-K+-Cl−cotransport increased sharply in response to hypotonic stress. Although in numerous cell systems, including endothelia (39), erythrocytes (1), and Xenopus oocytes (48), cotransport has been found to be inhibited by hypotonicity, this finding is not unprecedented. Lytle and Forbush (24) reported that hypotonicity increases [3H]benzmetanide binding and NKCC1 phosphorylation in shark rectal gland, an effect attributed to a decrease in [Cl−]i, although the functional correlate of this finding in terms of ion translocation was not addressed. Experimentally, it is difficult to dissociate changes in [Cl−]ifrom changes in cell volume. However, we suspect that cotransport is stimulated not by cell swelling per se, but rather by the events brought about by cell swelling, specifically KCl extrusion. Comparison of time course data shows that the increased rate of86Rb and125I efflux induced by hypotonicity wanes after ∼8 min (suggesting that RVD has largely been accomplished), whereas the stimulated rate of cotransport does not reach its peak until 10 min and appears to persist. Although we did not directly measure [Cl−]ior cell volume in the present study, it seems reasonable to expect that hypotonicity indeed decreases [Cl−]i, since extracellular Cl−concentration is reduced and 125I efflux is enhanced. Isosmotic substitution of Cl− but not Na+ also activated cotransport, although such manipulations also likely induced cell shrinkage. Cell shrinkage occurs to a lesser extent or not at all when rather than gluconate is used as the replacement anion (11). However, the failure of isotonic buffer to stimulate cotransport in our experiments could also be attributable to an inhibitory interaction of this species with the external- or internal-facing conformation of the cotransporter (12, 16, 51) or to inhibition of a putative anion-sensitive kinase, such as was the case for one of the two kinases identified by Treharne et al. (49).
The mechanism by which cotransporter function is influenced by the actin cytoskeleton remains obscure and may be indirect. Inhibition of MLCK by ML-7 suppresses hypertonic stimulation of cotransport but not phosphorylation of NKCC1 (21). This is consistent with our previous finding that the actin stabilizer phalloidin attenuates cAMP-dependent activation of cotransport but not [3H]bumetanide binding (33). We previously demonstrated that cytochalasin D activates Na+-K+-Cl−cotransport in T84 cells (29), and in the present study, cytochalasin D was found to attenuate hypertonic but not hypotonic activation of cotransport. Jessen and Hoffmann (20) noted similar behavior in Ehrlich ascites cells, where cytochalasin B activated basal cotransport but attenuated hypertonic activation. In contrast, phalloidin attenuated only hypotonic but not hypertonic activation of Na+-K+-Cl−cotransport. This implies that the signal transduction cascades required for hypertonic activation in T84 cells require the presence of an intact or organized filament system but does not involve active cytoskeletal remodeling per se. Indeed, hypotonicity but not hypertonicity elicits F-actin rearrangements reminiscent of the phalloidin-sensitive cytoskeletal reorganization previously demonstrated in cAMP-stimulated T84 cells (46); these changes resemble hypotonic actin remodeling observed by others in shark rectal gland and Ehrlich ascites cells (20, 53). Hypotonic stimulation of cotransport appears to require this actin remodeling, since it is attenuated by phalloidin. Hypotonic stimulation of cotransport may depend in part on stimulation of bumetanide-insensitive K+ and Cl− efflux pathways, both of which were blocked in phalloidin-loaded cells. How a change in filament structure affects the function of ion transport proteins is poorly understood, but increasing numbers of examples of such regulation have been identified (4, 5, 43).
Hypotonic and cAMP-dependent activation of the T84 cotransporter are likely related at least in part to a decrease in [Cl−]i. This raises the speculative possibility that the [Cl−]i-sensitive regulatory site could also be F-actin dependent. If this is true, this cytoskeletal dependence of the Cl− sensor could account for our previous finding that phalloidin inhibited cAMP-stimulated cotransport more profoundly in the HT-29 subclone that expressed the regulated Cl− efflux pathway than in the undifferentiated parent line (33). Of interest in this regard is the recent finding that stimulated Cl− efflux appears to be required for the actin remodeling that accompanies cytokine-elicited neutrophil activation (37). Moreover, Cl−-depleted buffer in the absence of cytokine has been shown to elicit neutrophil actin polymerization, protein phosphorylation, lysozyme secretion, and respiratory burst activity (9, 37). Similar to its aforementioned role as a possible kinetic regulator of ion transport pathways, [Cl−]icould modulate the activity of the kinases, phosphatases, or other regulatory proteins that subsequently affect actin nucleation cascades. Halide binding to heterotrimeric G proteins has been shown by Higashijima et al. (17) to affect intrinsic guanosine 5′-triphosphatase activity, and numerous GTP-binding proteins are known to affect cytoskeletal organization. Against this hypothesis, however, stands our observation in the present study that an isosmotic reduction in extracellular Cl− concentration does not appear in and of itself to cause a marked actin rearrangement, and stimulation of cotransport by this manipulation was only marginally if at all blunted by phalloidin. In the absence of direct measurements of [Cl−]iand cell volume, it is difficult to fully interpret these data, however. As stated above, part of the activation of cotransport by the isotonic gluconate buffer may involve cell shrinkage in addition to a lowering of [Cl−]i; phalloidin appeared to exert an intermediate effect on isotonic gluconate stimulation of cotransport compared with phalloidin’s augmentation of the hypertonic response and blunting of the hypotonic response.
In summary, we have shown in model T84 intestinal epithelial cells that Na+-K+-Cl−cotransport is activated by hypertonic stress and reduced extracellular Cl− concentration in the absence of a stimulus for transepithelial Cl− secretion. However, cAMP (forskolin stimulation) in this model elicits a far greater activation of cotransport than either hypertonic shrinkage or low extracellular Cl− concentration, even when hypertonic stress and low extracellular Cl− concentration stimuli are presented in combination. This implies that cAMP may well act through a pathway other than simply reduced cell Cl− and shrinkage. Our previous findings that cAMP-dependent activation of cotransport is associated with F-actin remodeling raise the speculative consideration that the additional pathway could involve the cytoskeleton. In this context, we found that activation of cotransport by hypertonic stress and by reduced extracellular Cl− concentration is not associated with obvious actin rearrangements, and in neither case is the response diminished by phalloidin-induced actin stabilization. In contrast, we showed that a hypotonic stimulus activates Na+-K+-Cl−cotransport in phalloidin-sensitive fashion and also induces F-actin remodeling reminiscent of that previously observed with cAMP. Activation of cotransport under these conditions may reflect an associated fall in [Cl−]idue to reduced extracellular Cl− concentration as well as hypotonic-stimulated KCl efflux. Our findings emphasize the complex interrelationship among [Cl−]i, cell volume, and the cytoskeleton in the regulation of Na+-K+-Cl−transport and, by extension, epithelial Cl− secretion.
This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-48010 and by the George H. A. Clowes, Jr., MD, FACS, Memorial Career Development Award from the American College of Surgeons (to J. B. Matthews).
Address for reprint requests: J. B. Matthews, Dept. of Surgery, Beth Israel Deaconess Medical Center, East Campus, 330 Brookline Ave., Boston, MA 02215.
Some of these data have appeared in abstract form (47).
- Copyright © 1998 the American Physiological Society