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Am J Physiol Gastrointest Liver Physiol 276: G491-G498, 1999;
0193-1857/99 $5.00
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Vol. 276, Issue 2, G491-G498, February 1999

Electrogenic Na+ transport in rat late distal colon by natural and synthetic glucocorticosteroids

Ingo Grotjohann1, Jörg-Dieter Schulzke2, and Michael Fromm1

1 Institut für Klinische Physiologie and 2 Medizinische Klinik I Gastroenterologie und Infektiologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, D-12200 Berlin, Germany


    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The potency of in vitro-added corticosteroids to stimulate electrogenic Na+ absorption (JNa, the Na+ absorptive short-circuit current blockable by 10-4 M amiloride) was determined in rat late distal colon. JNa was determined 8 h after steroid addition from the drop in short-circuit current caused by 10-4 M amiloride. The concentration dependency of JNa was obtained for seven corticosteroids and compared with that established for aldosterone. Apparent mineralocorticoid potencies as determined from apparent Michaelis-Menten constant (Km) values were as follows: aldosterone 1.2 nM >> RU-28362 20 nM = deoxycorticosterone 20 nM > deoxycortisol 36 nM >=  dexamethasone 37 nM >> corticosterone 170 nM > cortisol 210 nM. These steroids exhibited Vmax values of 9-13 µmol · h-1 · cm-2 and similar concentration dependencies. Hill coefficients were between 1.6 and 2.1, suggesting cooperative effects between activated receptors. We conclude that corticosteroids exhibit graded mineralocorticoid potency instead of a sharp partition into exclusive groups of mineralocorticoid and nonmineralocorticoid hormones. The low apparent Km value of RU-28362 for mineralocorticoid action and the need for high concentrations of the mineralocorticoid antagonist mespirenone to block this response indicated that JNa in a native mammalian epithelium can be mediated by the glucocorticoid receptor. Glucocorticoid receptor-specific amounts of RU-28362 in combination with mineralocorticoid receptor-specific amounts of aldosterone or of the mineralocorticoid antagonist spironolactone showed cooperative action, suggesting a heterodimeric activation of JNa by the glucocorticoid receptor and mineralocorticoid receptor.

short-circuit current; corticosterone; RU-28362; spironolactone; heterodimerization


    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE WIDELY ACCEPTED MODEL of corticosteroid hormone action on electrogenic Na+ absorption in rat distal colon implies an activation of mineralocorticoid receptors (MRs) with a concomitant inhibition of electroneutral NaCl absorption (11), whereas the activation of the glucocorticoid receptor (GR) leads to an activation of electroneutral Na+ absorption with concomitant inhibition of electrogenic Na+ absorption (3). However, all mineralocorticoid (MC) and glucocorticoid (GC) hormones bind to both receptors, with MR and GR having different affinities. Higher concentrations of the synthetic "glucocorticoid" hormone dexamethasone activate electrogenic Na+ absorption as efficiently as aldosterone by binding to the MR as well (5). The main GC hormone of the rat, corticosterone, has a higher affinity for the MR than for the GR (35). The MC specificity of a certain tissue is guaranteed by the presence of the enzyme 11beta -hydroxysteroid dehydrogenase (11beta -HSD), which inactivates GCs by oxidation of the 11beta -OH group (7, 9, 18). However, the mechanisms responsible for the MR specificity in tissues expressing both MR and GR are not completely understood and are just beginning to emerge (14). This simple pattern of MC action was challenged by a recent study of Lomax and Sandle (22) showing that the GR-specific synthetic hormone RU-28362 induces electrogenic Na+ absorption via the GR in rat late distal colon. Because this observation is contrary to the existing model of electrogenic Na+ absorption, we tried to find a general explanation for the MC action of natural and synthetic corticosteroid hormones.

MRs and GRs are closely related members of the steroid receptor family that share a common mechanism of action (for review, see Ref. 40). Without ligand, the receptor is bound to a heteromeric complex that includes a 90-kDa heat shock protein and several smaller peptides. When steroid binding occurs, these associated proteins dissociate and the receptor undergoes a conformational change that exposes the DNA binding domain and a specific nuclear localization signal (NLS). Exposing the NLS is necessary for the transport of the receptor into the nucleus. Anti-MCs like spironolactone (2) do not induce a sufficient conformational change, which exposes the NLS and impairs nuclear translocation so that only a small portion of ligand-bound receptors is transferred to the nucleus (10, 28). In the nucleus, steroid-activated receptors bind as dimers to the glucocorticoid response element (GRE), which despite its name does not seem to differentiate between activated MR or GR (39, 40). The receptor dimerization and the binding to the GRE are highly cooperative (8). In tissues expressing both GR and MR, the occurrence of functional heterodimers with inhibitory or activating effects has been observed (20, 38), thereby enhancing the functional diversity of corticosteroid action (37). Because MR and GR are identical through the entire dimer interface region (21), there is no reason to assume that in colon heterodimerization of GR and MR does not take place. Therefore, a differentiated model of steroid binding with the occurrence of MR-MR, GR-GR, and MR-GR dimers has to be considered for rat colon as well.

The purpose of the present study was to determine the acute effects of in vitro-added natural and synthetic glucocorticosteroids on amiloride-sensitive colonic Na+ transport, as measured in standard short-circuit current (Isc) experiments. This study presents the complete concentration dependency for a large set of steroids in in vitro assays in the same colonic segment under equal conditions. We investigated the action of the steroids as they arrived at the steroid receptor within the target tissue, making no attempts to block the activity of 11beta -HSD (7, 9, 18). It turned out that the kinetic appearance of the tested steroids is very similar. They exhibit graded MC potency instead of a sharp partition into exclusive groups of MC and non-MC. Experiments combining aldosterone and RU-28362 corroborated the cooperative effects of GR and MR in the induction of electrogenic Na+ absorption. These results suggest an activation of electrogenic Na+ absorption in rat distal colon with participation of heterodimers between MR and GR.


    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Tissue preparation. Untreated male Wistar rats (200-350 g) had free access to a standard rat diet (Altromin 1320) and tap water ad libitum. The animals were killed by inhalation of a saturated atmosphere of diethylether, according to the guidelines of the Society of Laboratory Animal Science. The colon was removed, rinsed with Ringer solution, and "totally" stripped of serosa and muscle layers, as described previously (13, 33). The segment used in this study was the late distal colon, which is defined as the distal 4-5 cm of the intestine, as measured from the anus. Two tissue pieces located just proximal and distal to the lymph node, situated at ~2.5 cm from the anus, were obtained from each late distal colon.

Electrophysiological measurements. Epithelia were mounted into conventional Ussing chambers equipped with water-jacketed gas lifts (32). The exposed area was 0.54 cm2; the circulating fluid was 10 ml on each side. The bathing fluid consisted of (in mM) 140.5 Na+, 5.4 K+, 1.2 Ca2+, 1.2 Mg2+, 123.8 Cl-, 21 HCO-3, 2.4 HPO2-4, 0.6 H2PO-4, 10 D-(+)-glucose, 0.5 beta -OH-butyrate, 2.5 glutamine, and 10 D-(+)-mannose plus 50 mg/l azlocillin. Azlocillin (Securopen, Bayer) did not affect Isc at the concentration used. The solution was gassed with a mixture of 95% O2 and 5% CO2 and had a pH of 7.4. All experiments were performed at 37°C. Isc (µmol · h-1 · cm-2) and tissue conductance (mS/cm2) were recorded using automatic clamp devices (AC-microclamp, f+p Datensysteme, Aachen, Germany). The bath resistance was measured before each single experiment and was subtracted automatically.

After all tissues were mounted, steroids were added to both sides of the chamber. The steroids were dissolved in 96% methanol in such a way that 10 µl added to the 10 ml of bathing fluid gave the desired final concentration. Methanol alone had no measurable effect on Isc. Eight hours after steroid addition, 10-4 M amiloride was added to the mucosal side. The drop in Isc was recognized as the electrogenic Na+ absorption (JNa). All drugs were purchased from Sigma, except mespirenone, which was a generous gift of Prof. W. Losert (Schering, Berlin, Germany), and RU-28362, which was kindly provided by Roussel Uclaf (Romainville, France). To achieve quantitatively comparable results over a period of more than 1 yr, control experiments of a known result (3 × 10-9 M aldosterone) were regularly fit in the running series of experiments and preparative techniques were tuned if necessary.

Kinetics. For those steroids exhibiting complete concentration dependency patterns, kinetic parameters were evaluated. Michaelis-Menten constant (Km) and Hill coefficients were determined from Hill plots. Maximal velocity (Vmax) was determined by least-square best fit by varying Vmax for linearity of the plot. Due to the logarithmic y-axis, an inexact Vmax would only disturb the Hill curve at maximum substrate concentration but would not have much effect on the determination of Km.

Dimer formation in Fig. 5 combining aldosterone and RU-28362 was calculated using the software package Gepasi3 (24, 25). The forward and backward rate constants for binding of aldosterone to MR (K1 = 8.61 × 105 M-1 · min-1, K-1= 1.64 × 10-4 min-1, respectively) and of RU-28362 to GR (K1 = 2.00 × 106 M-1 · min-1, K×1= 3.32 × 10-4 min-1) and the apparent dissociation constant (Kd) for aldosterone at the GR (~11.89 nM) were taken from a study using dog brain and pituitary (27), but the data are well within the ranges reported for the rat (27). For binding of RU-28362 to the MR, a Kd value of 1 µM was chosen arbitrarily, but this Kd is negligible in experiments combining RU-28362 with aldosterone. The concentration of MR and GR was calculated from the number of receptors per surface cell determined in the study of Schulman et al. (Ref. 31; MR = 7,228 · cell-1, GR= 20,857 · cell-1) and an estimate of the surface cell volume from the same study (4.2 × 10-12 liters). Because MR and GR are identical through the entire dimer interface region (21), the same rate constants were arbitrarily chosen for all possible receptor combinations (K1 = 107 M-1 · min-1, K-1 = 10-2 min-1), leading to homo- or heterodimers (MR-MR, GR-GR, MR-GR). MR-MR homodimers or MR-GR heterodimers, but not GR-GR homodimers, lead to MC action. Production or degradation of the receptors is not regarded.

Statistics. Results are given as means ± SE. The results were subjected to ANOVA. Significances were calculated using Student's unpaired t-test or the Mann-Whitney U test, as appropriate. For post hoc test for pairwise multiple comparisons between means, Tamhane's T2 test implemented in the program package SPSS was utilized. P < 0.05 was considered significant.


    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Concentration dependencies. Most steroids tested produced a typical Isc time course similar to that of aldosterone (13). About 2 h after in vitro addition of the respective steroid, Isc started to increase and reached maximum values after 7-8 h. The amiloride-induced drop in Isc after 8 h (JNa) was determined in all experiments and served as a measure of MC efficiency. A drop in Isc to values close to zero indicates that most of the Isc is Na+ absorption.

In a previous study, a detailed concentration dependency for aldosterone was established (13). In this study, concentration dependencies of GC agonists, natural corticosteroids, and their metabolites were obtained (Fig. 1). In Fig. 1, each data point represents a group of independent 8-h experiments. A concentration of 10-5 M was generally used as a maximum, and the concentration that produced no more significant JNa was used as a minimum. It can be seen from Fig. 1, B-F, that six of the glucocorticosteroids exhibited sigmoidal concentration dependencies of similar shape and similar maximum JNa between 10 and 13 µmol · h-1 · cm-2. For comparison, the concentration dependency of aldosterone obtained earlier (13) is given in Fig. 1A. However, the lowest concentration at which JNa was induced was between 20- and 200-fold higher for the six glucocorticosteroids than for aldosterone. Thus dexamethasone, RU-28362, deoxycortisol, deoxycorticosterone, cortisol, and corticosterone exert concentration-dependent patterns that are similar to that of aldosterone. There were no significant differences in Vmax. However, the concentration dependencies apparently did not follow simple Michaelis-Menten kinetics, i.e., the variation in JNa was completed within a concentration range of two orders of magnitude instead of four orders of magnitude, as expected for Michaelis-Menten kinetics.


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  		Fig. 1.   Concentration dependency of several natural and synthetic corticosteroids. A: aldosterone (from Ref. 13). B: dexamethasone. C: RU-28362. D: deoxycortisol. E: deoxycorticosterone. F: cortisol. G: corticosterone. H: 18-hydroxycorticosterone. All values are given as means ± SE; number of tissues tested is given on top of the error bars. JNa, electrogenic Na+ absorption.

Corticosteroids of minor MC activity (18-hydroxycorticosterone, 11-dehydro-B, 5alpha -H-4,4-dihydro-B, and 6beta -hydroxy-B) did not display a complete sigmoidal concentration dependency at concentrations up to 10-5 M (for 18-hydroxy-B, see Fig. 1H), but the shape of this incomplete curve may be compatible with that of the other steroids tested.

Hill plot. Kinetic results were obtained from Hill plots (Fig. 2). The slopes of these plots indicate the Hill coefficients, which were similar for all steroids tested. Numerical data are given in Table 1. It turned out that Vmax (range 9.1 ± 1.4 to 12.7 ± 1.3) as well as the Hill coefficients (range 1.6 to 2.1) showed no systematic variation. In contrast, regarding the apparent Km, three groups of substances can be defined (cf. Fig. 2). The first is aldosterone with an apparent Km of 1.2 nM. A second group exhibiting apparent Km of 20-37 nM consists of dexamethasone, RU-28362, deoxycortisol, and deoxycorticosterone. The third group is represented by corticosterone and cortisol, which produced apparent Km values of 170 and 210 nM. An estimated Km for 18-hydroxycorticosterone would be in the range of 10 µM, thus outside the physiological range for corticosteroids.


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  		Fig. 2.   Hill plot of the concentration (C) dependencies shown in Fig. 1. Kinetic data are given in Table 1. All steroids show roughly the same Hill coefficient (~2) and partition into 4 groups in regards to the Michaelis-Menten constant values. Aldosterone is the most potent steroid with mineralocorticoid action, followed by the synthetic steroids dexamethasone and RU-28362; the metabolic precursors of corticosterone and cortisol together form the second group. The third group consists of corticosterone and cortisol, followed by a fourth group that showed no complete concentration dependency curve in the physiological concentration range, here represented by 18-hydroxycorticosterone. Vmax, maximal velocity, v, actual velocity.

                              
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  		Table 1.   Kinetic data of corticosteroids

Dexamethasone and RU-28362 show similar Km values for MC action in this part of colon. Although the MC action of dexamethasone can be explained by crossover binding to the MR, RU-28362 is assumed to lack considerable affinity to the MR (36), suggesting an MC action with involvement of the GR.

Inhibition by mespirenone. Mespirenone is a spirolactone with about three times higher anti-MC effectiveness compared with spironolactone (23). To test whether the anti-MC could block the effect of aldosterone and the six glucocorticosteroids on JNa in a similar way, mespirenone was added at the beginning of the experiment. The respective steroids were added at submaximal concentrations at the same time (Table 2). Mespirenone prevented the MC effect of aldosterone as well as that of the glucocorticosteroids. Whereas the effect of 3 nM aldosterone on JNa could be completely inhibited by 1 µM mespirenone (data not shown), the effect of GCs in concentrations leading to a submaximal JNa were only abolished at a concentration of 10-4 M, which is more than four orders of magnitude higher than the Kd of mespirenone for the MR (23). The best investigated member of the spirolactone family, spironolactone itself, possesses a Kd value for the GR of ~2.6 × 10-7 M (27). Therefore, 10-4 M mespirenone is most probably sufficient to block effectively not only the MR but the GR as well. This suggests an involvement of the GR in the MC response of GCs seen in these experiments.

                              
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  		Table 2.   Inhibition of mineralocorticoid effect of steroids by mespirenone

Na+ conductance. The effect of amiloride on Isc is caused by a block of the apically located epithelial Na+ channel. Thus Isc changes are paralleled by changes in epithelial conductance [in this case Na+ conductance (GNa)]. Activation or inhibition of the Na+-K+-ATPase also influenced the epithelial GNa. When the Na+-K+-ATPase is activated in addition to Na+ channels being opened, a block of electrogenic Na+ absorption by amiloride would be accompanied by a larger change in GNa than when the ATPase is not activated.

With regards to the present study, a cumulative plot of all data of JNa vs. the corresponding partial ion GNa revealed a linear relationship for all steroids tested and at all concentrations of these steroids (Fig. 3). The intercept of +0.012 is obtained by subtracting the intercept obtained in our previous study on aldosterone (13) from the result of the linear regression of the GC data in this study. This means that there is principally no different action of all investigated corticosteroids regarding the opening of Na+ channels and the influence on the Na+-K+-ATPase in these in vitro measurements, even compared with aldosterone, suggesting a common mechanism of action.


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  		Fig. 3.   Plot of tissue Na+ conductance (GNa) vs. amiloride-sensitive short-circuit current (Isc) (JNa) of all steroids. Data are means obtained between 2 and 7 h after steroid addition. The intercept with the abscissa results from subtracting the relation found for aldosterone alone (13). Isc measured in the single experiments reflects the same mode of action with all tested steroids regarding opening of Na+ channels and driving force.

Combination of aldosterone and RU-28362. To show a joint action of MR and GR in activation of electrogenic Na+ absorption, an MR-specific concentration of aldosterone (0.1 nM) was combined with a GR-specific concentration of RU-28362 (1 nM) in the same experiment (n = 10). Whereas the single application of aldosterone or RU-28362 alone at the respective concentrations did not evoke any significant electrogenic Na+ absorption after an 8-h incubation, the combination of both steroids caused a electrogenic Na+ absorption of 42 ± 6% of Vmax (Fig. 4). This resembles a JNa evoked by ~0.5-1 nM aldosterone alone. The result shows a cooperative action of both GR and MR in the activation of JNa.


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  		Fig. 4.   Cooperative action of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR). Aldosterone and RU-28362 were given in MR- and GR-specific concentrations, respectively. Single application did not lead to amiloride-sensitive Na+ absorption, whereas the combination of both steroids revealed a synergistic action of MR and GR in the induction of JNa (n = 10). A combination of 10 nM spironolactone with 1 nM RU-28362 showed synergistic effects of an MR antagonist and a GR agonist (n = 9). Aldo = 0.1 nM aldosterone; RU = 1 nM RU-28362; Spi = 10 nM spironolactone; Vmax was ~11 µmol · h-1 · cm-2. P values indicate significances of JNa vs. zero. ns, Not significant.

Combination of spironolactone and RU-28362. Because the spirolactone mespirenone needed a high concentration not specific for MR to abolish the MC action of RU-28362, a possible positively synergistic mechanism of spirolactones with RU-28362 was investigated. For these experiments, the well-characterized spironolactone was used. A GR-specific concentration of RU-28362 (1 nM) was combined with an MR-specific concentration of spironolactone (10 nM). Although both steroids alone did not lead to a significant electrogenic Na+ absorption, the combination yielded 6.3 ± 1.5% of Vmax (n = 9) (Fig. 4). This means that an MR antagonist and a GR agonist can synergistically initiate a small but significant MC action.


    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

In rat distal colon, Na+ absorption under unstimulated conditions is an electroneutral process (4). JNa can be induced by chronic hyperaldosteronism for 3 days, and this is accompanied by a complete inhibition of electroneutral NaCl absorption (4). Alternatively, JNa can be stimulated by addition of physiological concentrations of aldosterone in vitro, and in this case no influence on electroneutral NaCl absorption can be observed (13). GC-induced Na+ absorption has been investigated in many studies, but most of them used in vivo application of the hormones or Na+ deprivation of the animals. To avoid regulatory effects after hormone application, many in vivo studies make use of adrenalectomized rats. However, because there is still some risk of other systemic regulatory mechanisms after hormone application and adrenalectomy, in this study a complete in vitro assay was applied.

Using adrenalectomized rats is not necessary for in vitro measurements of JNa because 1) control rats did not show any amiloride-sensitive Na+ absorption and 2) a former study (13) demonstrated that the effect of aldosterone on JNa is easily reversible within a few hours after removal of aldosterone from the bathing solution.

MC action by natural corticosteroids. Deoxycorticosterone is an intermediary substance in the chain of steroid formation in between progesterone and corticosterone. In analogy, deoxycortisol is intermediary to 17-hydroxyprogesterone and cortisol. The concentration dependencies (Fig. 1, C and D) indicate that deoxycorticosterone was slightly more potent than deoxycortisol. Both steroids are about one order of magnitude more potent than the main natural GCs in rats or humans (corticosterone or cortisol) and therefore are potent MC hormones. The MC action of deoxycorticosterone has been shown before (26, 34). The physiological role in JNa is not clear yet, but their systemic concentration may be only transiently elevated during synthesis of other hormones.

Corticosterone is the main GC in rats and mice, whereas cortisol is predominant in most other mammals. In regards to apparent MC potency, corticosterone and cortisol form one group that exerts similar Km values, ~2 × 10-7 M (Fig. 1, E and F). This is one order of magnitude higher than the Km of the synthetic GCs and two orders of magnitude higher than that of aldosterone. The Km value for corticosterone in this study (170 nM, Table 1) is similar to that found in a study (42) that used in vivo infusion techniques in adrenalectomized rats (330 nM).

Some corticosteroids are subject to enzymatic transformation, which alters MC potency, e.g., by the enzyme 11beta -HSD, which is present in distal colon and transforms corticosterone and cortisol to the respective 11beta -OH metabolites (7, 9). Because we did not alter the physiological action of 11beta -HSD, the Km values for corticosterone and cortisol obtained in this study refer to physiological conditions.

In the rat, corticosterone plasma concentrations in vivo span between 10-7 and 10-6 M (12), and this approximates the range in which this hormone varies Na+ transport in the colonic segment used in this study. Corticosterone may therefore be a physiological regulator of electrogenic Na+ absorption in rat late distal colon. The physiological relevance may be an accelerated start of conductive Na+ absorption by a transient rise in corticosterone concentration in course of the aldosterone synthesis (42). This may be an important aspect in stress situations, as could be shown for the stress induced by surgery (12). Here, the determined corticosterone levels are sufficient to explain JNa in this colonic segment before the final aldosterone response. This result is in accord with those obtained in avian cecum (16) in which corticosterone may contribute to the regulation of Na+ absorption as well.

Dexamethasone and RU-28362. Dexamethasone is a well-investigated synthetic GC (6, 19, 29, 30) with a relatively high affinity for the MR in rat (Kd of ~10 nM; Ref. 35). Whereas high concentrations of dexamethasone applied in vivo induce both electroneutral Na+ absorption and JNa (5), GR-specific concentrations of dexamethasone were reported to induce only electroneutral Na+ absorption (1) and even inhibit JNa (3). Therefore, all effects of dexamethasone regarding JNa were attributed to crossover binding of dexamethasone to MR, if the hormone is used in higher concentrations. In the present study, 10-7 M dexamethasone induced Na+ transport similarly to aldosterone; however, the Km value is shifted toward a 30-fold higher concentration of 37 nM.

RU-28362 is a synthetic steroid nominally devoid of affinity to MR (36). Therefore, it serves as an ideal tool for performing mechanistic studies on steroid receptor function. Experiments that used RU-28362 in adrenally intact rats showed that this steroid in moderate doses (70 µg · 100 g body wt-1 · day-1) induces only amiloride-insensitive Isc in rat distal colon (17) and, when applied for 7 days, stimulates electroneutral NaCl absorption in contrast to the same concentration of aldosterone, which induces JNa (41). Higher doses (600 µg · 100 g body wt-1 · day-1 for 3 days) of RU-28362 in the same animal model induces amiloride-sensitive Na+ absorption in the segment immediately ahead of the last lymph node (5). This result was attributed to a small degree of crossover binding to MR by RU-28362.

However, with the use of the segment between the last lymph node and the anus of adrenalectomized rats, JNa could be observed even with a moderate dose of RU-28362 (70 µg · 100 g body wt-1 · day-1 for 7 days) (22), whereas the segment before did not show any response. Because the specific MR antagonist RU-28318 did block JNa induced by aldosterone but not that induced by RU-28362, GR was made responsible for the RU-28362-induced JNa. However, the question arose why this phenomenon should be restricted to the very last colonic segment, which is the most susceptible to MC effects (6). The hypothesis that GR alone is responsible for the JNa has to assume a different regulation by steroid receptors in the last colonic segment.

In the present study, we were able to show a complete concentration dependency for RU-28362-induced JNa with an apparent Km value of 20 nM (Fig. 1C). The last colonic segment showed slightly larger responses than the preceding segment (data not shown), but this difference was not significant because of a large variety between individual tissues. This means that RU-28362-induced JNa is not restricted to the most caudal colonic segment (22) but probably needs a somewhat higher concentration of the GC in the preceding segment. This situation resembles that found for aldosterone (13), thereby suggesting a common mechanism for the induction of JNa in rat distal colon by all investigated steroid hormones.

Cooperative effects. All six steroids for which complete concentration dependency curves could be obtained (i.e., aldosterone, dexamethasone, RU-28362, deoxycorticosterone, cortisol, and corticosterone) showed Hill coefficients of ~2 (Table 1). This suggests a strong cooperative effect regarding the stimulation of electrogenic Na+ absorption. The combination experiments with MR-specific concentrations of aldosterone and GR-specific concentrations of RU-28362 showed that this cooperative effect includes both steroid receptor types. These results lead to a model of MC action in rat distal colon that includes activating heterodimers of MR and GR. In Fig. 5, we calculated the number of respective homo- and heterodimers occurring during the combination experiment with 0.1 nM aldosterone plus 1 nM RU-28362, as shown in Fig. 4 (for details, see METHODS). The Kd for receptor dimerization was arbitrarily chosen as 1 nM, which is supposed to follow random statistics. RU-28362 is nearly exclusively bound to GR as well as aldosterone to MR under these conditions. The amount of activated MR does not allow formation of MR-MR homodimers within 8 h, but the abundance of activated GR can account for Na+ absorption after 2 h via formation of MR-GR heterodimers. GR-GR homodimers are supposed to stay without function regarding JNa in this time range. For RU-28362 alone, this means that a Kd value for its binding to MR in the range of 10 µM is sufficient to explain its activating effect on JNa via receptor heterodimerization.


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  		Fig. 5.   Calculation of dimer formation during combined action of 0.1 nM aldosterone and 1 nM RU-28362. Formation of homo- and heterodimers during the experiment shown in middle of Fig. 4 is visualized. The calculation allowed equal probabilities for the formation of homo- or heterodimers (for details, see METHODS). MR-MR homodimers and MR-GR heterodimers are thought to induce JNa, whereas GR-GR homodimers do not have any influence. In this case, JNa crucially depends on the formation of MR-GR heterodimers.

The interpretation of our results including heterodimerization fits a finding by Garty et al. (15) who injected Xenopus oocytes with RNA from toad bladders so that they expressed amiloride-sensitive Na+ channels. The number of channels expressed could be increased synergistically by the combined administration of an MR agonist and a GR antagonist. This example shows that heterodimerization may also occur between an agonist- and an antagonist-occupied receptor, leading to a receptor heterodimer with transactivation potency.

One objection comes from the microelectrode study of Lomax and Sandle (22), who showed that infusion of the MR antagonist RU-28318 together with RU-28362 did not block the induction of JNa by RU-28362. This seemed to indicate that RU-28362 induces JNa via GR only without participation of MR. However, the experiment is not conclusive regarding this point in light of the possibility of GR and MR heterodimerization. The mechanism by which most of the MR antagonists exert their effect is activating MR without exposing the NLS, thereby preventing the transport of the MR to the nucleus (28). This block of nuclear translocation is not complete, but usually a small amount of MR carrying the MR antagonist is still present in the nucleus (10). This concentration is too small to activate Na+ absorption by itself, but it could serve as a partner in heterodimerization with activated GR bearing RU-28362, thereby promoting heterodimeric induction of JNa. A combination experiment of an MR-specific concentration of spironolactone (10 nM) with a by-itself ineffective concentration of RU-28362 (1 nM) resulted in a small but significant electrogenic Na+ absorption (Fig. 4), further confirming this possibility. In this particular combination, spironolactone even would act as a partial agonist.

Role of GR homodimers. Within the model calculations, the MC action has been attributed to MR-MR and MR-GR dimers, leaving the GR-GR homodimers without function in this regard. The inhibitory function of GC on JNa observed by other groups (3) may need a longer time to be established, similar to the inhibitory action of aldosterone on electroneutral Na+ absorption (11), which was also not observed in our experiments. On the other hand, this inhibitory action of GC may only occur in an in vivo situation and needs other cofactors not provided in our experiments because the inhibitory effect of a GR-specific concentration of dexamethasone was reported to take place within 30 min (3), an effect never observed in the in vitro experiments.

In conclusion, we have shown that in rat distal colon aldosterone, natural corticosteroids, and synthetic GC induce electrogenic Na+ absorption of the same size and with similar kinetics, differing only in their Km values. This very homogeneous picture suggests a common mode of action. Inhibitory effects of GC on MC action reported by many investigators in in vivo studies could not be observed in these in vitro studies. Corticosterone may be a potent inductor of JNa under physiological conditions, e.g., in stress situations. Because of the strong cooperative effects of GR and MR in the induction of JNa, a model of MC action was proposed that was based on receptor heterodimerization between GR and MR.


    ACKNOWLEDGEMENTS

The superb technical assistance of Anja Fromm, Ursula Lempart, and Sieglinde Lüderitz is gratefully acknowledged.


    FOOTNOTES

This study was supported by the Sonnenfeld-Stiftung.

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. §1734 solely to indicate this fact.

Address for reprint requests: M. Fromm, Inst. f. Klinische Physiologie, UKBF der Freien Universität Berlin, D-12200 Berlin, Germany.

Received 14 August 1998; accepted in final form 20 October 1998.


    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 276(2):G491-G498
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