Early aldosterone effect in distal colon by transcriptional regulation of ENaC subunits

H. J. Epple1,2, S. Amasheh1, J. Mankertz2, M. Goltz3, J. D. Schulzke2, and M. Fromm1

Departments of 1 Clinical Physiology and 2 Gastroenterology, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, 12200 Berlin; and 3 Department of Xenotransplantation, Robert Koch-Institut, 13353 Berlin, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Aldosterone-induced sodium absorption is mediated by the epithelial Na+ channel (ENaC). It is thought that the "early effect" is not based on genomic regulation of ENaC expression, because ENaC subunit transcription was reported to start later than Na+ transport. We investigated electrogenic Na+ absorption (JNa) and, in identical tissues, mRNA expression of ENaC subunits in early (EDC) and late (LDC) distal colon of the rat. In both segments, 8-h in vitro incubation with 3 nM aldosterone enhanced expression of beta - and gamma -ENaC mRNA and induced JNa. JNa was 10 times higher in LDC than in EDC. alpha -ENaC mRNA was unchanged in EDC, whereas it decreased in LDC. In LDC, beta - and gamma -ENaC mRNA was induced 1 h after aldosterone addition, whereas JNa became apparent >1 h later. Downregulation of alpha -ENaC mRNA did not take part in acute regulation because it started after a lag time of 3 h. Time correlation of beta - and gamma -ENaC induction and JNa stimulation suggests that the early aldosterone effect on Na+ absorption in distal colon is caused by transcriptional upregulation of beta - and gamma -ENaC expression.

epithelial sodium channel; segmental heterogeneity; mineralocorticoid receptor; glucocorticoid receptor; heterodimer


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ALDOSTERONE IS THE KEY HORMONE in the regulation of Na+ homeostasis. Its Na+-saving action is mediated by the activity of the amiloride-sensitive epithelial Na+ channel (ENaC) located in the apical membrane of aldosterone-responsive tissues such as kidney collecting duct and late distal colon (LDC) (i.e., in human sigmoid and rectum). The importance of proper regulation of ENaC activity is highlighted by human diseases caused by gain or loss of ENaC function due to gene mutations (Liddle disease or pseudohypoaldosteronism type I, respectively) (20). Therefore, the regulation of ENaC activity by aldosterone has been extensively studied by different electrophysiological and biochemical means in a wide variety of mammalian and amphibian epithelia (18).

According to a widely accepted model, ENaC activity [i.e., electrogenic Na+ absorption (JNa)] in mammalian distal colon is controlled by the mineralocorticoid receptor (MR) (2, 18). Therefore, many functional studies of the action of mineralocorticoid hormones have been performed in this tissue (see, e.g., Refs. 12 and 15). It was demonstrated that the action of aldosterone on Na+ transport is dependent on both intact transcription and translation machinery (for review, see Ref. 17). However, the aldosterone-induced protein(s) responsible for the acute induction of electrogenic sodium absorption have not been identified so far (26).

The recent molecular cloning of the three subunits forming the pore of the ENaC (termed alpha -, beta -, and gamma -ENaC; Refs. 4 and 5) brought new vigor into the search for the molecular mechanism of aldosterone action. Two independent studies found that endogenous aldosterone stimulation enhances beta - and gamma -ENaC mRNA expression in rat distal colon (21, 24). In light of these results, it was suggested that ENaC activity is regulated by aldosterone-dependent transcriptional control of its beta - and gamma -subunits. This straightforward model was challenged by a study on the time course of ENaC mRNA expression in rat distal colon (1). In this study, beta - and gamma -ENaC mRNA was upregulated no earlier then 3 h after the beginning of aldosterone administration. By comparison of data taken from the literature on the time course of JNa induced by aldosterone (3, 14-16), it was concluded that the induction of the early response to aldosterone must be independent from beta - and gamma -ENaC mRNA expression (1). Despite the obvious methodological problem of comparing time course data obtained in different laboratories (including our own) under different experimental conditions, the conclusions drawn were generally accepted (2, 29).

Other difficulties that might have distorted the interpretation of molecular or functional data obtained in rat distal colon concern the segmental heterogeneity of this tissue. In all studies on ENaC expression in rat distal colon published so far, the segment of the distal colon taken for the experiments was not further specified, on the assumption that the whole distal colon displays uniform properties. It has been demonstrated, however, that the very distal part of the colon is very sensitive to nanomolar concentrations of the mineralocorticoid, whereas a more proximal segment of distal colon exhibits only minor sensitivity. For that reason, rat distal colon was divided into two functionally distinct segments, termed early (EDC) and late (LDC) distal colon (16). In fact, distinct patterns of subunit expression have been found along the axis of the renal collecting duct (10).

In light of these results, we studied the effect of mineralocorticoid stimulation on ENaC expression in rat distal colon using a well-characterized in vitro model. Thus we were able to properly define the colonic segment under investigation and to add hormones at specified concentrations without possible interference from the complex regulatory mechanisms present in the in vivo situation. Most importantly, we determined channel activity and ENaC expression in identical epithelia, obtaining functional and molecular data from the same tissues after exactly defined intervals.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of epithelia. Untreated male Wistar rats (200-300 g), fed with a standard rat diet (Altromin 1320) and tap water, were anesthetized and killed by inhalation of ether. The colon was removed, rinsed with Ringer solution, and "totally" stripped of serosa and muscle layers as described previously (16). Two segments of the colon termed LDC and EDC were used in this study. Specimens of LDC were obtained from the very last part of the colon, located between the lymph node at the pelvic brim and the anus. To prepare this extraperitoneal segment of the colon, it was necessary to cut open the pelvic bones. EDC was obtained 6-7 cm proximal to the anus.

Steroid application and electrophysiological measurements. Epithelia were mounted into conventional Ussing-type chambers equipped with water-jacketed gas lifts. The exposed area was 0.54 cm2, and the circulating fluid was 10 ml on each side. The bathing fluid consisted of (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, 10 D(+)-mannose, 0.5 beta -OH-butyrate, 2.5 glutamine, and 50 mg/l azlocillin (Securopen, Bayer).

Short-circuit current (Isc, µmol · h-1 · cm-2) and tissue conductance (mS/cm2) were recorded using microprocessor-driven clamp devices (AC-microclamp, f+p Datensysteme, Aachen, Germany). The resistance of the bathing fluid between the voltage-sensing electrodes was measured before each experiment and taken into account. After mounting of the tissues and a 30-min equilibration period, steroids and other substances were added to both sides of the epithelium as indicated. For time course experiments, all epithelia including controls were kept in the Ussing chamber for 8 h (after equilibration) before JNa was determined. During this time, aldosterone was added to the bathing solution so that the indicated incubation times were achieved. All substances used were dissolved in 96% methanol so that 10 µl added to the bathing solution gave the desired final concentration. Methanol alone had no detectable effect on Isc and mRNA expression. At the end of the electrophysiological measurement, amiloride (10-4 M) was added to the mucosal compartment. The drop in Isc after addition of amiloride was assigned to JNa. Thus all results of amiloride-sensitive Isc are expressed as fluxes of monovalent cations (µmol · h-1 · cm-2).

Extraction and isolation of RNA. RNA was prepared from matched groups of epithelia (untreated controls and epithelia treated as specified) immediately after determination of JNa. The area of the epithelium exposed to the bathing fluid was cut out with a scalpel. For RNA extraction, three identically treated epithelia were pooled and placed into 6 ml of iced RNAzol (Boehringer Mannheim). The epithelia were then homogenized using an Ultra-Turrax (Ika-Werk, Janke & Kunkel). Extraction of total RNA was performed according to the instructions given by the manufacturer. In this way, 60-120 µg of total RNA could be extracted from three tissue preparations.

Northern hybridization. Aliquots of 10 µg of colon epithelium total RNA were separated on 1% agarose gels in 1× MOPS under denaturing conditions (2% formaldehyde). Nucleic acids were transferred to nylon membranes (Boehringer Mannheim) and ultraviolet cross-linked. The membranes were hybridized for 2 h at 68°C in Quik-Hyb (Stratagene) and 100 µg/ml herring sperm DNA with digoxigenin-labeled cDNA probes corresponding to alpha -, beta -, or gamma -ENaC and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). After stringency washes (2× SSC-0.1% SDS at room temperature and 0.5× SSC-0.1% SDS at 65°C, 2 times each), probes for ENaC subunit mRNA and GAPDH mRNA were detected using anti-digoxigenin antibody conjugated to alkaline phosphatase (Anti-Digoxigenin-AP, Boehringer Mannheim) and a chemiluminescent substrate (CDP-Star, Boehringer Mannheim) according to the instructions of the manufacturer. Hybridization intensity was quantified with luminescent imaging (LAS-1000, Fujifilm) using AIDA software (Raytest). The intensity of the GAPDH signal was used for normalization to detect differences between different lanes.

PCR. Probes were prepared by PCR with digoxigenin-labeled dUTP using a PCR digoxigenin probe synthesis kit (Boehringer Mannheim). cDNA clones for alpha -, beta -, and gamma -ENaC were kindly provided by B. Rossier (Dept. of Pharmacology, University of Lausanne, Lausanne, Switzerland), and cDNA clone for rat GAPDH was kindly provided by O. Huber (Inst. of Clinical Chemistry, Freie Universtät Berlin, Berlin, Germany). Sense and antisense primers were designed based on regions that showed significant sequence divergence between the respective ENaC subunits. The primer pairs were 5'-CACAGCAGGTGTGCATTCAC-3' (sense) and 5'- AGGTTGCACAGGAGGCTGAC-3' (antisense), extending from bases 1397 to 1416 and 1814 to 1795 of alpha -ENaC; 5'-CGGCTCCGACGTTGCCATTC-3' (sense) and 5'-TCTGGTCCCGCTCCTGAGACAG-3' (antisense), extending from bases 1158 to 1177 and 1510 to 1489 of beta -ENaC; 5'-CTCAAGCACATGATCTTGGGTAGCA-3' (sense) and 5'-TGGGAATACCATTTGGCAGGAGTGT-3' (antisense), extending from bases 2388 to 2412 and 2695 to 2671 of gamma -ENaC; and 5'-GACAACTCCCTCAAGATTGTCAG-3' (sense) and 5'-CTTCTTGATGTCATCATACTTGGC-3' (antisense), extending from bases 445 to 467 and 804 to 781 of rat GAPDH. PCR was performed for 45 s at 95°C, 1 min at 60°C, and 2 min at 72°C for 30 cycles. To control the result of the PCR, a small sample of the amplified product was electrophoretically separated on an agarose gel.

Materials. Aldosterone was purchased from Sigma (St. Louis, MO). RU-28362 was kindly provided by Roussel Uclaf (Romainville, France). PD-98059 was from Calbiochem (San Diego, CA).

Statistical analysis. Data are expressed as means ± SE. Statistical analysis was performed using Student's t-test. P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of aldosterone on rat EDC and LDC. After 8-h in vitro stimulation with 3 nM aldosterone, which is a concentration found in vivo under stimulated conditions (14), JNa was determined by mucosal addition of 10-4 M amiloride (Fig. 1A). As described previously (16), JNa in LDC was >10 times that of EDC (Fig. 1B; 14.2 ± 2.1 and 1.25 ± 0.4 µmol · h-1 · cm-2, respectively, n = 9 each), confirming the concept of functional diversity within the distal colon. In controls, there was no significant JNa in either EDC or LDC.


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Fig. 1.   Effect of aldosterone on electrogenic Na+ transport (JNa) in rat early (EDC) and late (LDC) distal colon. Twelve epithelia (3 for each group) were incubated with 3 nM aldosterone (aldo) or carrier (ctrl) in parallel. A: short-circuit current (Isc). After 8-h incubation, JNa was determined as amiloride-sensitive Isc. Depicted are representative Isc curves for each group. Drop in Isc after mucosal addition of 100 µM amiloride is totally accounted for by aldosterone-induced JNa (16). B: JNa in EDC and LDC. Data are means ± SE of n = 9 experiments for each group. *P < 0.05, ***P < 0.001 vs. control.

Directly after completion of the electrophysiological measurements, RNA was extracted from the tissues for Northern blot hybridization. In aldosterone-treated epithelia a marked increase of beta - and gamma -ENaC mRNA expression was found in both EDC and LDC (Fig. 2; P < 0.001, n = 9). In contrast to earlier in vivo studies (1, 21, 24), alpha -ENaC was not constitutively expressed in LDC. Instead, in this segment of the distal colon, alpha -ENaC mRNA was downregulated after 8 h of aldosterone incubation (65.7 ± 10% of controls, P < 0.05, n = 12; Fig. 1C). In EDC on the other hand, alpha -ENaC mRNA remained unchanged with or without aldosterone (aldosterone-treated EDC: 112.3 ± 12.4% of controls, n = 9). To further characterize this surprising finding, we investigated the effect of different aldosterone concentrations on expression of alpha -ENaC mRNA in EDC and LDC. Aldosterone was varied between 0.1 nM and 0.3 µM, because it has been shown that JNa is regulated by aldosterone within this concentration range (16). In EDC. the expression of alpha -ENaC mRNA remained unchanged compared with controls. In LDC however, 0.1 nM aldosterone sufficed to maximally suppress alpha -ENaC expression (Fig. 3). The different expression of alpha -ENaC mRNA in EDC and LDC added to the concept of a segmental heterogeneity within the distal colon.


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Fig. 2.   Effect of aldosterone (aldo) on ENaC subunit mRNA expression in rat EDC and LDC. A: Northern blots. RNA was extracted immediately after determination of JNa (Fig. 1). Total RNA from 3 epithelia in each group was pooled to average expression intensities. Blots are representative of at least 3 Northern blots detecting single subunits. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. B: densitometry. Intensity of ENaC subunit mRNA expression was normalized to GAPDH. Relative abundance of aldosterone-treated epithelia was expressed in percentage of signal obtained in controls treated with carrier only. Data are given as mean percentages ± SE determined from at least 3 Northern blots detecting single subunits. *P < 0.05 vs. control.



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Fig. 3.   Dose response of aldosterone on alpha -ENaC mRNA expression in rat EDC and LDC. A: Northern blots. Epithelia were incubated with aldosterone (aldo; concentrations as indicated) or carrier (ctrl) for 8 h. Blots are representative of 3 Northern blots detecting alpha -subunit in EDC or LDC. B: densitometry. Intensity of alpha -ENaC mRNA expression was normalized to GAPDH. Relative abundance of aldosterone-treated epithelia was expressed in percentage of signal obtained in controls treated with carrier only. Data are given as mean percentages ± SE determined from at least 3 Northern blots detecting alpha -ENaC mRNA. *P < 0.05 vs. control.

Time course. To achieve maximal resolution, we performed time course experiments in LDC. Epithelia were incubated with aldosterone for the time period indicated. After determination of JNa, RNA was extracted from the epithelia for quantification of ENaC expression as described in METHODS. Thus the molecular data obtained reflect the RNA expression at the time of the JNa measurement.

As shown previously (16), JNa started to increase between 2 and 3 h after addition of aldosterone, reaching maximum values 8 h after aldosterone incubation (Fig. 4). Interestingly, beta - and gamma -ENaC mRNA was found to be increased as soon as 1 h after aldosterone addition, more than 1 h before JNa started to rise (Fig. 5). This result is clearly compatible with a model of acute regulation of ENaC activity by transcriptional control of its beta - and gamma -subunit expression.


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Fig. 4.   Time course of aldosterone-induced JNa. Fifteen epithelia [3 for each time and 3 for controls (0 h)] were incubated with 3 nM aldosterone or carrier in parallel. After 1, 2, 3, or 8 h of aldosterone (3 nM) incubation, JNa was determined as described. In controls JNa was determined after 8 h incubation with carrier. Data are means ± SE of 6 experiments for each time point. *P < 0.05, *** P < 0.001 vs. control.



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Fig. 5.   Time course of aldosterone-induced ENaC subunit RNA expression. A: Northern blots. Immediately after determination of JNa (Fig. 4), RNA was extracted from epithelia for Northern blot hybridization. Total RNA from 3 epithelia for each group was pooled to average expression intensities. Blots are representative for at least 3 Northern blots detecting single subunits. B: densitometry. Relative abundance of aldosterone-treated epithelia was expressed in percentage of signal obtained in controls treated with carrier only. As tested in independent experiments, ENaC subunit expression in untreated epithelia was same as in epithelia incubated with carrier for 8 h (data not shown). Data are given as mean percentages ± SE determined from 3-5 Northern blots detecting single subunits (alpha -ENaC, beta -ENaC, gamma -ENaC).

The downregulation of alpha -ENaC mRNA only started after a lag time of 3 h, when JNa has already started to increase (Fig. 5). Thus alpha -ENaC apparently does not take part in the acute regulation of ENaC activity in this tissue. In further experiments, therefore, we concentrated on the correlation between JNa and expression of beta - and gamma -ENaC only.

Heterodimeric activation of ENaC mRNA expression. In a recent study, we demonstrated (19) stimulation of JNa in rat distal colon by the activated glucocorticoid receptor (GR) and cooperative stimulation of JNa by heterodimers of activated MR and GR. To investigate the effect of heterodimeric activation on ENaC mRNA expression, we combined a MR-specific concentration of aldosterone (0.1 nM) with a GR-specific concentration of the "pure" glucocorticoid RU-28362 (1 nM) in the same experiment. In these concentrations, aldosterone induced small JNa (1.3 ± 0.3 µmol · h-1 · cm-2, P < 0.001, n = 8), whereas RU-28362 did not induce significant JNa (0.3 ± 0.2 µmol · h-1 · cm-2;not significant, n = 7). The combination of both hormones evoked a clearly overadditive response (JNa 5.8 ± 1.0 µmol · h-1 · cm-2, n = 8) as reported previously (19) (Fig. 6A).


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Fig. 6.   Heterodimeric activation of mineralocorticoid and glucocorticoid receptor. A: JNa. Aldosterone (aldo) and RU-28363 (RU) were given in receptor-specific concentrations (0.1 nM for aldosterone and 1 nM for RU-28362). After 8-h incubation, JNa was determined as amiloride-sensitive Isc. Data are means ± SE of n = 6 experiments for each group. **P < 0.005, ***P < 0.001 vs. control; n.s., not significant. B: beta - and gamma -ENaC mRNA expression. Immediately after determination of JNa, RNA was extracted from epithelia for Northern blot hybridization. Total RNA from 3 epithelia of each group was pooled to average expression intensities. Blots are representative of at least 3 Northern blots detecting beta - or gamma -ENaC mRNA.

This positive cooperativity was reflected at the molecular level. Although aldosterone and RU-28362 only induced a small increase of beta - and gamma -ENaC mRNA when given alone, the combination of both substances strongly enhanced beta - and gamma -ENaC mRNA expression (Fig. 6B).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Time course. Despite substantial progress in the last few years, the molecular mediation of the acute action of aldosterone in mammalian distal colon is still unknown. Because this action is dependent on functional transcription and translation (17), several aldosterone-induced proteins have been proposed as potential mediators inducing JNa (see, e.g., Refs. 7, 11, 23, 25). However, the mRNA encoding for these proteins has been shown to be induced too late to explain the "early" action of aldosterone. On the other hand, "early" aldosterone-induced RNAs or proteins were identified by differential display PCR in the toad kidney cell line A6, but the regulatory role of these aldosterone-induced proteins in the intact mammalian target organ has not been elucidated so far (6, 26).

After the successful cloning of the cDNA encoding for the subunits composing the ENaC (4, 5), these subunits were obviously key candidates as possible mediators of the aldosterone response. Three studies investigated aldosterone-dependent ENaC subunit expression in rat distal colon (1, 21, 24). All of them made use of aldosterone stimulation in vivo. Additional electrophysiological measurements for ENaC activity were only performed in one study, demonstrating a parallel increase of gamma -ENaC expression and JNa after 14 days of salt deprivation, whereas alpha -ENaC mRNA remained unaltered (21). This result seemed to indicate a regulation of ENaC activity by transcriptional control of its gamma -subunit (expression of beta -ENaC was not investigated in this study). However, a subsequent study using implanted minipumps failed to demonstrate a significant increase in ENaC subunit mRNA expression during the first 3 h of aldosterone administration (1). Although no measurement of ENaC function was performed, the time course obtained in this study was used as an argument against a direct transcriptional control of ENaC activity in the acute action of aldosterone (2). Consequently, it was concluded that the short-term response to aldosterone is mediated not via expression of ENaC subunits but rather by different "early" aldosterone-induced regulatory proteins (6, 26, 29).

The results of the present study disprove these conclusions. The time courses of beta - and gamma -ENaC mRNA induction and JNa increase clearly demonstrate that the JNa increase lags behind that of beta - and gamma -ENaC mRNA expression and not vice versa as proposed earlier (1). Thus the time courses of beta - and gamma -ENaC mRNA expression and JNa found in the present study are entirely compatible with direct transcriptional control of ENaC activity by expression of its beta - and gamma -subunits, although our results do not, of course, exclude the participation of other mechanisms in the regulation of ENaC function. In this context, however, it should be mentioned that the time course of ENaC RNA induction in vitro presented in the present study is in accordance with former in vivo analysis of Na+ transport using the same colonic segment and a similar aldosterone concentration (13).

The experimental model used in the present study is probably better suited to resolve the time correlation between ENaC activity and mRNA expression during the acute aldosterone response than the models of earlier studies. Because in our study electrophysiological and molecular data were obtained from identical epithelia, these data reflect the functional state of the epithelia at a given time. One explanation for the delayed effect of previous in vivo studies using minipump infusion may be the only gradual increase of aldosterone concentrations at the target cells, which is slow compared with the immediate delivery in our in vitro setup. In the latter case, it is very difficult to estimate the time needed to reach an equilibrium of receptor occupancy. Thus conclusions based on correlation of molecular and functional time course should only be drawn if these data were obtained simultaneously using the same experimental system.

alpha -Subunit. In EDC alpha -ENaC was constitutively expressed independent from aldosterone as described for rat distal colon previously (1, 21, 24). In contrast, in LDC we observed aldosterone-dependent downregulation of alpha -ENaC mRNA. This is the first report of aldosterone-dependent downregulation of an ENaC subunit in the colon. To the best of our knowledge, there is only one study, using a rat parotid cell line, that reports downregulation of alpha -ENaC in a mammalian tissue (30). In this study a phorbol ester [12-O-tetradecanoylphorbol 13-acetate (TPA)] repressed alpha -ENaC expression, and this effect was abolished by pretreatment of the cells with an inhibitor of the extracellular signal-regulated protein kinase pathway (PD-98059; Ref. 30). In our experimental model using intact distal colon, however, neither TPA nor PD-98059 had any effect on alpha -ENaC expression (data not shown), suggesting different regulatory pathways for alpha -ENaC mRNA expression in rat distal colon and parotid cells. For two reasons, a regulatory role of alpha -ENaC in the acute response of the distal colon to aldosterone seems unlikely. First, downregulation of alpha -ENaC clearly occurred only after JNa was already induced. Second, alpha -ENaC mRNA was maximally suppressed by 0.1 nM aldosterone, which was shown to exert no measurable effect on JNa in a previous study (16).

Segmental heterogeneity. The functionally defined segmental heterogeneity of the distal colon with an aboral gradient of increasing aldosterone sensitivity (16) was extended by the different patterns of alpha -ENaC mRNA expression found in EDC and LDC. In the present study alpha -ENaC mRNA downregulation was found only in the most distal part of the LDC located between the anus and the lymph node regularly present at the pelvic brim [a segment termed LDC2 in a previous study from our laboratory (16)]. The reason why earlier in vivo studies failed to observe downregulation of alpha -ENaC may be that RNA was extracted from more proximal parts of the colon.

Heterodimeric activation. According to a generally accepted model, JNa in the distal colon can only be induced by activated MR (2, 18). However, two recent studies demonstrated stimulation of JNa by the GR-specific substance RU-28362 (19, 22). Moreover, it was shown that the combination of RU-28362 with aldosterone exerts a synergistic action on JNa, if receptor-specific concentrations of the respective hormones are used. To explain these results, heterodimerization between MR and GR was proposed (19). In the present study, we found positive cooperativity also on the molecular level. Receptor-specific concentrations of aldosterone and RU-28362 revealed a clearly cooperative effect on both JNa and beta - and gamma -ENaC mRNA expression. This finding indicates that the cooperative process takes place between receptor binding of the hormones and transcription of the beta - and gamma -ENaC genes. It is well described that activated steroid receptors bind as dimers to the steroid-responsive elements (27, 28) and that the process of dimerization and receptor binding is highly cooperative (8). Therefore, it seems very likely that the positive cooperativity of aldosterone and RU-28362 on JNa and ENaC subunit mRNA expression is caused by MR and GR heterodimerization.

As to the quantitative effects of aldosterone and the combination of aldosterone with RU-28362, the magnitude of JNa was fully accounted for by different levels of beta - and gamma -ENaC mRNA expression. In fact, under all experimental conditions used in the present study, independent of the respective hormones and concentrations used or the colonic segment investigated, beta - and gamma -ENaC mRNA expression always paralleled and preceded JNa. Thus, although other mechanisms may also be involved, ENaC expression must be considered as a key candidate mechanism in the acute regulation of ENaC function in the distal colon.


    ACKNOWLEDGEMENTS

The superb technical assistance of A. Fromm is gratefully acknowledged.


    FOOTNOTES

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 and other correspondence: M. Fromm, Dept. of Clinical Physiology, Universitätsklinikum Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany (E-mail: m.fromm{at}medizin.fu-berlin.de).

Received 8 October 1999; accepted in final form 16 December 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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