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
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ABSTRACT |
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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 - and
-ENaC mRNA and induced JNa.
JNa was 10 times higher in LDC than in EDC.
-ENaC mRNA was unchanged in EDC, whereas it decreased in LDC. In
LDC,
- and
-ENaC mRNA was induced 1 h after aldosterone addition,
whereas JNa became apparent >1 h later.
Downregulation of
-ENaC mRNA did not take part in acute regulation
because it started after a lag time of 3 h. Time correlation of
-
and
-ENaC induction and JNa stimulation suggests
that the early aldosterone effect on Na+ absorption in
distal colon is caused by transcriptional upregulation of
- and
-ENaC expression.
epithelial sodium channel; segmental heterogeneity; mineralocorticoid receptor; glucocorticoid receptor; heterodimer
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INTRODUCTION |
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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 -,
-, and
-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
- and
-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
- and
-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,
- and
-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
- and
-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.
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METHODS |
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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
-OH-butyrate, 2.5 glutamine, and 50 mg/l azlocillin (Securopen, Bayer).
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 -,
-, or
-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
-,
-, and
-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
-ENaC;
5'-CGGCTCCGACGTTGCCATTC-3' (sense) and
5'-TCTGGTCCCGCTCCTGAGACAG-3' (antisense), extending from
bases 1158 to 1177 and 1510 to 1489 of
-ENaC;
5'-CTCAAGCACATGATCTTGGGTAGCA-3' (sense) and
5'-TGGGAATACCATTTGGCAGGAGTGT-3' (antisense), extending from
bases 2388 to 2412 and 2695 to 2671 of
-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.
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RESULTS |
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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
104 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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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,
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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 · h1 · 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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DISCUSSION |
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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-Subunit.
In EDC
-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
-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
-ENaC in a mammalian tissue (30). In this
study a phorbol ester [12-O-tetradecanoylphorbol 13-acetate (TPA)] repressed
-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
-ENaC expression (data
not shown), suggesting different regulatory pathways for
-ENaC mRNA
expression in rat distal colon and parotid cells. For two reasons, a
regulatory role of
-ENaC in the acute response of the distal colon
to aldosterone seems unlikely. First, downregulation of
-ENaC
clearly occurred only after JNa was already
induced. Second,
-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 -ENaC mRNA expression found in
EDC and LDC. In the present study
-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
-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 - and
-ENaC mRNA expression. This finding indicates that the cooperative
process takes place between receptor binding of the hormones and
transcription of the
- and
-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.
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ACKNOWLEDGEMENTS |
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The superb technical assistance of A. Fromm is gratefully acknowledged.
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FOOTNOTES |
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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.
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