Mechanisms of Ageing and Development
98 (1997) 139 – 150

The role of 11i-hydroxysteroid dehydrogenase in
maturation of the intestine
I. Pohlova, I. Miks' ´k, J. Pacha *
´
ı
´
Institute of Physiology, Czech Academy of Sciences, Vıdenska 1083,
´ ' ´
14220 Prague 4 -Krc, Czech Republic
'
Received 16 November 1996; received in revised form 22 April 1997

Abstract
Glucocorticoids promote the development of many organs including intestine. At the
cellular level, the activity of glucocorticoids is regulated by 11i-hydroxysteroid dehydrogenase (11iHSD) which converts active glucocorticoids to inactive metabolites. As 11iHSD is
also expressed in the intestine, this enzyme may be an important regulator of intestinal
maturation. To investigate this, we have performed the systematic study of the development
of intestinal 11iHSD activity and its cofactor preference as well as of the effect of 11iHSD
inhibition by carbenoxolone on postnatal development of sucrase, alkaline phosphatase and
Na,K-ATPase in the intestine. The activity of 11iHSD was low in ileum of suckling rats and
signiﬁcantly increased during the weaning period. In colon, the activity was already high in
suckling rats and gradually rose during the postnatal development. 11iHSD activity was
undetectable in jejunum both in young and adult rats. At 14.5 nM corticosterone, colonic
11iHSD utilized predominantly NAD as a cofactor, but displayed signiﬁcant sensitivity also
to NADP. Ileal 11iHSD had similar sensitivity to both cofactors. With NAD as a cofactor,
ileal 11iHSD had a Km (59910 nM) compatible with the colonic enzyme (81 9 14 nM).
Carbenoxolone administration to suckling and weanling rats in vivo did not result in any
changes of sucrase activity in jejunum and ileum, alkaline phosphatase activity in ileum and
distal colon or Na,K-ATPase activity in ileum. However, carbenoxolone signiﬁcantly increased Na,K-ATPase activity in distal colon. Our results indicate that the high-afﬁnity type
of 11iHSD is expressed not only in colon but also in ileum and that 11iHSD is an
important factor in the regulation of tissue levels of active glucocorticoids in developing
colon but not in the small intestine. © 1997 Elsevier Science Ireland Ltd.
* Corresponding author. Tel: + 420 24752440; fax: +420 24719571.
0047-6374/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved.
PII S 0 0 4 7 - 6 3 7 4 ( 9 7 ) 0 0 0 7 9 - 1

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Keywords: Mineralocorticoid receptors; Glucocorticoid receptors; Corticosterone; Steroid
metabolism; Intestinal maturation; 11i-Hydroxysteroid dehydrogenase; Carbenoxolone

1. Introduction
Postnatal maturation of the rodent intestine is characterized by signiﬁcant
modiﬁcations of enzyme activities and transport properties. These developmental
changes in enzyme status and intestinal transport coincide with weaning period
and are inﬂuenced by the hormonal milieu of the pups [1,2]. Several lines of
evidence indicate that pituitary-adrenal system modulates intestinal development.
Adrenalectomy or hypophysectomy of rats attenuate developmental changes and
such alterations can be prevented by the administration of corticosterone [1].
Corticosterone, the plasma concentration of which increases just before the time
of weaning [3] has been shown to affect the development of various enzymes
including sucrase, alkaline phosphatase and peptidases in the small intestine [1,4].
Aldosterone, which has developmental proﬁle similar to corticosterone, induces
electrogenic amiloride-sensitive Na + transport in suckling and weanling rats [5].
11i-hydroxysteroid dehydrogenase (11iHSD), a microsomal enzyme responsible for the interconversion of glucocorticoids (corticosterone in the rat), to their
inactive 11-dehydro metabolites (11-dehydrocorticosterone in the rat), is considered to be a potential regulator of glucocorticoid and mineralocorticoid action
[6,7]. In glucocorticoid-target organs, it might modulate the access of endogenous
glucocorticoids to glucocorticoid receptors via 11-dehydrogenase or 11-oxo-reductase pathway. In mineralocorticoid-target tissue, it protects the non-selective mineralocorticoid receptors from glucocorticoid excess. Two species of 11iHSD exist:
one is low-afﬁnity and bidirectional (11iHSD1), the other is high-afﬁnity and
unidirectional (11iHSD2). 11iHSD1 has higher Km (vM) for its glucocorticoid
substrates, preference for NADP/NADPH as cofactors and seems to be an enzyme that produces active glucocorticoids from inactive metabolites in many
tissues [8,9]. On the other hand, 11iHSD2 is an enzyme, that has only 11i-dehydrogenase activity, has nanomolar afﬁnity for glucocorticoids and prefers NAD as
cofactor [10]. The presence of both isoforms has been demonstrated in multiple
organs including rat intestine [11,12]. The intestinal 11iHSD activity is not
distributed homogeneously along the intestine and its developmental proﬁle is not
identical in particular intestinal segments [13]. After birth 11iHSD activity is high
in caecum, proximal and distal colon and does not alter much until adulthood.
Quite contrasting developmental proﬁle is typical for ileum, where 11iHSD
activity is low during the ﬁrst two postnatal weeks and increases during weaning
period.
The aim of this study was to study cofactor dependence of 11iHSD in the
intestinal segments that express different developmental proﬁles of activity and to

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investigate whether 11iHSD plays a physiological role in the maturation of the
intestinal epithelium. Using the 11iHSD inhibitor, carbenoxolone [7], we examined
the possible role of 11iHSD in the developmental changes of sucrase, alkaline
phosphatase and Na,K-ATPase activities during weaning. These enzymes were
chosen because they are sensitive to corticosteroids and have distinct developmental
proﬁles. Sucrase and Na,K-ATPase activities increase during development in the
small and large intestine (1,14,15) whereas alkaline phosphatase decreases in ileum
[16].

2. Materials and methods

2.1. Animals
Wistar rats were bred and maintained on a 12-h light: 12 h darkness cycle and
received tap water ad libitum. The day of birth was designated day 0 and
approximately 24 h after birth the litter size was reduced to 8 pups which were kept
with the dams until they are 30 days old. To examine the effect of carbenoxolone
on sucrase, alkaline phosphatase and Na,K-ATPase activities, each litter was
divided into two groups. One group received i.p. injections of carbenoxolone (60
mg/kg body wt. day in 0.9% saline) or vehicle only (0.9% saline) from day 12 to day
23. Rats were killed by decapitation and segments of the intestine (jejunum, ileum,
colon) were removed, rinsed with ice-cold saline, opened longitudinally and used
for enzyme activity studies.

2.2. Assay of 11iHSD acti6ity
The intestinal segments were homogenized in 0.2 M sucrose (1:4 w/v) using a
Polytron homogenizer. The homogenate was centrifuged at 1000× g for 10 min and
the supernatant was assayed for protein concentration using the method of Bradford [17]. 11iHSD activity was determined by measuring the rate of conversion of
corticosterone to 11-dehydrocorticosterone in assay tubes containing 250 vl of
intestinal homogenate (1 mg of protein), 750 vl of buffer (100 mM KCl, 50 mM
TRIS-HCl, pH 9.0) and 40 vl NAD or NADP (ﬁnal concentration 400 vM). After
10 min preincubation at 37°C 1.3 vCi of [3H]corticosterone (ﬁnal concentration
14.5 nM) was added and the reaction continued 45 min. Preliminary studies
indicated that using this amount of tissue protein the rate of reaction was linear
within this time. The reaction was terminated by cooling of samples, which were
then centrifuged 20 min (3000 × g). The supernatant was loaded onto C18 reverse
phase Sep-Pak columns (Waters, Milford, MA, USA) and steroids were quantitatively (98%) eluted in 2 ml methanol. The samples were evaporated to dryness
under nitrogen at 40°C, reconstituted in 100 vl methanol, injected onto a steel
cartridge (125 ×4 mm internal diameter) packed with LiChrospher 100 RP-18 (5
vm, Merck, Darmstadt, Germany) and analyzed by high performance liquid
chromatographic system (Waters, Milford, MA, USA). The samples were separated

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using a linear methanol-water gradient from 45:55 (v/v) to 65:35 (v/v) in 15 min and
isocratic washing with 100% methanol for 10 min at a ﬂow rate 1.0 ml/min.
Column temperature was held at 45°C. The elution of 3H-labelled steroids were
monitored by on-line radioactive detection using radioisotope detector with solid
cell (Beckman Type 171, Fullerton, CA, USA). After subtraction of background
radioactivity, integrated counts within peaks were analyzed by Apex Version 3.1
software (DataApex, Prague, Czech Republic). The activity of 11iHSD was
expressed as the percentage distribution of radioactivity among the HPLC peaks of
corticosterone and 11-dehydrocorticosterone in each chromatogram.
Dependence of 11iHSD activity on pH was measured as mentioned above using
pH range of the buffer 7.0 – 9.5. Reaction was started after 10 min temperature
equilibration by adding [3H]corticosterone and continued for another 45 min.
To determine the Km of 11iHSD for corticosterone, aliquots of intestinal
homogenates (ileum 250 vg prot, colon 125 vg prot) were incubated for 10 min
(colon) or 20 min (ileum) at 37°C with 14.5 nM [3H]corticosterone and 0.0–1000
nM unlabeled corticosterone and ﬁxed concentration of NAD (400 vM). The data
were plotted according to the Lineweaver-Burk double-reciprocal linear transformation of the Michaelis-Menten equation and the least squares best ﬁt computer
program was used to calculate the Km and Vmax values.

2.3. Assay of sucrase
Sucrase was assayed in mid-jejunum and mid-ileum. For this purpose the
segments were slit lengthwise and scraped with a steel spatula to remove mucosa.
Homogenate of mucosa was prepared in 9 volumes of 154 mM KCl using a Teﬂon
homogenizer. Sucrase activity was determined in 60 mM sodium maleate buffer
(pH 6.0) with 300 mM sucrose at 37°C. The reaction was stopped by boiling and
the glucose liberated was measured with the glucose oxidase reagent. Corrections
were made for endogenous glucose in the tissue and in the substrate. Sucrase
activity was expressed as vmoles of glucose produced per hour and mg of protein.
Protein was measured by the method of Bradford [17].

2.4. Assay of Na,K-ATPase
Na,K-ATPase activity was measured in crude homogenate as the ouabain-sensitive release of inorganic phosphate from ATP [14]. Brieﬂy, the mucosa was scraped
and homogenized with a Teﬂon pestle in nine volumes of the solution containing
(mM): 30 TRIS-HCl; 250 sucrose; 5 Na2EDTA; pH 7.3. Samples of homogenate
were preincubated at 37°C for 10 min in a solution containing (in mM): 100 NaCl;
100 TRIS-HCl; 20 KCl, 5 MgCl2; pH 7.3 with or without ouabain (ﬁnal concentration 2 mM). The reaction was started by addition of ATP (ﬁnal concentration 3.2
mM) and continued for 30 min. After stopping the reaction by trichloroacetic acid,
the released inorganic phosphate was assayed. Na,K-ATPase activity was calculated
as the difference between ATPase activity without and with ouabain and expressed
as vmol of inorganic phosphate per mg of protein per hour.

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2.5. Alkaline phosphatase
Alkaline phosphatase was measured according to Murer et al. [18]. Samples of
homogenate were added to a mixture containing (in mM): 50 glycine-NaOH buffer
(pH 10.5); 2 MgSO4 and 2.5 ZnSO4. After a 10-min preincubation at 37°C the
reaction was initiated by addition of p-nitrophenyl phosphate as substrate (ﬁnal
concentration 5.5 mM). The reaction was stopped with 2 M NaOH. The activity
was expressed as vmol of released p-nitrophenol per mg of protein per hour.

2.6. Statistical analysis
The data are presented as the means9 S.E.M. Group data were compared using
Student’s t-test. Statistical signiﬁcance was set at PB 0.05.

3. Results
The developmental proﬁle of 11iHSD activity (Fig. 1) and cofactor preference
were investigated in homogenates of three intestinal segments - jejunum, ileum and
colon. Colon is the site of a high 11iHSD activity which increases only moderately
during development. In contrast, ileum of suckling rats has very low activity which
is much higher in weaning period and adulthood. The conversion of corticosterone
is absent in jejunum. At 14.5 nM corticosterone, the colonic 11iHSD has a clear
preference for NAD over NADP activity in both young and adult animals. The
enzyme used NADP to dehydrogenate corticosterone at 50% rate compared to that
observed when NAD was used as a cofactor. The rate of conversion was very low
in the absence of cofactors in all three segments. (Fig. 1). The effect of pH on the
activity of 11iHSD is shown in Fig. 2. Unlike the NAD-dependent 11iHSD from
cultured human colonic cells T84, which has a pH optimum of approximately seven
[19], the rat colonic enzyme had a pH optimum of about nine. Carbenoxolone (0.1
M) strongly inhibited 11iHSD in both young (829 9%) and adult (889 10%) rats.
The kinetic analysis of 11iHSD activity in ileum and colon revealed the presence
of high-afﬁnity 11iHSD in both intestinal segments (Table 1). Maximal capacity
(Vmax) was, however, attenuated in ileum.
To investigate whether 11iHSD could play a role in the developmental changes
of enzymes that are known to be sensitive to corticosteroids, we examined Na,KATPase and alkaline phosphatase activities in ileum and distal colon and sucrase
activity in jejunum and ileum. As carbenoxolone was demonstrated to be efﬁcient
to inhibit 11iHSD (see above), the developmental changes of these enzymes were
examined in the presence and absence of carbenoxolone in 24-day-old rats. The
reason for the study of jejunum, which has no dehydrogenase activity (Fig. 1), was
the ﬁnding of considerable 11-oxo-reductase activity in adult jejunum [20]. The data
in Tables 2 and 3 show that carbenoxolone did not change neither the activity of
alkaline phosphatase in colon and ileum nor the activity of sucrase in jejunum and
ileum. In contrast to sucrase and alkaline phosphatase, carbenoxolone administra-

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Fig. 1. 11i-hydroxysteroid dehydrogenase activity in the presence of 14.5 nM corticosterone and its
dependence on cofactors (NAD or NADP). Data are means 9S.E.M. (n =5). The activity was expressed
as conversion of corticosterone to 11-dehydrocorticosterone. Signiﬁcantly different from the data
obtained in the presence of NADP (*PB 0.05).

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Fig. 2. Effect of pH on activity of 11i-hydroxysteroid dehydrogenase in distal colon of adult rats
expressed as conversion of corticosterone to 11-dehydrocorticosterone in the presence of NAD.

tion resulted in signiﬁcant changes of distal colonic Na,K-ATPase but not of ileal
Na,K-ATPase (Table 3). As shown in Fig. 3, the sucrase and Na,K-ATPase
activities rose in intact rats during postnatal development whereas alkaline phosphatase activity declined.

4. Discussion
In the previous study we have demonstrated the different developmental proﬁle
of 11iHSD in ileum and colon and shown that corticosteroids stimulate 11iHSD
activity in immature and adult intestine [13]. This study is the ﬁrst attempt to
evaluate the possible role of 11iHSD action in maturation of the intestine. Since
Table 1
Kinetic parameters of ileal and colonic 11iHSD of young rats
Tissue

Km (nM)

Vmax (pmol/min×mg protein)

Ileum
Distal colon

599 10
819 14

66 96
8 91

Kinetic study was performed in the presence of NAD as described in Section 2, 24-day-old rats were
used. Values are means 9 S.E.M. of four separate experiments.

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Fig. 3. Developmental changes of sucrase, Na,K-ATPase and alkaline phosphatase activities in jejunum,
ileum and distal colon of intact rats during weaning. Each point is the mean9 S.E.M. (sucrase:
n = 10–17; Na,K-ATPase: n= 6–8; alkaline phosphatase: n = 8 – 10).

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Table 2
Effect of in vivo carbenoxolone administration on the activity of sucrase in jejunum and ileum
Controls
Jejunum
Ileum

Carbenoxolone

5.349 0.79 [14]
1.619 0.36 [15]

6.71 90.65 [15]
1.81 90.28 [22]

The data are mean 9 S.E.M., sucrase activity in vmol glucose/(h×mg protein), numbers of animals in
parentheses. Controls, 24-day-old rats that were i.p. treated with vehicle since day 12; carbenoxolone
treated rats received the drug in a dose 60 mg/(kg body wt. day) since day 12 of life.

glucocorticoids are essential for maturation of intestine [1] and 11iHSD regulates
the availability of glucocorticoids in target tissues and cells [21–23], 11iHSD might
play an important role in the intestinal development. In addition, 11iHSD has
been proposed to protect mineralocorticoid receptors from binding glucocorticoids
in mineralocorticoid-target tissue including distal colon [28] and immature distal
colon has signiﬁcantly increased Na + absorption due to stimulation of electrogenic
Na + transport pathways by aldosterone [5]. However, the precise physiological role
of 11iHSD during development remains speculative.
We have demonstrated that the developmental proﬁle of 11iHSD activity in
ileum is different from the proﬁle in caecum and proximal or distal colon. The ileal
developmental proﬁle was comparable with that of 11iHSD1 isoform in liver [24],
whereas the proﬁle in the large intestinal segments was very similar to the proﬁle in
kidney [25] which is the major site of 11iHSD2 biosynthesis in adult animals [26].
The present results demonstrate clear-cut difference in cofactor preference in ileum
and distal colon and indicate the existence of both isoforms in the intestine. Km for
rat liver 11iHSD1 is approximately 1 vM [9] and NAD has no signiﬁcant effect on
its activity [27]. In contrast, 11iHSD2 in colonic T84 cells has Km 11 nM and clear
preferences for NAD over NADP [19]. As we used substrate concentration 14.5
nM, our data seem to reﬂect the presence of 11iHSD2 in both ileum and distal
colon. This conclusion is supported by the kinetic analysis (Table 1) which has
demonstrated the presence of a high-afﬁnity ileal and colonic 11iHSD2 similar to
that recently reported in adult rat and human surface colonocytes [12]. The ‘mixed’
Table 3
Effect of in vivo carbenoxolone administration on the activities of alkaline phosphatase and Na,KATPase in ileum and distal colon of young rats
Enzyme

Treatment

Ileum

Distal colon

Alkaline phosphatase

Controls
Carbenoxolone
Controls
Carbenoxolone

17.1 9 3.0 [9]
17.7 94.6 [11]
6.55 9 0.69 [14]
6.22 91.05 [15]

3.69 0.6 [11]
3.890.5 [15]
4.03 90.59 [13]
5.71 9 0.42* [16]

Na,K-ATPase

Alkaline phosphatase in vmol of p-nitrophenol produced per hour and mg protein; Na,K-ATPase
activity in vmol of phosphate produced per hour and mg protein. For further details see Table 2.
*Signiﬁcantly different from control animals (PB0.05).

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cofactor preference probably reﬂects the presence of both isoforms of 11iHSD
in the intestine as was recently demonstrated in distal colon of adult rats [12].
According to these ﬁndings 11iHSD2 is localized predominantly in the surface
colonocytes which are responsible for Na + transport and are target cells for
mineralocorticoid action, whereas 11iHSD1 is expressed predominantly in
subepithelial cells. The question which is not yet resolved is the reversibility of
colonic 11iHSD1 isoform. Whorwood et al. [12] observed in isolated colonic
subepithelial cells 11-oxo-reductase activity in the presence of 500 nM 11-dehydrocorticosterone but we have not seen any 11-oxo-reductase activity in slices of
ileum and colon in the presence of 1.45 vM 11-dehydrocorticosterone [13,14]. In
contrast, Marhefka et al. [20] detected 11-oxo-reductase activity in slices of duodenum, jejunum and ileum of adult rats. At present it is no explanation for
these differences.
The results discussed above support the view that intestinal isoforms of
11iHSD possess dehydrogenase activity and perhaps also oxo-reductase activity,
i.e. they can increase or decrease glucocorticoid exposure to the glucocorticoid
and mineralocorticoid receptors. However, the physiological signiﬁcance of these
processes remains to be fully deﬁned. Postnatal maturation of the rodent intestine is characterized by signiﬁcant modiﬁcations of enzyme status during the
weaning period when it is modulated by corticosteroids [1,2]. To test the functional role of 11iHSD in developing intestine, we have investigated the effect of
11iHSD inhibitor carbenoxolone on the activity of three enzymes with large
developmental changes during the weaning period which are sensitive to corticosteroids. These enzymes were sucrase, the activity of which substantially increases
during weaning in jejunum and ileum [1]; alkaline phosphatase, whose activity
decreases in ileum during weaning [16] and Na,K-ATPase whose activity roses
postnatally in colon [14] and in ileum [15]. Even if sucrase and alkaline phosphatase activities are increased by glucocorticoids [1,29], we have not observed
any signiﬁcant changes of their activity in carbenoxolone-treated animals. In
contrast, carbenoxolone administration signiﬁcantly increased colonic but not
ileal Na,K-ATPase. This is in agreement with the ﬁndings that, in immature rats,
colonic Na,K-ATPase is increased by mineralocorticoids [31] and glucocorticoids
[30] but not with the data of Zemelman et al. [15] who demonstrated the stimulatory effect of glucocorticoids on Na,K-ATPase in immature ileum. Similar
effect of carbenoxolone on Na,K-ATPase was also observed in adult kidney
[28,32]. The absence of the effect of carbenoxolone in ileum indicates that
11iHSD has a protective effect on Na,K-ATPase only in mineralocorticoid-sensitive tissue. The activity of 11iHSD in colonocytes of the immature rats may
protect these cells from glucocorticoids and support the mineralocorticoid regulation of Na + transport via Na + channels and Na,K-ATPase [5,31]. The developmental increase of colonic Na,K-ATPase seems to be mineralocorticoid-and
11iHSD-dependent whereas the glucocorticoid-dependent development of intestine does not seem to be inﬂuenced by this enzyme.

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Acknowledgements
This study was supported by Grants No. 305/94/1719 and No. 306/96/1289 from
the Grant Agency of the Czech Republic. The authors thank Dr. J. Zicha (Institute
of Physiology, Czech Acad. Sci., Prague) for reading the manuscript and valuable
advice. The technical assistance of Mrs. R. Somolova is greatly appreciated.
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