The research activities of the
laboratory lie in the biology of epithelial cells with particular
emphasis on the transport mechanisms of the intestinal epithelium. The
intestinal epithelium, which is in nocstant and rapid renewal,
represents an attractive system for the study of mechanisms involved in
the determination of the cell state. Within its fundamental unit, the
cryptvillus axis, are two main distinct populations of cells: the
proliferating and poorly differentiated cells localised in the crypts,
and the mature enterocytes in the villi of the small intestine or in
the intestinal surface of the large intestine. Due to the presence of
undifferentiated cells and enterocytes of different stages of
maturation the digestive and transport properties of the epithelium are
not distributed homogeneously along the cryptvillus axis (vertical
gradient). Similarly, heterogeneous distribution of the digestive and
transport functions exists also along the intestine from the duodenum to
the rectum (horizontal gradient). It is known that the regulation of
epithelial cell growth and digestive and transport functions is
susceptible to various influences both along the vertical and the
horizontal axes. The regulation of these gradients is a subject of many
integrative mechanisms involving hormone, nervous, immune and dietary
changes. In principal there are two patterns of this regulation specific or nonspecific reversible regulation and
irreversible mostly
developmental regulation reflecting the three major changes in
functional demands of the gut: (1) the gut is substituted for the
placenta, (2) weaning is associated with major qualitative changes in
the diet and (3) the growing organism requires quantitatively more
nutrients and ions than in adulthood. Therefore, many of the digestive
and transport functions undergo profound changes during maturation,
which have important implications for the regulation of fluid and
electrolyte
balance in infants.
The experimental activities in the laboratory
focus on three
research projects. First, the development of cellular mechanisms of
absorptive and secretory pathways and maturation of their signalling
systems. Second, understanding the cellular and molecular mechanisms by
which corticosteroid hormones regulate intestinal functions. The third
research program is directed at understanding the complex interactions
that exist between the structural and functional differentiation of the
renal tubular epithelium using chick mesonephros as a model of the
functional
embryonic kidney. The goal of these three programs is to understand at
the cellular and molecular level how solutes move across epithelial
cell membranes and how this movement is regulated to suit the needs of
the organism during development.
Our studies led to the conclusion that ion and water transport
undergoes marked agedependent changes during the suckling and weaning
period and that sucklings and weanlings are in relative lack of NaCl in
the developing body. The studies have established heterogeneity of
colonic sodium and potassium transport relative to developmental and
spatial distribution. We have shown that increased colonic sodium
absorption of the immature intestine reflects the presence of
electrogenic amiloridesensitive sodium transport via epithelial sodium
channels localized in the apical membrane of colonic enterocytes. The
increased sodium absorption is associated in immature intestine with
increased potassium secretion and both transport pathways decrease or
disappear after weaning. The increased colonic sodium absorption is
accompanied by an enlargement of the enterocyte membrane surfaces. The
main regulatory signal for these developmental changes is aldosterone,
but thyroid hormones play a distinct permissive
role in this phenomenon. Hypothyroidism blocks the stimulatory effect
of aldosterone and replacement therapy of hypothyroidism with
triiodothyronine restores the effect of aldosterone. The target of
thyroid hormones is the apical sodium channel and not basolateral sodium
pump (Na,K-ATPase). In addition, we have established that the immature
intestine is more sensitive to corticosteroids than the adult intestine.
This phenomenon seems to be associated with increased plasma
concentration of thyroid hormones in sucklings and weanlings. At the
molecular level we have further analysed the permissive role of thyroid
hormones in aldosterone effects and showed that this effect is
localized beyond the transcriptional step of sodium channel regulation.
As mentioned above the effect of aldosterone in the colon is associated
with increased potassium secretion and further studies have established
the mechanism by which potasium ions are transported across the apical
and basolateral membrane. Potassium ions penetrate into the cell across
the basolateral membrane through furosemidesensitive Na/Cl/K
cotransporter and exit the enterocytes through apical barium-sensitive
potassium channels.
We found that corticosteroid effects in the developing intestine
correlate with dramatic changes in the activity of an enzyme, 11ß-hydroxysteroid
dehydrogenase (HSD). This enzyme metabolises glucocorticoids to
biologically inactive forms and thereby allows aldosterone and
glucocorticoids to occupy their respective intracellular receptors.
Whereas the activity of ileal 11HSD is low during the suckling period
and rises severalfold during weaning, the activity of colonic 11HSD is
high in the large intestine since birth. This discrepancy in the
developmental pattern supports (1) the specificity of aldosterone
effect and efficiency of mineralocorticoid target tissue (large
intestine) since birth and (2) facilitates the maturation effect of
glucocorticoids
in the small intestine during early postnatal life. The detailed
studies of prereceptor modulation of corticosteroid signals in the
intestine established not only marked developmental changes but also
modifications of 11HSD in some pathological states such as hypertension.
In addition, we have been able to demonstrate species differences -
20-hydroxysteroid
dehydrogenase instead of 11HSD is involved in prereceptor modulation of
corticosteroid signals in the avian intestine.
The embryological studies characterized the effect of some chemicals
and xenobiotics on nephron growth, maturation and induction of anomalies
in the renal tubular morphology (cystic dilatation). We showed that
cystic dilatation is accompanied by changes in the amount of some
structural proteins and by defect in the enzyme supply of the dilated
tubules.
Primary emphasis, at the present time, is to define the
developmental changes in establishing the gradient of transport
functions along the cryptvillus axis in immature intestine. By
electroctro- physiological techniques (patch-clamp, Ussing chambers)
and dual-wavelength fluorescence method (pH and Ca2+ intracellular
indicators) coupled with digital imaging technique we are able to
monitor the distribution of transport proteins along the cryptvillus
axis and to study their regulation. Most recently we applied RT-PCR to
study the regulation of transport proteins at the transcriptional level.
The work of the laboratory has been supported by grants from the
Grant Agency of the Czech Republic, Academy of Sciences and Charles
University.
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