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Ontogenic appearance of three fatty acid binding proteins in the rat stomach.

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THE ANATOMICAL RECORD 229:51-60 (1991)
Ontogenic Appearance of Three Fatty Acid
Binding Proteins in the Rat Stomach
SHOICHI ISEKI, TATSUO KANDA, MASAHIRO HITOMI, A N D TERUO O N 0
Department of Anatomy, Kanazawa University School of Medicine, Kanazawa (S.I.1, and
Department of Biochemistry, Niigata Universit.y School o f Medicine,
Niigata (T.K., M.H., T.O.),Japan
ABSTRACT
With the use of specific antibodies against three structurally different fatty acid binding proteins (FABPs),viz, liver FABP (L-FABP),heart FABP
(H-FABP), and intestinal FABP (I-FABP),the localization and relative amount of
the immunoreactive proteins were determined by immunoblotting and immunocytochemistry in the gastric epithelium of rats during prenatal and postnatal
development. H-FABP immunoreactivity was first detected at embryonic day 20
(E201, with predominant localization in the parietal cells, whereas I-FABP immunoreactivity was detected at the day of birth in the surface mucous cells. Both
immunoreactivities were continuously localized in the same cell types with increasing intensity into adulthood. In contrast, the immunoreactivity for L-FABP
showed remarkable changes in intensity and localization during development of
t h e rat stomach. It was first detected in the surface mucous cells of E19. In the first
2 weeks of postnatal life, i.e., the suckling period, L-FABP immunoreactivity
reached a peak in intensity and was localized not only in the surface mucous cells,
but also in some of the parietal cells, brush cells, and endocrine D cells. In the
following few weeks of weaning, the reactivity of surface mucous cells and parietal
cells disappeared, leaving only a small amount of total L-FABP immunoreactivity
in the adult stomach, which was localized exclusively in the brush cells and D cells.
These results revealed t h a t the appearance of the three types of FABPs in the r a t
stomach is specific to cell types and developmental stages.
Fatty acid binding proteins (FABPs) represent a
class of low-molecular-weight cytosolic proteins
present in a variety of mammalian tissues and having
high binding affinity for long-chain fatty acids (Ockner
e t al., 1972; Glatz and Veerkamp, 1985; Sweetser e t al.,
1987). In the rat, three structurally different FABPs
have been identified, isolated, and named liver FABP
(L-FABP; Ockner e t al., 1982; Takahashi e t al., 1983),
heart FABP (H-FABP; Fournier e t al., 1978; Said and
Schultz, 1984), and intestinal FABP (I-FABP; Alpers e t
al., 1984). These proteins may function in t h e uptake,
intracellular transport, and metabolism of free fatty
acids and their acyl-CoA esters. The occurrence of the
three FABPs in rat tissues has been studied biochemically in terms of the tissue-specific expression of the
mRNAs (Gordon et al., 1985; Heuckeroth e t al., 1987)
and of the proteins (Bass and Manning, 1986; Paulussen e t al., 1989). According to those studies, L-FABP is
abundantly expressed in the liver and intestine, HFABP in the heart and muscle, and I-FABP in the intestine. It should be noticed that the stomach is one of
the few organs which express all three types of FABPs
to some extent, and therefore is suitable to investigate
the specific roles of different FABPs in different cell
populations of the same organ. However, immunohistochemical evidence for the localization of FABPs in
particular cell types of the gastric epithelium is limited. Studies using adult rat stomachs have described
the localization of L-FABP in brush cells and D cells
c
1991 WILEY-LISS. INC
(Iseki and Kondo, 1989, 1990) and of H-FABP in parietal cells (Kanda e t al., 1989). In the present study, we
have utilized specific antibodies to L-, H-, and I-FABPs
to elucidate the ontogenic changes in the relative
amount and localization of the three FABPs in the rat
stomach.
MATERIALS AND METHODS
Antibodies
L-, H-, and I-FABPs were purified from rat materials, antisera against them were raised in rabbits, and
the IgG fractions were purified as described previously
(Takahashi e t al., 1983; Iseki e t al., 1989; Kanda e t al.,
1989, 1990). Anti-L-FABP antibody was further puritied by affinity chromatography using a n antigen column prepared with Sephadex 4B and L-FABP. For negative control experiments, antibodies were absorbed
overnight by 100 Lgiml of corresponding FABPs prior
to use. For anti-L-FABP antibody, the pass-through
fraction of the affinity chromatography was also used
as a control.
Received February 6. 1990: accepted May 17, 1990.
Address reprint requests to Dr. S. Iseki, Dept. Anatomy, Kanazawa
University School of Medicine, Kanazawa. Ishikawa 920, J a p a n .
52
S. ISEKI ET AL.
Animals
Male Wistar rats were used in the present study except for the homogenization of fetal rat stomachs,
which was performed without identification of the sex
of animals. Rats of the following ages were used: embryonic day 18 (E18), E19, E20, E21, postnatal day 0
(newborn, about 8 hours after birth), 3,7,10,14,17,21,
24,28,35,42, and 56 (adult).The day of the appearance
of spermatozoa in the vaginal smear was counted a s
EO. The animals were grown with free access to mother’s milk and chow diet in the first 3 postnatal weeks.
At 21 days of age, they were separated from their mothers and subsequently grown on standard chow and water. The animals were sacrificed in the morning either
by decapitation (prenatal animals) or by transcardial
perfusion with cold physiological saline under Nembutal anesthesia (postnatal animals).
lrnrnunochernical Studies
Immediately after the sacrifice of animals, whole
stomachs were removed, cleared of contents by cold
physiological saline, and homogenized in ice-cold 10
mM potassium phosphate, pH 7.4, containing 150 mM
KCl. At least ten stomachs were combined to obtain the
fetal materials. The homogenates were centrifuged for
105,OOOgto yield a cytosolic protein preparation. In the
adult rats, cytosols of the liver, jejunum, and heart
were also prepared.
The cytosolic protein samples (5 pg) were separated
by electrophoresis in 15% (W/V) acrylamide slab-gels
at pH 8.8 in the presence of 0.1% (WIV) SDS (Laemmli,
1970). The proteins were then transferred electrophoretically to nitrocellulose membranes (Schleicher &
Schuell) according to Towbin et al. (1979). After blotting, the membranes were incubated for 2 hours in
phosphate-buffered saline (PBS) (10 mM sodium phosphate, pH 7.4, 150 mM NaCl) containing 5% (WIV)
bovine serum albumin (BSA). Blots were then incubated for 2 hours at room temperature with the antibodies against FABPs diluted in buffer A (0.05% VIV
Tween 20 and 1% BSA in PBS). After washing with
buffer B (0.05% Tween 20 in PBS) for 30 minutes, the
blots were treated for 1hour with biotinylated antirabbit IgG (Dako) diluted at 1:200 in buffer A. They were
then washed again with buffer B and subsequently incubated for 1hour with streptavidin-conjugated horseradish peroxidase (Dako) diluted at 1:600 in buffer A.
After washing with buffer B, the immunoreaction was
visualized by incubation of the membranes with 0.1
mg/ml of diaminobenzidine tetrahydrochloride and
0.01% (VIV) H 2 0 2in 50 mM Tris-HC1, pH 7.6.
lrnrnunocytochernical Studies
For preparing the histological specimens, postnatal
animals were perfused transcardially first with physiological saline and then with 4% (WIV) paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The stomach
specimens were excised and immersed in the same fixative for a n additional 4 hours. The stomach specimens
from the decapitated prenatal animals were also fixed
by immersion in the same fixative. Three animals of
each age were used for histology. After a rinse with
phosphate buffer, all tissue blocks were immersed overnight in 30% (WIV) buffered sucrose at 4°C.
For light microscopic immunohistochemistry, thick
(15 pm) frozen sections were cut by a cryostat and
mounted on gelatin-coated glass slides. They were incubated with the antibodies against FABPs diluted
with PBS overnight a t room temperature. The sites of
the immunoreactions were visualized by the peroxidase-antiperoxidase (PAP) method (Sternberger,
1974). Details of the immunohistochemical procedures
have been described previously (Iseki et al., 1989).
For electron microscopic immunocytochemistry, the
cryostat sections immunostained by the PAP method
were postfixed in 0.5% (WIV) Os04 in 0.1 M phosphate
buffer for 15 minutes and stained e n bloc with 1% (WI
V) uranyl acetate for 20 minutes. They were then dehydrated in a n alcohol series and embedded in Epon
812 (Serva) to obtain ultrathin (0.1 pm) sections for
electron microscopy.
RESULTS
lrnrnunochernical Analysis of Cytosols
The specificity of the antibodies raised against the
three FABPs was examined by Western blot analysis of
cytosols from various tissues of adult (56 day) rats. As
shown in Figure 1, reactivity with anti-L-FABP antibody was prominent in the liver and jejunum in the 14
kD region, whereas it was negligible in the heart and
stomach. Anti-HI-FABP antibody formed a clear protein band of 15 kD in the stomach a s well as in the
heart. Reactivity with anti-I-FABP antibody in the 15
kD region was observed primarily in the jejunum and
also in the stomach at a much lower intensity. These
results confirmed the FABP-type specificity of the
three antibodies used in the present study and also
were consistent with the previous biochemical result
that the major type of FABP in the adult rat stomach is
H-FABP (Kanda et al., 1989).
Next, whole stomachs of rats from various stages of
development were homogenized and the cytosolic fraction was analyzed by Western blots using the three
antibodies (Fig. 2). The reactivity of the 14 kD protein
band to anti-L-FABP antibody, which was scarcely detectable a t E18, showed a marked increase in intensity
after birth, reaching a peak at 7 days, and remained
high in the first 2 weeks of postnatal life. It then declined substantively in intensity at 21-28 days and became scarcely detectable at 42-56 days after birth. It
was noted that the reactivity was transiently stronger
a t 28 days than a t 21 days. In contrast, reactivity to
anti-H-FABP in the 15 kD region was continuously
present from birth to adulthood and increased substantially in intensity between 14 and 21 days. I n the immunoblot using anti-I-FABP antibody, the intensity of
reaction in the 15 kD region increased gradually until
21 days after birth and remained constant thereafter.
lrnrnunocytochernical Localization of Three FABPs in the
Developing Stomach
The immunoreactivity for L-FABP was first detected
in the gastric mucosa of E19, with localization to the
superficial epithelium (Fig. 3a). The immunopositive
epithelial cells were arranged with the negative cells
in a mosaic manner. Immunoelectron microscopy of the
L-FABP-reactive cells identified them a s the surface
mucous cells by their apical cytoplasm containing mucous globules (Fig. 3b). Although the immunoreactive
-
H-FABP
L FABP
Mr
(kD)
53
FABPs I N DEVELOPING RAT STOMACH
I =FABP
4329-
L
J
H
S
L
Fig. 1. Western blots of cytosolic proteins from various organs
shown by immunostaining for FABPs. Samples of total cytosolic proteins (5 pgilane) from the liver (L), jejunum (J),heart (HI, and stomach (S)of adult rats were subjected to SDS/polyacrylamide-gel elec-
Ageldaysl
El8
PO
3
7
J
H
S
L
J
H
S
trophoresis followed by electrophoretic transfer to nitrocellulose. The
blots were immunostained with the antibodies against L-, H-, and
I-FABPs.
10
14
21
28
42
56
L- FABP
-
H FABP
I -FABP
Fig. 2. Western blots of cytosolic proteins from the stomachs in
various developmental stages shown by immunostaining for FABPs.
Samples of total stomach cytosolic proteins (5 pgilane) from rats a t
ages of E l 8 to 56 days were electrophoresed, blotted, and immunostained for L-, H-, and I-FABPs. The immunopositive protein bands of
14 kD (L-FABP) and 15 kD (H- and I-FABPs) are shown.
material was distributed throughout the cytoplasm, a
circumscribed intense reaction was noted around the
empty spaces representing the glycogen areas, which
are abundant in this cell type a t prenatal stages (Helander, 1969b). No immunoreaction was observed in
the parietal cells, mucous neckichief cells.
Approximately 8 hours after birth, L-FABP-immunoreactive cells still constituted only a part of the total
surface mucous cell population in both the fundic and
pyloric regions (Fig. 4a). The glycogen areas were no
longer present, but instead large lipid droplets were
noted in the cytoplasm (Fig. 4b). No circumscribed enrichment of the immunoreaction was observed around
the lipid droplets of reactive cells. Occasionally, solitary L-FABP-immunoreactive cells with a quite different figure from that of surface mucous cells were encountered in the superficial epithelium of the newborn
gastric mucosa (Fig. 5a). These cells were smaller than
the surface mucous cells and had a barrel-like shape
with a constricted apical portion protruding to the gastric lumen. In appropriate sections, a thin cytoplasmic
process was seen extending from the basal portion of
the cell toward the basement membrane. In immunoelectron micrographs, no mucous globules were found
in the apical cytoplasm of these cells (Fig. 5b). Instead,
they had closely packed large microvilli, approximately 0.7 pm long, straight microfilaments extending
from the cores of the microvilli, and numerous round
vesicles 50-300 nm in diameter in the supranuclear
cytoplasm (Fig. 5c). By such apical features, these cells
were identified as the brush cells (Isomaki, 1973;
Nabeyama and Leblond, 1974; Iseki and Kondo, 1989).
The immunoreactive material appeared diffusely in
the cytoplasmic matrix of the brush cells without association with subcellular structures. A careful observation of the newborn gastric epithelium failed to reveal
any brush cells that were immunonegative for LFABP.
54
S.ISEKI ET AL
FABPs I N DEVELOPING RAT STOMACH
Through 3 to 14 days after birth, i.e., in the suckling
period, the immunoreactivity for L-FABP was observed
in nearly all superficial epithelial cells and also in the
cells located in the gastric pits and upper portions of
the gastric and pyloric glands (Fig. 6a). In immunoelectron microscopy, all surface mucous cells, including relatively immature ones in the gastric pits, a s well a s the
parietal cells located in the pits and upper portions of
the gastric glands were immunoreactive (Fig. 6b). The
mucous neck cells and chief cells, which were indistinguishable in the early postnatal days, were immunonegative for L-FABP. The reactive brush cells were
found only occasionally throughout the first 2 postnatal weeks. In addition, starting around 7 days after
birth, another type of immunoreactive cell became evident in the lower portions of the gastric and pyloric
glands (Fig. 7a). By immunoelectron microscopy, they
were identified a s the endocrine D cells by their characteristic basal granules, 200-300 nm in diameter,
with limiting membranes very closely applied to moderately electron-dense cores (Figs. 7b, c; Alumets et al.,
1977). Empty spaces seemingly representing lipid
droplets were often found in this cell type during the
suckling period. By a careful survey of the gastric epithelium by immunoelectron microscopy, the D cells
without immunoreaction for L-FABP were also found.
The immunoreaction product appeared evenly in the
entire cytoplasmic matrix of reactive D cells without
association with subcellular structures, although it
could not be determined conclusively if the cores of the
endocrine granules were free of the reaction.
From 17 to 28 days after birth, i.e., in the weaning
period, there was a progressive decrease in the intensity of L-FABP-immunoreactivity in the surface mucous cells, leading eventually to no immunostaining of
55
this cell type by 28 days (Figs. 8-11). The reactivity of
the parietal cells located in the gastric pits also decreased progressively in this period. However, the parietal cells located in the neck region of the gastric
glands showed a transient rise in the reactivity to antiL-FABP antibody a t 24-28 days (Fig. lo), probably
accounting for the quantitative result of the immunoblot analysis (Fig. 2). A remarkable rise in the number
of immunoreactive brush cells was noted after 17 days,
reaching the adult value (approximately a cell in every
five gastric pits; Iseki and Kondo, 1989) by 28 days
after birth. A group of immunopositive brush cells in
the distal wall of the groove between the forestomach
and glandular stomach (gastric groove) became apparent around 24 days (Fig. 9). The number of immunoreactive D cells also increased progressively in this period. After 28 days, the immunoreactivity in the neck
parietal cells disappeared rapidly. As a consequence, in
the mature stomach of 42-56 days postpartum, the reactivity to anti-L-FABP-antibody was found exclusively in the brush cells located in the superficial epithelium and gastric pits and in the endocrine D cells
located in the gastric glands (Fig. 11).
Reactivity to anti-H-FABP antibody was first detected in the stomach of E20 (Fig. 12a) and localized
exclusively in the parietal cells, which were so recognized by the presence of intracellular secretory canaliculi (Fig. 12b; Helander, 1969a). In this stage, there was
a marked heterogeneity in the parietal cell population
in terms of the intensity of immunoreaction. Within
the first postnatal week, all parietal cells in the stomach became intensely immunoreactive for H-FABP and
remained so to adulthood (Fig. 13). No reactivity to
anti-H-FABP antibody was detected in any cell types
other than the parietal cells. The immunoreaction
product was distributed evenly throughout the cytoplasmic matrix of the parietal cells, whereas interior of
the intracellular canaliculi, mitochondria, endoplasmic
reticulum, and Golgi apparatus was free of the reaction
Fig. 3.a: Light micrograph of the gastric mucosa of E l 9 treated by (Fig. 12c). Furthermore, no regional enrichment of the
immunohistochemistry for L-FABP. Immunoreactive epithelial cells immunoreactivity was discerned in the cytoplasmic
are seen in a mosaic pattern. x 300. b: Immunoelectron micrograph of
portion adjacent to any of those subcellular structures.
the El9 gastric mucosa. A surface mucous cell intensely immunoreThe reactivity to anti-I-FABP antibody was first deactive for L-FABP is shown. The cytoplasmic immunoreactivity is
enriched in intensity around the glycogen area (Gy). Arrows indicate tected in the newborn gastric mucosa (Fig. 14a) and
localized exclusively in the surface mucous cells (Fig.
the mucous granules. x 13,700.
14b). The immunopositive cells were intermingled with
Fig. 4.a: Light micrograph of the newborn gastric mucosa immu- the negative cells a s in the case of L-FABP. This mosaic
nostained for L-FABP. Immunoreactive cells occur in the surface
arrangement of I-FABP-immunoreactive cells was reepithelium in a mosaic pattern. M, muscularis mucosae. ~ 3 0 0b.
Immunoelectron micrograph of the newborn gastric mucosa. L-FABP- placed after 3 days by eventually all the surface muimmunoreactive surface mucous cells are shown. The reaction product
cous cells lining the luminal surface of fundic and pyis distributed evenly in the entire cytoplasm. L, lipid droplets.
loric
mucosa reactive to the antibody. These cells were
x 5,500.
continuously immunoreactive into adulthood (Fig. 15).
Fig. 5. a: Light micrograph of the newborn gastric mucosa immu- However, the relatively immature cells of the same
nostained for L-FABP showing an immunoreactive brush cell (arrow). type, located in the deep regions of the gastric pits,
The basal cell process of the brush cell is not contained in this parremained immunonegative for I-FABP. The immunoticular section. x 600. b: An immunoelectron micrograph showing a
L-FABP-immunoreactivebrush cell found in the newborn gastric mu- reactive material was distributed evenly throughout
cosa. Note large microvilli (Mv)protruding to the gastric lumen and
the cytoplasmic matrix of the reactive surface mucous
the absence in apical cytoplasm of the mucous granules characteristic
cells.
of the surface mucous cells. An empty space (asterisk) presumably
Incubation of the sections with the three control anrepresents a lipid droplet. In this particular section, the immunoreaction appears stronger in the apical and lower lateral cytoplasm, tibodies preabsorbed by corresponding types of FABPs
brought about no immunostaining of any portion of the
probably due to uneven penetration of the antibody. ~ 9 , 8 0 0 c:
.
Higher magnification of the apical portion of the brush cell shown in
stomach, confirming that the observed immunoreacb. Note large microvilli (Mv), extension of the core microfilaments
tions were specific to the three FABPs. Table 1 sum(Mf) deep into the cytoplasm, bundles of intermediate filaments (IF),
and numerous vesicles (V). D, desmosomes; Mi, mitochondrion. marized the occurrence of the three FABPs in various
cell types in the developing stomach described above.
x 21,000. Scale bar = 500 nm.
56
S. ISEKI ET AL.
Fig. 6. a: Light micrograph of the 7 day gastric mucosa immunostained for L-FABP. Immunoreactivity covers the entire surface epithelium and also extends to the upper portions of the gastric glands.
M, muscularis mucosa. x 300. b Immunoelectron micrograph of the 7
day gastric mucosa. Not only surface mucous cells (SM) but also the
parietal cells located in the upper portions of gastric glands (arrows),
are immunoreactive for L-FABP. X 1,700.
Fig. 7. a: A light micrograph of the 7 day gastric mucosa immunostained for L-FABP, showing an immunoreactive D cell (arrow)in the
DISCUSSION
In the suckling rat stomach, considerable hydrolysis
of milk fat by lingual lipase is known to take place
(Hamosh, 1979) at a pH optimum around 5.0, followed
by absorption of fatty acids by the surface mucous cells
(Helander and Olivecrona, 1970). The absorbed fatty
acids are stored in large lipid droplets, which are not
associated with limiting membranes derived from endoplasmic reticulum or Golgi apparatus, unlike the absorptive epithelial cells of the small intestine. The
present immunochemical and immunocytochemical evidence for the occurrence of L-FABP in the surface mucous cells during the suckling period and its disappear-
lower portion of gastric gland. x 600. b: An immunoelectron micrograph of the 7 day gastric mucosa. A L-FABP-immunoreactive D cell
found in the lower portion of gastric gland is shown. Note numerous
round endocrine granules in the cytoplasm apposing the basement
membrane (BM).Empty spaces (asterisks)presumably represent lipid
droplets. The immunoreaction product occurs evenly throughout the
cytoplasm. Chief cells ( C ) are immunonegative. x 8,300. c: Higher
magnification of the basal portion of the D cell shown in b. Note the
endocrine granules with limiting membrane very closely applied to
the core. x 22,000. Scale bar = 500 nm.
ance after weaning suggests that L-FABP is somehow
involved in the absorption of milk fat by the gastric
epithelium of suckling rats. On the other hand, I-FABP
is also demonstrated immunohistochemically in the
surface mucous cells throughout the postnatal development of rats, without relation to suckling and weaning.
The amount of I-FABP in the gastric epithelium is
probably very low, judging from the relative intensity
of immunoreactivity in the immunoblot. The physiological significance of I-FABP in the surface mucous
cells remains unclear.
The parietal cells are first recognized in the gastric
mucosa of the rat at E19-E20 both morphologically
FABPs I N DEVELOPING RAT STOMACH
57
Fig. 8. Light micrograph of the 21 day gastric mucosa immunostained for L-FABP. Both the number of immunoreactive surface mucous cells and the intensity of the reactivity are decreased compared
with the 7 day gastric mucosa. Note the strongly immunoreactive
brush cells (arrowheads). x 300.
Fig. 10.A light micrograph of the 24 day gastric mucosa immunostained for L-FABP. Intense immunoreactivity is no longer present in
the surface mucous cells. Brush cells (B) in the gastric pits and D cells
(D) in the gastric glands are immunoreactive. Also note that many
parietal cells (P) located in the neck region of gastric glands are intensely to moderately reactive for L-FABP. x 300.
Fig. 9. A light micrograph of the 24 day gastric mucosa immunostained for L-FABP. The junctional region between the forestomach
(F) and the glandular stomach is shown. Note a cluster of immunoreactive brush cells in the distal wall of the gastric groove ( G ) .
x 300.
Fig. 11. Light micrograph of the 56 day gastric mucosa immunostained for L-FABP. Immunoreactivity is no longer present in the
surface mucous cells or neck parietal cells and is exclusively localized
to the brush cells (B) and D cells (D). x 300.
and by their carbonic anhydrase activity (Helander,
1969a). They differentiate progressively after birth a s
shown by the continuous decline of the pH of gastric
contents. The present results demonstrate that HFABP occurs in the parietal cells from very early
stages of their development and is continuously expressed in this cell type. Thus, H-FABP is a good histochemical marker of parietal cells. The relative
amount of H-FABP, a s shown by the immunoblot analysis, appears to increase in coincidence with the weaning of animals.
In the context of the functional role of H-FABP, past
studies have demonstrated that H-FABP of the rat is
widely expressed in the energy-consuming organs such
as the heart, muscle and kidney (Bass and Manning,
1986; Paulussen et al., 1989). It is generally believed
that H-FABP acts in these tissues as a n intracellular
carrier of fatty acids to mitochondria for 6-oxidization
(Fournier et al., 1978; Glatz and Veerkamp, 1985). Because the parietal cells of the stomach probably undergo active energy consumption, a s suggested by the
presence of numerous mitochondria in their cytoplasm,
it is plausible that H-FABP is involved in this cell type
in the utilization of plasma-free fatty acids as energy
sources. On the other hand, the present study also reveals L-FABP to be expressed in a subpopulation of the
58
S.ISEKI ET AL.
Figs. 12-1 5
59
FABPs IN DEVELOPING RAT STOMACH
TABLE 1. Ontogenic appearance of the immunoreactivity for three FABPs in the rat stomach
FABP
type
L
H
I
Cell type
Surface
mucous cell
Parietal cell
Brush cell
D cell
Parietal cell
Surface
mucous cell
El8
19
20
PO
7
-'
+-
+
++
+
+
+
++
+
~
-
-
-
-
++-
-
-
+
+
-
-
-
+
-
~
Age (days)
14
21
24
28
42
56
-
-
++
+
+
+
-
-
++
+
+
++
+
++
+
++
+
++
+
++
+
++
+
+
+
+
'The intensity of the immunoreactivity and the number of immunoreactive cells are synthetically expressed a s
parietal cells throughout the suckling period followed
by its transient increase after weaning. The functional
significance of L-FABP in the parietal cells remains
unclear.
In addition to the two cell types described previously,
the present study reveals two minor cell types in the
gastric epithelium to express L-FABP. The D cells,
identified by their characteristic endocrine granules
that contain somatostatin, are known to appear in a
limited number in the rat gastric epithelium a few days
after birth and to increase in number linearly from 10
days to 3 weeks (Alumets et al., 1977). The present
results suggest that D cells express L-FABP from the
early stages of their ontogenic development. However,
it should also be noted that only a subpopulation, about
two thirds in the adult case (Iseki and Kondo, 19901, of
the total D cell population in the stomach are immunoreactive for L-FABP throughout development. The
brush cells, also termed tuft cells, fibrillovesicular cells,
or caveolated cells, represent a rarer but distinct cell
population found in the endoderm-derived mucosal epithelia including the gastric epithelium (Isomaki,
1973; Nabeyama and Leblond, 1974; Iseki and Kondo,
~
-
-
++
++
+
++
+
++
+
+
to + t
1989). The nature of this cell type is largely unknown,
and very little information is available on its ontogeny.
The present work reveals for the first time that the
brush cells appear in the rat gastric mucosa as early a s
the day of birth, with intense immunoreactivity for LFABP. Unlike D cells, all brush cells in the stomach
seem to be immunoreactive. Based on this selective
reactivity, it is concluded that the frequency of brush
cells remains low in the rat gastric epithelium during
the first 2 weeks of postnatal development but increases remarkably in the following 2 weeks. This is
coincident with the end of the suckling period and the
beginning of the weaning period. In the adult rat stomach, L-FABP, although low in amount in the whole
stomach homogenate, is exclusively expressed in D
cells and brush cells, as reported previously (Iseki and
Kondo, 1990). The occurrence of L-FABP in these minor cell types, which are generally considered not to be
involved in active fat absorption, implies further aspects of the role played by L-FABP. The present study
indicates that structurally different FABPs are expressed in the rat stomach, depending on the cell types
and developmental stages.
ACKNOWLEDGMENTS
Fig. 12 a: Light micrograph of the E20 gastric mucosa immunostained for H-FABP. Immunoreactive cells are mainly found in the
developing gland epithelium. M, muscularis mucosa. x 300. b Immunoelectron micrograph of the E20 gastric mucosa. Two parietal cells
identified by t h e intracellular secretory canaliculi (asterisks) a r e
shown, one with intense and the other with weaker immunoreactivity
for H-FABP. A surface mucous cell (SM) is immunonegative. x 6,100.
c: Higher magnification of the intensely H-FABP-immunoreactive
parietal cell shown in b. Note that the immunoreaction product is
distributed evenly in the cytoplasmic matrix, but the interior of the
membranous organelles, such a s intracellular canaliculi (IC), mitochondria (Mi), endoplasmic reticulum (ER) and Golgi apparatus (G),
is free of the reaction. x 17,000.
Fig. 13. A light micrograph of the 56 day gastric mucosa immunostained for H-FABP. Parietal cells in the entire thickness of the mucosa a r e intensely immunoreactive. M, muscularis mucosae. x 150.
Fig. 14. a: A light micrograph of the newborn gastric mucosa immunostained for I-FABP. Immunoreactive cells a r e found in the surface epithelium. x 300. b Immunoelectron micrograph of the newborn gastric mucosa. Surface mucous cells with various intensities of
I-FABP-immunoreactivity are shown. The reaction product occurs
evenly throughout t h e cytoplasm. x 5,500.
Fig. 15. Light micrograph of the 56 day gastric mucosa immunostained for I-FABP. Immunoreactivity is localized exclusively to the
surface mucous cells. x 300.
The authors are indebted to Mr. S. Yamazaki for photographic work. This work was supported in part by
Grant-in-Aid 01570006 from the Ministry of Education, Science and Culture, Japan.
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