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Restricted mobility and endocytosis of anionic sites on newborn rat jejunal brush border membranes.

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THE ANATOMICAL RECORD 202:61-71 (1982)
Restricted Mobility and Endocytosis of Anionic Sites
on Newborn Rat Jejunal Brush Border Membranes
Department of Anatomy, Indiana University School of Medicine,
Indianapolis, Indiana 4622.3
The distribution and mobility of anionic sites on the
microvillous surface of newborn rat jejunal absorptive cells were studied using
polycationic ferritin (PCF)as a visual probe and were compared with anionic sites
previously described for adult jejunum. Segments from 5- to 26-day-oldrats were
incubated in PCF for 5 minutes either before or after fixation for electron
microscopy. From days 5 to 20, anionic sites were distributed diffusely along the
lengths of the microvilli and did not show random translational mobility. In contrast, microvilli examined from animals at weaning (21 to 26 days) resembled
those from adults in which most binding sites were capable of lateral mobility
and were induced by PCF to cluster into discrete patches. The diffuse pattern
was altered by cortisone administration, paralleling a premature reduction in the
endocytic apparatus of the cell.
The difference in mobility of anionic sites with age coincides with differences in
absorptive function. Evidence is presented showing that in the neonate binding
of PCF to the microvilli was followed with time by endocytosis into an apical
system of tubules for intracellular transport, incorporation into coated vesicles,
and release through the lateral cell surface. The results suggest that endocytosis
is accomplished by a mechanism that includes a directionally controlled move
ment for the selective internalization of PCF binding sites from the membranes
of the microvilli to those of the tubular cytoplasmic channels.
It was shown previously in absorptive cells
from the adult rat jejunum that receptors bearing anionic sites are distributed in a diffuse,
random pattern along the length of the
microvillous surface, as observed by the binding of polycationic ferritin (PCF)to fixed cells
(Jersild and Crawford, 1978). PCF also can be
used on living cells at physiological pH, and
has been useful in determining the degree of
lateral mobility of PCF receptors and their
capacity for redistribution through electrostatic coupling (Danon et al., 1972). When
applied to the unfixed membranes of living absorptive cells, PCF induced a redistribution of
anionic sites into discrete patches separated
by areas of unlabeled membrane. Other anionic
sites formed a nonmobile cap of PCF binding
sites over the tips of the microvilli (Jersild and
Crawford, 1978).
During the first 3 weeks of postnatal
development in the rat, the composition of
brush border enzymes and other proteins and
glycoproteins differs considerably from that in
0003-276X/82/2021-0061$03.500 1982 Alan R. Liss. Inc.
the adult. This reflects differences in diet and,
in some species, in the ability for endocytosis
of intact proteins, including antibodies, in the
neonate (Brambell, 1970;Galand and Forstner,
1974; Seetharam et al., 1977). Endocytosis is
accomplished by an extensive system of
tubules and vesicles present throughout the
apical cytoplasm (Kraehenbuhl and Campiche,
1969; Rodewald, 1973). A change from these
and other features of the neonate to those of
the adult absorptive cell occurs at the time of
weaning about the end of the third week, and is
normally completed by 24 days of age. The
changes can also be induced prematurely by
cortisone (Yeh and Moog, 1975, 1977).
The present study was undertaken to examine the distribution and mobility of anionic
sites on the brush border surface of the
newborn rat jejunum through weaning. The
results show considerable difference from
those obtained in the adult in the distribution
Received March 30, 1981. accepted June 5, 1981.
of PCF binding sites and in their lateral mobility that parallel basic functional differences
between the neonatal and adult jejunum.
PBS for 15, 20, 30 or 60 minutes (5 animals).
Care was taken not to affect the blood supply
to the segment. All incubations were stopped
by rinsing the tissue in PBS and then transferring the excised tissue to glutaraldehyde.
In control experiments, 1 mglml native ferritin (Calbiochem,La Jolla, California) replaced PCF for incubation of unfixed or prefixed
specimens on days 5, 10, and 15 after birth.
Native ferritin was also injected into ligated
segments for continuous in vivo exposure for
60 minutes. Other control samples of unfixed
specimens were incubated in 1 mglml poly-Llysine (Miles Laboratories, Inc., Elkhart, Indiana) for 5 minutes at room temperature to
block anionic sites, then rinsed and incubated
in PCF as usual (Skutelsky and Danon, 1976).
Some fixed specimens were treated before
PCF incubation with chloroform-methanol for
20 hours according to the method of Suzuki
(1965) for ganglioside extraction in aldehydefixed tissues. Methylation was performed by
treatment of fixed tissues with 0.05 N HC1 in
absolute methanol for 16 hours at room temperature before PCF incubation (Fisher and
Lillie, 1954). Portions of these samples were
saponified with 1.0% KOH in 80% ethanol (Ito,
1965). Control tissues were exposed solely to
absolute methanol under the same conditions.
In another series, cortisone acetate (Merck,
Sharp and Dohme, West Point, PA.) was administered intraperitoneally in saline to
neonates at 50 pglgm body weightlday (Yeh
and Moog, 1975), or 5 mglrat on day 1 (Halliday, 1959). Data on these two groups were
essentially the same and, therefore, will be
presented collectively. Littermates served as
saline-injected controls for each experimental
condition. Animals were injected and sacrificed as presented in Table 1.
Rats of Wistar strain were purchased from
Harlan Industries, Indianapolis, Indiana. A
total of 20 animals were examined on days 5,8,
10, 12, 15, 17, 19, 21, 22, 23, 24, and 26 after
birth. Immediately after sacrifice of each
animal, 5-mm segments of proximal jejunum,
starting from the ligament of Treitz, were
removed, opened longitudinally, and allowed
to form an inverted roll. These were used for all
in vitro studies following a thorough saline
rinse and blotting to remove surface debris and
Initial fixation was in 3% glutaraldehyde,
buffered to pH 7.4 with 0.1 M sodium
cacodylate, for 4 hours at room temperature.
Secondary fixation in 1%cacodylate-buffered
Os04for 1 hour was followed by a graded series
of ethanol dehydration and embedment in
Epon 812 for electron microscopy. Sections
were stained briefly with lead citrate.
Segments were incubated in phosphatebuffered saline (PBS) containing 1 mglml
polycationic f e r r i t i n ( P C F ) (Miles
Laboratories, Inc., Elkhart, Indiana) for 5
minutes at room temperature either before or
after initial fixation. This concentration of
PCF was used to directly compare with results
obtained previously in the adult jejunum (Jersild and Crawford, 1978). It was shown in that
study that mucus has a greater affinity for
PCF than anionic sites on the microvillous surface of unfixed cells, and that only mucus
shows binding at concentrations lower than 1
mglml. Incubations for longer than 5 minutes
were performed in ligated segments of jejunum in vivo at 1or 3 mglml PCF. After an inRESULTS
itial flush with PBS, the PCF was injected into
the lumen for a continous exposure of 15,45,or Distribution of anionic sites from days 1 to 20
Following a 5-minute incubation of living,
60 minutes (three animals), or for a 5-minute
pulse followed by a flush and chase in PCF-free unfixed intestinal segments in PCF, labeled
TABLE 1. Influence
of cortisone on the distribution of brush border anionic sites and endocytic tubules
Anionic sites'
Number of
Age treatment
Age at
Pat ched
present '
'Littermates in each experiment were injected with saline. All retained neonatal characteristics and are combined here.
or absent (-).
'PCF labeling density in diffuse or patched sites. varying from abundant (++++)to sparse (f)
'Tubules, as they typically appear in numbers in neonates before weaning (++ +).or in adults (+).
*Asterisks = data is a s presented in cortisone experiments -see footnote one.
anionic sites were dispersed over the lateral membranes were cut transversely. Binding of
surfaces of absorptive cell microvilli, either PCF to the microvilli was greatest on the cells
uniformly or interrupted by short, unlabeled of the distal (apical)villus and became generalsegments of membrane in an apparently ran- ly lighter or was lacking on cells of the proxdom manner. The tips of the microvilli usually imal villus, beginning a variable distance from
were not reactive throughout this period (Fig. the villous tips. All absorptive cells in reactive
I). Diffuse labeling also occurred for variable regions of the distal villus showed binding in
distances along the membranes of some of the contrast to an intermixing of labeled and
tubules that extend from the base of the unlabeled cells seen previously in adults (Jermicrovilli or along those of deeper profiles (Fig. sild and Crawford, 1978).
A similar diffuse arrangement of anionic
1).The bound PCF appeared to be suspended
among the filaments of the surface coat cover- sites was observed on microvilli of cells fixed
ing the membranes of the microvilli and with glutaraldehyde before incubation in PCF.
tubules. This is especially evident where the Fixed tissue treated with methanol or
Fig. 1. Microvilli of an absorptive cell from the distal
villus of a 10-day-old rat. Incubation was for 5 minutes in
PCF before fixation. Numerous binding sites are distributed
in a diffuse, random pattern over the length of the microvilli.
Some gaps in labeling occur locally. Binding of PCF to the
surface coat is evident where the membranes are cut
transversely. The surface coat a t the microvillous tips is not
labeled. One surface tubule and a vesicular profile in the terminal web zone show diffuse. membrane-bound labeling. The
tubule is labeled only along the upper part of its length (arrow). Other tubules are not labeled. X 70.000.
chloroform-methanol displayed the same binding characteristics as that of untreated
specimens. Tissues exposed to methanol-HC1
prior to incubation showed no evidence of PCF
binding. Subsequent saponification of a portion of this tissue resulted in a return to the
original binding pattern. Preincubation in
poly-L-lysine eliminated or greatly reduced
subsequent binding with PCF. There was no
retention or binding observed following incubation of fixed or living cells with native ferritin.
While most cells on the distal villus continued to show abundant, diffuse binding up to
day 20, some others showed a variable reduction in the number of binding sites along the
microvillous length between days 15 and 20. In
a few cases, cells were observed in which the
PCF clustered into small patches similar to
that seen in older animals (see below). Such
Fig. 2. Microvilli of an absorptive cell from a postweaned
rat in which the cells were fixed in glutaraldehyde before incubation in PCF for 5 minutes. PCF is distributed in a random, diffuse pattern primarily along the upper two-thirds of
their length. The microvillous tips are more heavily labeled
with PCF, which forms a distinct cap over each. X 57,000.
cells at times were observed on the villus proximal to cells with diffuse binding sites.
Distribution of anionic sites from days 21 to 26
Diffuse binding was observed only rarely in
living absorptive cells incubated for 5 minutes
with PCF following day 20, and not at all after
day 23. Rather, the binding pattern observed
throughout this 6-day period was more consistent with that found in adult animals studied
previously (Jersild and Crawford, 1978). When
cells were fixed before incubation in PCF, the
anionic sites were distributed in a diffuse pattern along the lateral microvillous surface (Fig.
2). When incubation preceded fixation, however, a striking redistribution of anionic sites
along the lateral surface occurred in which
discrete patches of densely packed ferritin
were separated by portions of membrane with
little or no binding. The tips of the microvilli
Fig. 3. Microvilli of an absorptive cell from the distal
villus of a 22-day-old rat. Incubation was for 5 minutes in
PCF before fixation. PCF is organized as a dense cap over
each microvillous tip similar to that in prefixed specimens
(Fig. 21. Along the length, the PCF is drawn into clusters of
various sizes, leaving the remaming membrane relatively
free of binding sites. X 57,000.
tained when the cells were continually exposed
to PCF. Whereas the interior of the cell showed
a progressive increase in labeling over the
60-minute test period examined, the microvilli
showed a marked decrease in PCF binding,
whether the PCF was presented as a pulse (Fig.
4) or continuously (Fig. 5). During this time,
some PCF also could be found in multivesicular bodies in the apical cytoplasm (Fig. 6). In
contrast to the diffuse pattern of PCF binding
in the endocytic tubules, PCF within these
bodies was largely in dense clusters with no apparent association with the membranes.
Influence of hormones on anionic sites
Some of the coated vesicles throughout the
The results of this study are summarized in apical region also contained PCF, presumably
Table 1. After injections of cortisone over a received through direct membrane continuity
period of 2 days, absorptive cells showed a with the tubules (Fig. 7). By 20 minutes, small
similar binding pattern to that observed in amounts of PCF were observed in the lateral
neonatal absorptive cells during the first 20 intercellular spaces, often in an area where
days described above. Injections over a period coated vesicles carrying PCF apparently had
of 4 to 5 days, beginning on day 8 or 10 after fused to the plasma membrane (Fig. 7). The
birth, respectively, resulted in a marked reduc- characteristic appearance of PCF in these
tion in diffuse labeling with PCF and a con- spaces was in the form of tight clusters, often
comitant decrease in number of endocytic in paracrystalline array, in which most of the
tubules. A sparse, but detectable, patching of ferritin appeared detached from the membrane
PCF-bound receptors was present in some surf ace.
specimens, but large numbers of clustered
binding sites, as seen in the adult, were not
achieved with any of the injection sequences.
I t was shown previously that electrostatic
Treatment with cortisone also did not enhance binding of PCF is specific for surface anionic
the development of dense caps of PCF binding sites on absorptive cell microvilli of adult rat
sites at the microvillous tips, as seen in many jejunum (Jersild and Crawford, 1978). These
cells from postweaned animals (Figs. 2, 3). results were confirmed in the present study for
Saline-injected littermates from all ex- jejunal absorptive cells of the neonatal rat.
periments continued to show diffuse labeling Although the nature of the anionic sites was
of the microvilli and the presence of numerous not determined precisely, the loss of binding
apical endocytic tubules.
sites upon treatment with methanol-HC1and
their reappearance following saponification
Pulse-labeling and long-term labeling
suggest that binding of PCF chiefly reflects
with PCF
the distribution of exposed carboxyl groups
Cells examined at the end of a 5-minute pulse (Revel, 1964). The clear association of most
with PCF are the equivalent of the earlier PCF with filaments of the surface coat and the
5-minute incubation studies. I t should be em- resistance of PCF binding sites to extraction
phasized here that diffusely labeled anionic with chloroform-methanol provide evidence
sites only extended into some of the tubules of that the binding is primarily related to the
the terminal web and, in these, labeling usually presence of surface-coat glycoproteins,
did not extend the full depth of the tubule (Fig. although other membrane proteins cannot be
1).Ferritin that could be characterized as free excluded. I t seems unlikely that external
in the lumen of these channels or as tight agents such as mucus, which does avidly bind
clusters along the membrane was only rarely PCF, could account for the diffuse binding patobserved. No evidence was obtained for the up- terns observed on the membrane in the neonate. It is also doubtful that mucus-bound
take and transport of native ferritin.
Following a 15- or 20-minute chase in PCF- PCF could account for the cell-specificclusters
free medium after pulsing with PCF, a much of PCF observed in postweaned rats. Morelarger number of tubular channels throughout over, binding sites in mucus have a greater afthe apical cytoplasm showed diffuse mem- finity for PCF than sites on the microvillous
brane binding (Fig. 4). Similar results were ob- surface of unfixed cells. At concentrations of
also frequently were covered by a dense cap of
ferritin particles (Fig. 3). The number of labeled cells per villus varied considerably. As
demonstrated in adult animals (Jersild and
Crawford, 1978), labeled cells alternated in an
apparently random fashion with cells that did
not bind PCF. Surface pits and apical tubules,
which are a part of the endocytic apparatus of
the neonatal absorptive cell, were reduced to
scattered profiles typical of adult animals.
PCF labeling of these profiles following
5-minute incubation was not observed.
PCF below that used in this study (e.g., 0.3
mg/ml),microvilli remain totally unlabeled and
subsequent cross-linking with labeled mucus
to produce the characteristic patches or caps
does not occur (Jersild and Crawford, 1978).
In the adult rat, it was shown previously
that most anionic sites of the microvillous surface are laterally mobile and can be induced by
Fig. 4. Apical portion of an absorptive cell from the upper villus of a 10-day-old rat. PCF was pulsed for 5 minutes
in viva, then chased with PBS for 20 minutes. Binding to the
microvilli is diffuse; however, large segments of the membrane are unlabeled. Most endocytic tubules in and below
PCF to cluster into discrete patches (Jersild
and Crawford, 1978). In contrast, PCF does
not induce a clustering of anionic sites under
similar conditions in the newborn. Rather, as
shown in the present study, these sites retain a
diffuse distribution. This suggests that these
binding sites do not possess lateral mobility,
and that subsequent patching on exposure to
the terminal web zone also show diffuse or scattered labeling, most of which is membrane bound. There is no evidence
for clustering of PCF including that in the endocytic pits at
the base of the microvilli. X 51,000.
PCF is prevented by certain restraining
mechanisms to be considered below.
The diffuse pattern was seen up to the time
of weaning, although there was a tendency
toward a reduction in number of anionic sites
in some specimens during the third postnatal
week. By the 24th day, anionic sites present on
the microvilli, with the exception of those at
their tips, consistently formed patches in the
presence of PCF, reflecting a distinct change in
Fig. 5. Base of microvilli and apical cytoplasm of an a b
sorptive cell from the distal villus of a 10-day-oldrat. The intestine was exposed continuously to PCF for 45 minutes in
vivo. The microvilli show widely dispersed labeling with
PCF. Greater concentrations of PCF still occur locally a t the
base of the microvilli and in endocytic pits (arrows). Endocytic tubules show abundant, diffuse, membranebound
PCF. X 61,000.
their lateral mobility at this time. The
possibility that patching of binding sites was
induced by fixation (Burry and Wood, 1979)is
negated by the diffuse binding pattern seen on
cells fixed prior to PCF exposure. These
features of anionic binding are comparable to
those maintained through adult life (Jersild
and Crawford, 1978).The change in behavior of
anionic sites with time is suggested to reflect
differences in absorptive cell function in the
newborn and adult animals.
A number of changes have been shown to occur in absorptive cell function and associated
structure during the same period of time.
Among these are a loss of ability to ingest intact proteins and antibodies (Halliday, 1955;
Jones, 1972) concomitant with an extensive
reduction in the endocytic system of tubules
(Clark, 1959; Rodewald, 1973;),and changes in
the composition of brush border enzymes and
other proteins, including a decrease in lactase
and an increase in other hydrolases (Galand
and Forstner, 1974; Seetharam et al., 1977).
These changes occur in a relatively short
period that extends from about the 19th to the
23rd day in the rat and are under the control of
the adrenal cortex. In this respect, these
changes can be induced prematurely in the
suckling rat by the administration of glucocorticoids (see review by Yeh and Moog, 1975).In
the present study, cortisone administered over
4 to 5 days likewise produced premature
changes in anionic sites, reflected primarily as
a marked decrease in PCF-binding, indicating
that this feature of the neonatal absorptive cell
is also under hormonal control. The lack of
response after only 2 days of cortisone treatment may reflect the inability of fully matured
cells to respond to the hormone. A respor:se instead may require replacement with crypt cells
Fig. 6. A rnultivesicular body, 20 minutes after a
5-minute pulse with PCF, contains an internal cluster of
PCF. X 51,000.
induced during differentiation as shown for
other features controlled by hormones (Clark,
1959, 1971; Doell et al., 1965).Ths view is supported by the observation that occasional cells
with patched PCF binding sites could be seen
on villi in a migratory position proximal to
cells that had diffuse anionic sites.
The concomitant loss of detectable
microvillous anionic sites and the marked
reduction in endocytic tubules observed in the
present study following cortisone administration suggest a relationship between these sites
and endocytosis. Indeed, evidence presented
here clearly demonstrates that considerable
PCF is absorbed and transported through the
cell by an endocytic process. In fact, the pattern of uptake and transport, the time course
for traversing the cell, and the sensitivity to
cortisone are very similar to that described for
antibody transport in the newborn rat
(Rodewald, 1973; Waldmann and Jones, 1973).
For this, the proximal small intestine has been
shown in important studies by Rodewald
(1976, 1980)to be the site for selective absorption of antibodies by a process that involves
the binding to receptors covering the
microvillous membrane.
Binding of PCF to the cell membrane of the
microvilli meets the first requirement for selective uptake as originally suggested by
Brambell (1970). While the selective uptake
mechanism has been shown to be fairly specific
for certain antibodies (Rodewald, 1973;
Waldmann and Jones, 1973), it is clearly evident in the present study that uptake is not
restricted to them but may include other
molecules that are capable of binding to
suitable receptors in the cell membrane, such
as PCF. It cannot be stated at this time
whether or not PCF is binding to the receptors
that transport antibodies into the cell. The
binding of PCF and its subsequent transport
may indicate, however, another mechanism by
which certain potential antigens enter the
body of the young in addition to their uptake
as immune complexes (Abrahamson et al.,
Binding of ligands to the cell surface is
known to stimulate pinocytic activity (Silverstein et al., 1977). For some cell types, ligand
binding has been reported to induce crosslinking and subsequent clustering of diffusely
distributed and apparently freely diffusible
receptors which are then internalized as concentrated patches (Schekman and Singer,
1976; Schreiner and Unanue, 1976; Willingham et al., 1979). This contrasts with the
results in the present study in which the
anionic sites on the microvilli of neonatal jejunum were not passively mobile and were not
redistributed into clusters following the binding of PCF. Yet with time after a 5-minute
pulse-labeling period, there was a large in-
crease in the number of endocytic tubules
labeled in the apical portion of the cell accompanied by a striking decrease in labeling of the
microvilli. It is of interest that labeling of the
microvilli also decreased with time during continual exposure to PCF. It thus appears that
. . - ..
. . .- .
. _
Fig. 7. Deep apical cytoplasm and lateral boundary b e
tween two cells from a 10-day-oldrat following a continuous
in vivo exposure to PCF for 45 minutes. PCF is present in
small coated vesicles (short arrows). In larger coated vesicles, two of which are attached to the cell membrane, the
PCF is seen to still be membrane bound (largearrows). Other
PCF is in the lateral intercellular space where i t forms dense
clusters. X 54,000. In the inset, diffuse, membrane-bound
label is present in an endocytic tubule and in an attached
coated vesicle. X 89,000.
binding sites can be temporarily depleted during the endocytic process. The results at this
time suggest that these sites interact with
other membrane of submembrane structures
(Edelman, 1976;Nicolson, 1976)which prevent
their passive mobility. When bound to a
suitable ligand, these sites then are conveyed
from the microvilli to the cell interior by a
directed movement that channels them into
tubular components of the system in which
they are further transported. A transmembrane system of directional control over binding site movement (Nicolson, 1976) is
therefore proposed which would ensure their
passage into subjacent tubules of considerable
length. It may also be an important
mechanism for the maintenance of the integrity of the structure and other functional activities of the microvilli during the event of endocytosis in a manner similar to that described
for other cells (Oliver and Berlin, 1976). For
this, the core actin filaments and the cross
filaments connecting them to the microvillous
membrane (Mukherjee and Staehelin, 1971;
Mooseker and Tilney, 1975)may play some important roles as yet not recognized in neonates.
Furthermore, the maintenance of diffuse binding within the endocytic tubules may indicate
a regulated movement of membrane-binding
PCF in these structures as well.
I t has been suggested that binding of proteins to microvillous receptors not only aids in
their selective uptake, but also protects them
from lysosomal degradation (Brambell, 1970).
In the present study, a small amount of PCF
was sometimes observed within multivesicular
bodies as internal clusters rather than diffusely bound to the membranes. This appearance is
very similar to that previously observed in
small quantities in the adult jejunum where its
transport was terminated (Jersild and
Crawford, 1978). It seems likely that it
represents the uptake of PCF unbound or only
loosely bound to membranes and ultimately
destined for degradation by the lysosomal activity found in these bodies (Kraehenbuhl and
Campiche, 1969).
This project was supported by grant 5S01
RR-05371 awarded by the Division of
Research Resources, NIH, DHEW. The author
gratefully acknowledges the skillful technical
assistance of Mrs. Vera McAdoo and the
secretarial assistance of Mrs. Deborah
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site, anionic, rat, restricted, brush, border, membranes, newborn, mobility, endocytosis, jejunal
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