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Interstitial cells of Cajal in human gut and gastrointestinal disease

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MICROSCOPY RESEARCH AND TECHNIQUE 47:325–335 (1999)
Receptors in Interstitial Cells of Cajal: Identification
and Possible Physiological Roles
MARIA-GIULIANA VANNUCCHI*
Department of Anatomy, Histology and Forensic Medicine, Section of Histology ‘‘E. Allara,’’ University of Florence, Florence, Italy
KEY WORDS
neural transmission; Kit-receptor; NK1 receptor; nitric oxide; somatostatin
ABSTRACT
Interstitial cells of Cajal (ICCs) are specialized cells of the gastrointestinal tract
forming distinct populations depending on their location in the gut wall. Morphological observations
and functional data have led to the hypothesis of two functions for the ICCs: (1) as pacemakers of the
rhythmic activity; (2) as intermediaries in neural inputs to the muscle. The identification of specific
receptors on the ICCs has represented an important step in the knowledge of these cells.
Immunohistochemical labeling of these receptors provided information on both ICC morphology and
contacts (particularly those with nerve endings) and on the functions of these cells. All ICC possess
the Kit receptor, which represents the best tool to identify these cells under the light microscope. It
has been demonstrated that this receptor is essential for ICC differentiation, and, by using mutant
mice lacking the Kit-related gene, it has been possible to discriminate among all the ICC those with
a primary role as pacemakers. The ileal ICC, in particular those at the deep muscular plexus,
express the tachykinin receptor NK1 and a subtype of somatostatin receptors and contain nitric
oxide synthase. All these data support a primary role of these ICC in neural transmission. Microsc.
Res. Tech. 47:325–335, 1999. r 1999 Wiley-Liss, Inc.
INTRODUCTION
One century ago Ramon y Cajal first described thin,
elongated and branched cells within the gastrointestinal wall that, on the basis of their shape and location,
he interpreted as peculiar neuronal cells. Although
Cajal identified these cells using a light microscope
(LM) after staining with methylene blue, the definitive
technique to unequivocally identify the ‘‘interstitial
cells of Cajal’’ (ICC) is the transmission electron microscopy (TEM). TEM allows one to see the peculiar cytological features and the ‘‘interstitial’’ position of the ICC
between nerve endings and smooth muscle cells. These
ultrastructural aspects led several authors to suggest
that the ICC could be involved in intestinal pacemaker
activity and neural transmission.
In the last decade, giant steps have been made in the
knowledge of the ontogeny, structural characteristics,
and related functions of ICC. It has been shown that
these cells possess receptors responsible for (1) their
development and differentiation (Kit receptor); (2) their
putative role in excitatory (NK1 receptor) and inhibitory (NO) neural transmission; and (3) their putative
ability to respond to hormones (somatostatin receptor).
KIT RECEPTOR
The kit receptor (kit-r) is encoded by the protooncogene c-kit located on the W (white spot) locus of
chromosome 5 of mouse; it is a tyrosine kinase similar
to other receptors for growth factors whose interactions
with the ligands initiate signaling via kinase cascade to
the cell nucleus. The discovery that ICC express the
kit-r has represented a fundamental advance in the
study of these cells.
r 1999 WILEY-LISS, INC.
Kit-r and ICC Identification
As previously stressed, ICC are identifiable unequivocally only under the TEM. The fact that the kit-r in the
gut is expressed by ICC provides a way to identify these
cells under the LM. Although other cells such as the
mast-cells are kit-r positive, their shape and localization should avoid confusing them with ICC. Therefore,
it is generally accepted that the kit-r immunolabeled
cells are ICC (Lavin et al., 1998). In this regard
however, it is necessary to consider some concern that
the kit-r study alone could lead to erroneous conclusions. First, owing to the great number of kit-r
antibodies commercially available, the possibility of a
low affinity or specificity of some of them has to be kept
in mind when ICC are studied using the LM only. To
avoid wrong interpretations, it is necessary to use
different kit-r antibodies whenever possible. Secondly,
absence of kit-r labeling might be associated with the
presence of otherwise normal ICC (Klüppel et al.,
1998). Finally, kit-r positive labeling may not necessarily imply a structural and functional integrity of ICC. In
conclusion, although the kit-r immunohistochemistry
maintains its importance in LM investigations, the
study of ICC, especially in pathological conditions, still
requires the contribution of the ultrastructural examination.
Abbreviations used: ICC-DMP ⫽ ICCs within the deep muscular plexus region
of the small intestine; ICC-MP ⫽ ICCs within the myenteric plexus region of
stomach, small intestine and colon; ICC-IM ⫽ ICCs intramuscular of the lower
esophagus and pyloric sphincter regions, stomach, and colon; ICC-SM ⫽ ICCs
along the submucosal surface of the circular muscle bundles of the colon.
*Correspondence to: Maria Giuliana Vannucchi, Department of Anatomy,
Histology and Forensic Medicine, Section of Histology ‘‘Enrico Allara,’’ Viale G.
Pieraccini, 6 I-50139 Florence, Italy. E-mail: gvannucchi@server1.pharm.unifi.it
Received 22 February 1999; accepted in revised form 1 August 1999
326
M.-G. VANNUCCHI
Kit-r and the Various ICC Populations
Combined morphological and functional studies of
ICC have demonstrated the existence of distinct populations of ICC in each gut region and muscle layers, and
distinct roles in intestinal motility have been attributed
to each population (Faussone-Pellegrini, 1992; Thuneberg, 1989). The results obtained in studies on kit-r
seem to confirm such a hypothesis. In the ileum, for
example, where two types of ICC (ICC-MP and ICCDMP) have been described, the investigations done in
W mutant mice and in anti-kit-r treated animals have
shown significant differences between the two ICC
populations: (1) in sensitivity to kit-r expression to
reach complete differentiation during the early stages
of postnatal life (Torihashi et al., 1995); (2) in the
persistence of the ICC-DMP and the loss of the ICC-MP
in adult mice with the W mutation (Ward et al., 1994;
Torihashi et al., 1997). Consistent with these observations, we report here a different kit-r labeling distribution and intensity between the two ileal ICC populations of three different animal species (see Experimental
Procedures).
Kit-r and ICC Ontogeny and Differentiation
The use of kit-r molecular biology and immunohistochemistry in grafts of intestinal segments (Young et al.,
1996) or in quail-chick chimeras for vagal neural crest
(Lecoin et al., 1996) have demonstrated that ICC: (1)
originate from gut mesenchyma and not from the
neural crest cells that colonize the gut; (2) develop in
the absence of neurons that are considered the source of
the stem cell factor (Steel factor), the ligand for the
kit-r. Various attempts to evaluate the importance of
kit-r in ICC differentiation have demonstrated that,
although kit-r is already expressed by these cells in the
embryo, its presence is not necessary for their migration, proliferation, survival (Bernex et al., 1996), and
early differentiation (Klüppel et al., 1998). On the
contrary, the presence of kit-r becomes necessary after
birth (2–5 days) to guarantee cell division and ICC
network formation (Klüppel et al., 1998). At this time,
the cells are close to myenteric neurons and nerve
endings that express the Steel factor (Torihashi et al.,
1997). The binding of the ligand to the ICC receptor
might be important for cell proliferation after birth.
Kit-r and Pacemaker Function
The observation that treating mice with kit-r antibodies led to the loss of the rhythmic activity in the mouse
intestine (Maeda et al., 1992) suggested that cells
located in the intestine normally contain the kit-r and
are responsible for the slow-wave activity. Two groups
showed by immunohistochemistry that the ICC express
kit-r and that these cells disappear in the anti-kit-r
treated (Torihashi et al., 1995) or W mutant mice
(Huizinga et al., 1995; Ward et al., 1994). Besides,
electrophysiological recordings confirmed that the lack
of ICC was associated with the loss of the rhythmic
activity (Huizinga et al., 1995; Ward et al., 1994) and
demonstrated that neural inputs were also reduced,
although not abolished, suggesting a role for ICC in
neural transmission also (Torihashi et al., 1995) (see
below).
ICCS AND NEURAL TRANSMISSION
The consistent relationship of ICC with nerve endings on one side and with the smooth muscle cells on the
other side suggested that these cells are potential
intermediaries in autonomic neurotransmission (Daniel
and Posey-Daniel, 1984). Indeed, electrophysiological
studies (Berezin et al., 1990; Huizinga et al., 1990;
Smith et al., 1989) have demonstrated that ICCs are
innervated and also possibly intercalated between nerve
endings and smooth muscle cells. Furthermore, a combined morphological and functional study of the ileum
of Steel mutant mice revealed a lack of the ICC-MP
network and of the electrical rhythmicity even though
ICC-DMP were maintained, as were the typical excitatory and inhibitory inputs (Ward et al., 1995). These
findings indicate that (1) ICC-MP are not essential for
mediating inputs from the enteric nervous system and
(2) the ICC-DMP are the primary mediators in neural
inputs. In another important study in animals treated
with kit-r antibody after birth, Torihashi et al. (1995)
found that several populations of ICC, such as all
ICC-MP of the gut, ICC-DMP of the ileum, ICC-SM and
ICC-IM of the colon, were reduced in number, and, in
parallel, the slow-wave activity was abolished and the
neural responses were reduced, although not abolished.
Altogether these findings confirm that ICC can mediate neural inputs and that some of the ICC populations
are more important than others in neural transmission.
TACHYKININ RECEPTORS
Three types of tachykinin receptors (NKr) have been
described that bind with different affinities a series of
neuropeptides called tachykinins including substance
P (SP), neurokinin A (NKA), and neurokinin B (NKB).
With the exception of the last, the tachykinins are
commonly synthesized and stored in enteric neurons
(Sternini et al., 1989). NKr are three distinct G-proteincoupled receptors (NK1r, NK2r, and NK3r with the
highest affinity to SP, NKA, and NKB, respectively)
(Maggi et al.,, 1993). Immunohistochemical studies
have shown that all the three receptors are detectable
in the gut (Grady et al., 1996) with a region-specific
distribution (Vannucchi and Faussone-Pellegrini, unpublished data). However, only NK1r has been observed on ICC.
NK1r and ICC-DMP
All ICC are intimately associated with nerve endings.
However, differences among the ICC types in the frequency of these contacts have been reported, i.e., between the ICC-MP and ICC-DMP in the small intestine
or between the ICC-MP and ICC-IM of the stomach.
These contacts are both more numerous and closer with
ICC-DMP and ICC-IM than with ICC-MP, both in
laboratory mammals and humans (Faussone-Pellegrini, 1992; Thuneberg, 1989). The presence of nerve
endings close to ICC-MP suggested a neural control of
slow-wave activity (Huizinga et al., 1997), in particular,
of the premature or delayed slow wave (Cayabyab et al.,
1997). Conversely, ICC-DMP and ICC-IM, which show
a larger number of contacts with nerve ending, could be
the intermediate step between enteric neurons and
smooth muscle cells in the control of gut motility. In this
regard, however, it has been reported that, in the dog
RECEPTORS IN INTERSTITIAL CELLS OF CAJAL
ileum, ICC-DMP produced slow waves of a different
character from those produced by the ICC-MP (Jimenez
et al., 1996). Indeed, immunohistochemical studies
have shown that ICC-DMP are closely apposed to SP
positive nerve endings and express its preferred receptor, the NK1r (Portbury et al., 1996; Sternini et al.,
1995; Vannucchi et al., 1997; Vannucchi and FaussonePellegrini, unpublished data). Recently, a combined
study of immunohistochemistry and pharmacology in
the guinea pig ileum, has demonstrated that the NK1r
on ICC-DMP is a true receptor since in the presence of
the agonist (SP), it aggregates on the plasma membrane and internalizes in the cytoplasm of ICC (Lavin
et al., 1998). Moreover, these authors quantified the
relationship between SP-IR nerve endings in the DMP
and NK1r-IR related ICC and demonstrated that up to
90% of these nerve endings are apposed closely to ICC.
It is noteworthy that neither NK2r nor NK3r antibodies
label ICC-DMP (Grady et al., 1996; Vannucchi and
Faussone-Pellegrini, unpublished data).
A study of the time course of appearance of NK1r on
ileal ICC during rat development has shown that
ICC-DMP express NK1r very early after birth and
reach the maximum intensity and distribution of IR at
1 week of postnatal life with no further changes in adult
life. Interestingly, TEM studies have shown that during
this period of life ICC-DMP acquire the adult features
and that their differentiative steps are temporally
related to those of the enteric plexuses (see Experimental Procedures).
Unexpectedly, ICC-IM of the stomach, to which a role
similar to that of the ICC-DMP has been attributed,
never stained with the NK1r antibody. However, it has
been recently reported that ICC-IM of the human
stomach can be stained with a monoclonal antibody to
NK1r (Smith et al., 1998). The possibility that the
antibody used does not recognize a slightly modified
NK1r in the stomach has to be considered.
NK1r and ICC-MP
In previous papers, it was reported that the NK1r-IR
at the MP of the small intestine was detectable on ICC
of the duodenum but not of the ileum (Portbury et al.,
1996). In a more recent work, Lavin et al. (1998) showed
that NK1r-IR was detectable in the cytoplasm of
ICC-MP of the ileum, but only after the receptor
internalization was induced by the agonist (SP), and
suggested that the absence of immunoreactivity in
resting conditions was due to a receptor conformation
that masked the site for antibody interaction. Besides,
they found that in the duodenum almost 40% of the
SP-IR nerve endings detected in the tertiary plexus of
the MP were closely apposed to ICC. In our laboratory,
using the same antibody, we detected NK1r-IR on
ICC-MP in laminae of rat and guinea pig ileum and
showed that SP-IR nerve endings of the tertiary plexus
of the MP were rarely close to the NK1r-IR ICC (see
Experimental Procedures). This finding suggests that
the difficulty in ICC-MP labeling by the available NKr1
antibodies might be due to the presence of few receptors
that concentrate after internalization, rather than to
changes in their conformation. A reduced number of
NK1r on ICC-MP would better fit with the scarcity of
closely apposed SP-IR nerve endings.
327
NK1r-IR was never observed in any ICCs population
of the colon, in guinea pig (Portbury et al, 1996), rat
(Grady et al., 1996), or mouse (personal observations).
NITRIC OXIDE TRANSMISSION
Nitric oxide (NO) is a gas that easily diffuses across
biological membranes (Moncada et al., 1991). Many of
its actions are mediated by activating soluble guanylate
cyclase. The regulatory step of its production is a
specific enzyme, nitric oxide synthase (NOS). Two types
of NOS have been identified in biological tissues: a
constitutive form and an inducible form. Both are
di-oxygenases that synthesize NO in the presence of
arginine, are cytosolic and require NADPH as a coenzyme. However, the constitutive NOS is calciumcalmodulin dependent and produces a picomolar amount
of NO, while the inducible NOS is calcium-independent
and produces larger amounts of NO when activated.
Several isoforms of both enzymes have been found
(Moncada and Higgs, 1993) but only the constitutive
NOS has been identified in neurons (neuronal NOS), as
well as in the endothelium, platelets, mesangial cells,
and, membrane-bound, in skeletal muscle. The activity
of constitutive NOS is normally inhibited at the resting
calcium concentration present in cells. Its activation is
dependent on events that increase the free calcium in
the cytoplasm.
NO and ICCs
The presence of NOS-positive neurons in the gastrointestinal tract has been amply reported (for review see
Lefebvre, 1995) and a loss of these neurons has been
associated with pathological conditions such as achalasia, congenital esophageal stenosis, hypertrophic pyloric stenosis (Mearin et al., 1993; Vanderwinden et al.,
1992), and an increase of them with idiopathic chronic
constipation (Cortesini et al., 1995). Furthermore, numerous NOS-containing nerve endings have been described in the DMP of the ileum, apparently surrounding unstained cells identified under the TEM as ICCDMP (Matini and Faussone-Pellegrini, 1997). A
significant increase in cGMP was reported in ICC of the
small intestine and colon in response to exogenous NO
(Shuttleworth et al., 1993; Young et al., 1993). The
availability of mutant mice for the kit-r has allowed
experiments to be carried out to demonstrate that ICC
mediate NO transmission. In fact, in W mutants, the
ICC-IM of the stomach fail to develop, and the NOdependent inhibitory neuroregulation is greatly reduced in spite of a normal presence and distribution of
the NOS-positive neurons and nerve fibers. At variance
with the results reported for the ileum, ICC-MP of the
stomach are unaffected by the kit-r mutation (Burns et
al., 1996). In a more recent study, the same group (Ward
et al., 1998) has demonstrated the importance of ICC-IM
in mediating nitrergic neurotransmission also at the
level of the lower esophageal and pyloric sphincters.
This group found that in W mutant mice the lack of
ICC-IM was associated with a marked decrease in the
response to nitrergic stimulation. In contrast to the
findings of Burns et al. (1996) in the fundus, the loss of
the ICC-IM in the sphincters did not completely abolish
the inhibitory response, and it was still possible to
slightly enhance muscle contractions adding NOS inhibitors. This finding suggested the existence of a
328
M.-G. VANNUCCHI
parallel nitrergic innervation of ICC-IM and of smooth
muscle cells at the sphincter levels. These data, and the
observation of loss of ICC in tissues from patients
affected by pyloric stenosis and achalasia, suggest an
association between the status of ICC-IM and sphincter
disorders.
NOS-Positive ICC
NOS-positivity has been described in the following
ICC types: ICC-SM of the canine colon (Xue et al.,
1994); ICC-MP of the rat ileum (Matini and FaussonePellegrini, 1997); ICC-IM of the mouse gastric sphincters (Ward et al., 1998), and ICC-DMP of the guinea-pig
ileum (see Experimental Procedures). The significance
of the presence of a constitutive NOS isoform in ICC is a
matter of debate and some hypotheses have been
proposed. Neural inputs into ICC can induce NO production, which in turn can cause smooth muscle relaxation
(Ward et al., 1998). A second possible role of NOS in ICC
could be to amplify inhibitory signals mediated by
nitrergic pathways since it has been observed that NO
increases calcium concentration in ICC, which leads to
NO production by ICC (Sanders, 1996; Ward et al.,
1998). A third possible function of NOS in ICC could be
to modulate neural transmission by NO action on
neurotransmitter release from nerve terminals, as has
been reported in the central nervous system (Garthwaite, 1991).
ICC AND SOMATOSTATIN (SOM) RECEPTORS
Som is a fourteen-amino-acid polypeptide widely
distributed in the nervous system as well as in the
endocrine tissues. It acts as a neuromodulator in the
nervous system and as a neurohormone in other organs. The presence of Som in the digestive system had
been well described (Costa et al., 1980; Ekblad et al.,
1988), but contrasting results have been obtained regarding its actions on intestinal smooth muscle. In fact,
it has been reported that through activation of inhibitory neurons and/or inhibition of excitatory neurons,
Som caused muscle relaxation in guinea-pig intestine
in vitro (Furness and Costa 1979), while it inhibited NO
release and thereby excited intestine circular muscle in
canine ileum in vivo (Jimenez et al., 1995). It has been
shown that rat ICC-DMP express a particular subtype
of Som receptors, sst2A, and are surrounded by Som-IR
nerve endings (Sternini et al., 1997). Interestingly,
although the entire gut was investigated, the positivity
for sst2A was present only on these ICC (besides the
neurons and some endocrine cells). Moreover, sst2A-IR
on ICC-DMP was confined to the plasma membrane
while it appeared to be located in the cytoplasm of
neurons and endocrine cells. These findings have been
interpreted as further support for the possibility that
Som influences smooth muscle activity by directly
activating sst2A-r-IR ICC-DMP.
ICCS AND OTHER RECEPTORS
Several immunohistochemical studies indicate that
other neurotransmitters may act on ICC. In particular,
it has to be mentioned that nerve endings positive for
ChAT (Vannucchi and Faussone-Pellegrini, 1996) or
VIP (Berezin et al., 1990; Matini et al., 1997) are
present at the DMP closely apposed to unlabeled cells
possibly identifiable as ICC. Indeed, the presence of
VIP receptors on ICC has been hypothesized on the
basis of electrophysiological data (Berezin et al., 1990).
At the present, however, the lack of specific antibodies
to identify these receptors does not allow confirmation
of the existence of any other receptor on ICC.
EXPERIMENTAL PROCEDURES
Tissue Peparation for Immunohistochemistry
Specimens of guinea-pig, rat, and mouse ileum were
cut to yield transverse and tangential sections following the procedures previously reported (Vannucchi et
al., 1997), and some specimens of rat and guinea pig
ileum were prepared as laminae containing either the
submucosa plus the circular muscle layer or the longitudinal muscle layer plus the myenteric plexus following
the procedure described in Portbury et al. (1996). All
the specimens were fixed in 4% paraformaldehyde in
0.1 M phosphate buffered saline (PBS) pH 7.4, for 4
hours at 4°C. Then, those to be frozen were placed in
30% sucrose in PBS, overnight at 4°C. The following
day these specimens were embedded in OCT compound
(Miles, Elkhart, IN) and frozen at ⫺80°C. Transverse
and tangential sections (14 µm thick) were obtained
from the frozen specimens with a cryostat. After fixation, the laminae were carefully washed in PBS. Both
cryostat sections and laminae were pre-incubated in
PBS containing 3% normal goat serum and 0.5% Triton
X-100 and incubated with primary antisera in moist
chambers.
Antibodies
To label the neurokinin receptor-1, NK1r polyclonal
antibody (a generous gift of Dr. P. Vigna) was used,
raised in rabbit against a synthetic fragment corresponding to the intracellular C-terminal portion of the
rat receptors (Grady et al., 1996; Vigna et al., 1994), at a
final dilution of 1:2,500 for 24 hours at 4°C. The kit
transmembrane receptor protein was labeled with the
kit-r (Ab-1) (Calbiochem, San Diego, CA) polyclonal
antibody raised in rabbit and used at the concentration
of 1 mg/ml for 48 hours at 4°C. Substance P was
identified by using a mouse anti-SP monoclonal antibody (SP14, a generous gift of Dr. J.Y. Couraud) at 1:400
overnight at 4°C. NOS anti-mouse monoclonal antibody
(nNOS) (Transduction Laboratories) was used at a final
dilution of 1:1,000 for 24 hours at room temperature.
Negative controls were performed by omitting the
primary antibodies or substituting them with a nonimmune rabbit or mouse serum in order to check the
specificity of the immunostaining. At the end of incubation, the polyclonal primary antisera in the sections
and laminae were revealed by using fluorescein (DTAF)conjugated pure goat anti-rabbit IgG (H⫹L; Jackson
Immuno-Research, West Grove, PA) or rhodamine antimouse IgG (Fab specific) TRITC conjugate (Sigma, St.
Louis, MO) secondary antibodies.
RESULTS AND DISCUSSION
ICC and KIT Receptors
Kit-r Distribution in ICC-MP and ICC-DMP of
Adult Guinea Pig, Rat, and Mouse Ileum. In the
guinea pig, kit-r-IR was detected in ICC-MP and in
ICC-DMP (Fig.1A), with the ICC-MP showing a higher
intensity of staining. ICC-MP had a round or triangular
body and two, sometimes three, rarely four, main
processes (Fig.1D). The surface of the body and processes was smooth and kit-r-IR was distributed over the
RECEPTORS IN INTERSTITIAL CELLS OF CAJAL
329
Fig. 1. Kit-immunoreactivity (IR) in transverse sections (A–C) and
in the lamina containing the longitudinal muscle layer (D). A: Guinea
pig. IR is detected in ICC-MP and ICC-DMP; the intensity of the
labeling is higher in ICC-MP. B: Rat. IR is detected only in ICC-DMP;
C: Mouse. IR is detected in ICC-MP and in ICC-DMP. The intensity of
labeling is higher in ICC-MP. D: Guinea-pig. IR is detected on the
ICC-MP body and processes and has a patchy distribution. A,B,C,
⫻1,000, scale bar ⫽ 10 µm. D, ⫻450, scale bar ⫽ 22 µm.
entire cell surface, although the intensity of labeling
was higher at the level of wide patches present on the
cell body and on the roots of the main processes. The
ICC-DMP had an oval or round body and 2–4 main
processes, two of which always originated at the opposite poles of the cell. The surface of both the body and
processes was smooth and kit-r-IR was detected over
the entire cell surface and showed a punctate aspect
(data not shown). In the rat, kit-r-IR was detected only
on the ICC-DMP (Fig.1B). The cells had an elongated
body and 2–3 main processes that ran parallel to the
muscle cells. The kit-r-IR was detected along the entire
cell surface and had a punctate appearance. In the
mouse, kit-r-IR was detected on ICC-MP as well as on
the ICC-DMP (Fig.1C); however, the ICC-MP showed a
higher intensity.
These data represent the first comparison of the kit-r
distribution in the ileal ICC of three of the animal
species most used in research. As reported before,
several kit-r antibodies are commercially available. We
used a kit-r antibody against the intracellular portion
of the receptor that, in our hands, demonstrated good
selectivity since other cells such as mast cells were very
rarely labeled. This antibody showed interesting intraand inter-species differences in kit-r expression. In
particular, in each species the labeling intensity and/or
distribution always differed between the two ileal ICC
networks, with the ICC-DMP showing a similar labeling in all the animals. Moreover, kit-r-IR at the MP had
a patchy organization while it was evenly distributed at
the DMP. These data are in agreement with those
reported for W mutant mice and anti-kit-r treated
newborns that indicate a diversity between the two
ileal ICC networks in kit-r sensitivity and dependence
during maturation and in the maintenance of the
differentiated state.
ICC and NK1 Receptors
Timing of Appearance and Distribution of NK1
Immunoreactivity (IR) and Tachykinergic-(SP/
TK)-IR Nerve Fibers in the ICC of Rat Ileum
During Ontogenesis and in Adult Rats, Mice, and
Guinea Pigs. In 18-day-old fetuses and in the newborn, NK1r-IR was detectable only at the MP region as
330
M.-G. VANNUCCHI
Fig. 2. Neurokinin 1 receptor immunoreactivity (NK1r-IR) in the
rat ileum during development. A: Five days of postnatal life. IR is
detected at the deep muscular plexus (DMP) on cells (asterisks) which
probably are interstitial cells of Cajal (ICCs). B: Seven days of
postnatal life. The IR at the DMP region is particularly intense. C:
Fourteen days of postnatal life. ICCs at the DMP have a spindle shape.
D: Adult rat. ICCs have a spindle shape and the IR has a punctate
aspect. mp ⫽ myenteric plexus; dmp ⫽ deep muscular plexus; ocl ⫽
outer circular muscle layer. A,B, ⫻400; C,D, ⫻1,000. Scale bar ⫽ 25
µm (A,B), 10 µm (C,D). Reproduced from Vannucchi et al. (1997) with
permission of the publisher.
faint patches on the neuronal contour. From 3 to 5 days
of postnatal life, the immunoreactivity became detectable in the DMP on non-neuronal cells, which probably
corresponded to ICC. At 3 days of postnatal life these
cells were dispersed. At 5 days (Fig. 2A), they formed a
continuous layer between the inner and outer portions
of the circular muscle layer. All these ICC had an
elongated shape and the IR was irregularly distributed
on their plasma membrane. At the end of the first week
of postnatal life, the IR at the DMP region was particularly intense and all ICC showed the same high degree
of labeling (Fig. 2B). These ICC were oriented in
parallel to the circular muscle cells, forming a one-cellthick layer. The ICC were spindle-shaped and most of
them had a labeling distributed uniformly over the
entire plasma membrane . By 14 postnatal days (Fig.
2C), most of the NK1r-IR ICCs in the DMP region were
similarly intensely and uniformly labeled. In the adult,
the ICC maintained their arrangement in rows parallel
to the circular muscle cells and their spindle shape and
showed similar intensity of labeling to those observed
during the suckling and weaning periods. The NK1r-IR,
however, was irregularly distributed along the cell
surface, with a punctate appearance (Fig. 2D). From 7
days of postnatal life to adult, no change in the intensity of labeling was observed. Similar to the rat, adult
guinea pig and mouse showed very intense NK1-IR at
the DMP. The IR had a punctate distribution over the
plasma membrane. In the rat (and mouse) the cell body
was elongated or spindle-shaped (Fig. 3A) and, in the
rat, thin and ramified branches were seen to protrude
laterally from the body and processes. In the guinea
pig, the body of these cells appeared oval- or roundshaped with two to four main processes; two of them
always originated at the opposite poles of the cell, and
the surface of both the body and processes was smooth
(Fig. 3C). In laminae obtained from guinea pig and rat
ileum, the NK1r-IR was detectable also on ICC-MP,
although these cells showed a very faint labeling (Fig.
3B,D).
In rat, from birth, SP/TK-IR varicose fibers could be
seen to emerge from the MP and to gradually penetrate
the circular muscle layer towards the DMP, reaching it
by 5 days (Fig. 4A). At 7 days of postnatal life (Fig. 4B),
RECEPTORS IN INTERSTITIAL CELLS OF CAJAL
331
Fig. 3. Neurokinin 1 receptor immunoreactivity (NK1r-IR) in
laminae of adult rat and guinea-pig ileum containing the circular
muscle layer or the longitudinal muscle layer plus the myenteric
plexus. A: NK1r-IR ICCs at the DMP of rat ileum. The cells have a
spindle or elongated body, the processes are thick and possess several
transverse and short branches. The cells appear regularly organized
in rows. C: NK1r-IR ICCs at the DMP of guinea-pig ileum. The cells
have a round or oval body and long and thin processes. The IR in both
animals has a punctate aspect. B,D: NK1r-IR ICCs at the MP of rat (C)
and guinea-pig (D) ileum. Both cells have a triangular shape and three
processes (asterisks) and the labeling is extremely faint. A,B,C,D,
⫻1,000. Scale bar ⫽ 10 µm.
SP/TK-IR varicose fibers were detected in the DMP
region. Between the first and third postnatal weeks, a
progressive enrichment in SP/TK-IR at the DMP was
observed (Fig. 4C). In the adult animal, the DMP
showed the most intense reactivity for SP/TK, now
appearing in transverse sections as a continuous line of
varicose nerve fibers (Fig. 4D). A similar labeling
distribution and intensity were observed in the ileum of
adult mice and guinea pigs.
These data demonstrate that the NK1r-IR on the
ICC-DMP and SP/TK-IR in nerve varicosities of the
DMP appeared very early after birth. Interestingly, the
intensity of the labeling for the NK1r was at its
maximum at 7 days of postnatal life, while that for
SP/TK reached the highest intensity in the adult age. It
has been reported that at 7 days of postnatal life the
ICC-DMP have already acquired adult features (Faussone-Pellegrini et al., 1996) and the high expression of
the NK1r with a peculiar distribution, different from
that observed in the adult animal, could imply a trophic
role of these receptors during a crucial period of the ICC
differentiation. Furthermore, immunohistochemical
studies in the rat ileum have demonstrated that at this
age the DMP shows NOS-IR, but not VIP or ChAT
labeling (Matini et al., 1997; Vannucchi and FaussonePellegrini, 1996). These data, together with the reported results on the presence of SP/TK-IR at this age,
also indicate that, very early in postnatal life, the DMP
contains both excitatory (SP) and inhibitory (NO) nerve
endings whose final targets seem to be ICC. The
difference between the earliest ages and the adulthood
in the NK1r distribution on ICC-DMP probably depends on a progressive membrane re-organization of
the same receptors in a cell that increases in size and
changes in shape.
Relationship Between the Tachykinergic Innervation and the Two Ileal ICC Populations. Simultaneous labeling with NK1r (Fig. 5A,B) or with the kit-r
polyclonal antibodies (Fig. 5C) and the SP monoclonal
antiserum revealed that, in the laminae obtained from
guinea-pig and rat ileum, numerous SP-IR varicose
nerve fibers were closely apposed to the ICC-DMP body
and processes and some of these fibers surrounded the
entire cell body, especially in the guinea pig (Fig. 5B). In
332
M.-G. VANNUCCHI
Fig. 4. Substance P/tachykinins-immunoreactivity (SP/TK-IR) in
the rat ileum during development. A: Five days of postnatal life. IR
varicose fibers (asterisks) emerging from the myenteric plexus, reach
the DMP region. B: Seven days of postnatal life. The varicose IR fibers
participate in the formation of the DMP and surround unlabeled cells.
C: Twenty-one days of postnatal life. IR varicose fibers become to be
uniformly distributed in the DMP region (asterisks). D: Adult rat. The
DMP shows an uniform and intense IR. mp ⫽ myenteric plexus;
dmp ⫽ deep muscular plexus; ocl ⫽ outer circular muscle layer; icl ⫽
inner circular muscle layer. A,B, ⫻1,000. Scale bar ⫽ 10 µm, C,D,
⫻400. Scale bar ⫽ 25 µm. Reproduced from Vannucchi et al. (1997)
with permission of the publisher.
contrast, in MP, most of the SP-IR nerve fibers were far
from the ICC, and SP-IR nerve varicosities were rarely
seen near these cells (Fig. 5C).
The observation that no other ICC population in the
gut but those in the ileum express the NK1r-IR (Portbury et al., 1996; personal observation) suggests that
the NK1r-IR labeling represents a immunohistochemical marker to identify the ileal ICC. The marked
difference in the labeling intensity between the ICCDMP and ICC-MP confirms that they are two distinct
ICC populations, and indicates that NK1r-IR is particularly useful for identifying the ICC-DMP. The different
NK1r labeling intensity of these cells has an important
functional significance and supports the hypothesis
that the two ICC populations play different roles. In
fact, using the simultaneous labeling, it was observed
that the intense NK1r-IR ICC-DMP were close to many
SP positive nerve varicosities in both rat and guinea pig
ileum, while the faint NK1r-IR ICC-MP were close to
very few SP-positive nerve varicosities. These findings
indicate that the ICC-DMP are implicated in the neurotransmission, whereas ICC-MP, considered as pacemak-
ers (Huizinga et al., 1995; Ward et al., 1994), are little
involved in neurotransmission.
ICC and NOS
NOS-IR was detected in guinea-pig ileum, in the
cytoplasm of non-neuronal cells located at the level of
the DMP that were identified as ICC (Fig. 6).
The presence of NOS in some ICC populations has
also been reported in other species (see Introduction),
and the possible significance of this enzyme in ICC is
discussed both in the Introduction and in the Concluding Remarks.
CONCLUDING REMARKS
Kit-r immunohistochemistry is the best tool to identify the ICC under the LM. The kit-r is involved in the
development and maintenance of the differentiated
state of ICC after birth. However, among ICC, ICCDMP seem to depend on the presence of kit-r for their
differentiation only partially and during a very short
period of postnatal life; besides, in adult W mutant mice
these cells are normally organized. The existence of
RECEPTORS IN INTERSTITIAL CELLS OF CAJAL
Fig. 5. Neurokinin 1 receptor (NK1r) (green) and Substance P (SP)
(red-orange) in the laminae of adult rat and guinea-pig ileum containing the circular muscle layer or the longitudinal muscle layer plus the
myenteric plexus. A: NK1r-IR ICCs at the DMP of the rat ileum with
closely apposed SP-IR varicose fibers. B: NK1r-IR ICCs at the DMP of
Fig. 6. NOS-immunoreactivity (IR) in transverse sections of guineapig ileum. The ICC-DMP (icc-dmp) appeared intensely labeled. x1000.
Scale bar ⫽ 10 mm.
other, unidentified, local factors whose presence during
the earlier stages of postnatal life could compensate the
kit-r impairment and guarantee the ICC differentiation, has been hypothesized to explain this diversity.
333
the guinea-pig ileum are closely apposed to SP-IR varicose fibers that,
in some instances, surround the entire cell body. C: NK1r-IR ICCs at
the MP of guinea-pig ileum and SP-IR varicose fibers of the tertiary
plexus. A–C: ⫻950, scale bar ⫽ 10 mm.
The observation that very early after birth ICC-DMP
express as much NK1r as in the adult, has suggested
that this receptor could also have a trophic function
during the earlier stages of life affecting the differentiation and network formation of this ICC population. Still
not clear is the significance of the kit-r presence on ICC
precursors from the first stages of the embryonic life up
to birth. During this period of prenatal life it has been
demonstrated that (1) the absence of kit-r does not
apparently affect the normal development of ICC; and
(2) ICCs develop normally during embryogenesis even
in the absence of neurons that are the source of the kit-r
specific ligand, the Steel factor.
The presence of sst2A receptor on ICC confirms the
complexity of the roles played by these cells that can
also be intermediaries in Som effects on the gut muscles.
The interesting observations that some receptors
such as kit-r and NK1r, which belong to two different
classes of receptors, are expressed by ICC very early
during ontogenesis, before the corresponding ligand
becomes necessary for cell differentiation (Kitr and
Steel factor) or is available in the synaptic vesicle
(NK1r and SP), need special attention. They suggest
that the commitment to synthesize the appropriate
molecules for the differentiation or for the functional
acquisition by the cell is independent of the presence of
the specific ligand and could be genetically determined,
or is due to local agents that act at less differentiated
stages. TEM investigations have shown that ICC are
334
M.-G. VANNUCCHI
consistently close to nerve endings from the very first
stages of development and could be the source of agents
able to induce the receptor expression. Indeed, these
contacts between the ICC-blast and the nerve endings
have been interpreted as the means by which some of
the blasts present become ICC instead of smooth muscle
cells or fibroblasts. On the other hand, it could also be
hypothesized that the ability of the differentiating ICC
to express these receptors is, in turn, the way to
influence the expression of the chemical codes in the
closely apposed nerve endings.
ACKNOWLEDGMENTS
The author thanks Dr. L. Corsani in performing
immunohistochemistry and P. Guasti and L. Calosi for
assistance in preparing the photographs.
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