Cell Tissue Res (1999) 297:111–117
© Springer-Verlag 1999
REGULAR ARTICLE
Ute Gröschel-Stewart · Michelle Bardini
Tim Robson · Geoffrey Burnstock
P2X receptors in the rat duodenal villus
Received: 11 January 1999 / Accepted: 8 March 1999
Abstract Immunohistochemical techniques were performed on freshly frozen sections of the duodenum of the
rat using specific polyclonal antibodies to unique peptide
sequences of P2X1–7 receptors. Of the antibodies to the
seven known P2X receptor subtypes that mediate extracellular signalling by nucleotides, three reacted with discrete
structures in the duodenal villus of the rat. Anti-P2X1 reacted with the capillary plexus in the intestinal villus,
which did not extend to the crypt region, suggesting that
nucleotides may be involved in the uptake and transport of
metabolites. Anti-P2X5 immunostained the membranes of
the narrow “stem” of villus goblet cells, where the nucleus
and cell organelles reside, possibly influencing synthesis
and release of mucins. P2X7 receptor immunoreactivity
was only seen in the membranes of enterocytes and goblet
cells at the tip of the villus, where cells are exfoliated into
the lumen, consistent with earlier findings that P2X7 is involved in apoptotic events. Thus, in complex structures
such as the intestinal villus, purinoceptors appear to participate in several and diverse signalling functions.
Key words P2X receptor · Immunohistochemistry ·
Duodenum · Rat (Sprague Dawley)
Introduction
Receptors responding to purine and pyrimidine nucleotides have been identified in both excitable and non-excitable cells, and purinergic signalling plays a role not
The support of Roche Bioscience in Palo Alto, USA, is gratefully
acknowledged.
U. Gröschel-Stewart
Institut für Zoologie, Technische Universität Darmstadt, Germany
M. Bardini · T. Robson · G. Burnstock (✉)
Autonomic Neuroscience Institute, Royal Free and University
College Medical School, Royal Free Campus,
Rowland Hill Street, London NW3 2PF, UK
e-mail: [email protected];
Tel: +44-171 830 2948; Fax: +44-171 830 2949
only in short-term events such as occur in neurotransmission and in secretion (see Burnstock 1997) but also in
long-term events such as embryonic development, cell
growth, proliferation and differentiation (Neary and
Burnstock 1996).
Two families of receptors to nucleotides are currently
recognized: a P2X receptor family, which are ligandgated ion channels, and so far seven subtypes have been
cloned and characterized; and a P2Y receptor family,
which are G protein-coupled receptors, and about nine
subtypes are established in vertebrates (Abbracchio and
Burnstock 1994; Burnstock and King 1996; Fredholm et
al. 1997; North and Barnard 1997). The distribution of
each subtype has been described largely on the basis of
in situ hybridization of mRNA (see Collo et al. 1996),
but some immunohistochemical localization of specific
antibodies has also been reported, mainly for P2X1,
P2X2 and P2X3 receptors (Vulchanova et al. 1996, 1997;
Cook et al. 1997; Chan et al. 1998; Llewellyn-Smith and
Burnstock 1998; Loesch and Burnstock 1998). P2X5 receptors have been claimed to be present in the brain,
heart, spinal cord and adrenal medulla (Garcia-Guzman
et al. 1997), and P2X7 receptors have been located principally on macrophages, monocytes, mast cells and microglia (Surprenant et al. 1996; Ferrari et al. 1997).
Rapid turnover rates are found in the epithelium of the
small intestine. The two most important compartments are
the crypt and villus. Crypts contain undifferentiated progenitor cells from which almost all other cell types arise,
such as the predominant absorptive cells (enterocytes) and
goblet cells as well as the more sparse neuroendocrine
cells and other cell types. The cells glide towards the villus tip, where they are finally ejected into the lumen. In
the rat, this whole process takes 3–4 days. The crypt cells
are thus the self-replenishing reservoir that constantly delivers new immature cells (see Madara 1991).
Since specific polyclonal antibodies to unique peptide
sequences of P2X1–7 receptors have recently become
available to us from Roche Bioscience (Chan et al.
1998), in the present study we have used these antibodies
in immunohistochemical studies in order to investigate
112
whether purines and P2 receptors are involved in signalling in the duodenal villus of the rat.
Materials and methods
Preparation of samples
The experiments were carried out on male or female SpragueDawley rats weighing 200–300 g. Principles of good laboratory
animal care were followed and animal experimentation was in
compliance with the specific national laws and regulations. The
animals were killed by a rising concentration of CO2 and cervical
dislocation, and subsequently were perfused, through the heart,
with 4% formaldehyde (in 0.1 M phosphate buffer) containing
0.2% of a saturated solution of picric acid (pH 7.4).
The duodenum was removed and postfixed in fresh fixative, as
above, for 2 h and finally cryoprotected in 7% sucrose, containing
0.05% merthiolate (Sigma), overnight. The tissue was embedded
in OCT compound (BDH) and frozen in isopentane pre-cooled in
liquid nitrogen. The tissues were sectioned at 12 µm, using a cryostat (Reichert Jung CM1800), and adjacent sections collected on
gelatin-coated slides and air dried at room temperature. The slides
were stored at –20ºC.
sites were blocked by a 20-min incubation with 10% normal horse
serum (NHS) in phosphate-buffered saline (PBS) containing
0.05% merthiolate (Sigma, Poole, UK).
The P2X antibodies were diluted to 5 µg/ml (determined as optimal by previous titrations) with 10% NHS; the myosin antibody
was used at 0.1 µg/ml. The specimens were then incubated overnight at room temperature with the primary antibodies. The secondary antibody was a biotinylated donkey anti-rabbit IgG (Jackson Immunoresearch, Pa., USA) used at 1:500 for 1 h, followed by the
extravidin peroxidase conjugate (Sigma) used at 1:1500 for 1 h. After washing, a nickel-diaminobenzidene (DAB) enhancement technique was used to visualize the reaction product. The specimens
were dehydrated to xylene and mounted using Eukitt (BDH).
The following control experiments were performed to establish
the specificity of the immunoreaction: omission of the primary antibodies; replacement of the primary antibodies with rabbit preimmune IgG or a non-reactive P2X antibody (here P2X6); and duplicate absorptions of the primary antibodies with their homologous peptide antigen.
The results were documented using the Edge R400 high-definition light microscope (Greenberg and Boyde 1997; Edge Scientific Instruments, Santa Monica, Calif., USA) with Kodak TMX
100 black-and-white film.
Results
Preparation of antibodies
The immunogens were synthetic peptides representing 15 receptor-type specific amino acids of the C-terminal part of the receptor:
1.
2.
3.
4.
5.
6.
7.
P2X1, amino acids 385–399 (ATSSTLGLQENMRTS)
P2X2, amino acids 458–472 (QQDSTSTDPKGLAQL)
P2X3, amino acids 383–397 (VEKQSTDSGAYSIGH)
P2X4, amino acids 374–388 (YVEDYEQGLSGEMNQ)
P2X5, amino acids 437–451 (RENAIVNVKQSQILH)
P2X6, amino acids 357–371 (EAGFYWRTKYEEARA)
P2X7, amino acids 555–569 (TWRFVSQDMADFAIL)
The peptides were covalently linked to keyhole limpet haemocyanin (KLH). Rabbits were immunized with the conjugated peptides
in multiple monthly injections (performed by Research Genetics,
Huntsville, Ala., USA).
Immunoglobulin G (IgG) fractions were isolated from the preimmune and immune sera (P2X1–7) following the method of
Harboe and Ingild (1973). The protein concentration was determined at 280 nm using an extinction factor of 1.43 for 1 mg/ml.
Rabbit polyclonal antibody to chicken smooth muscle myosin
was prepared as described elsewhere (Gröschel-Stewart et al.
1985).
The specificity of the P2X antibodies was verified by immunoblotting with membrane preparations from cloned P2X1–7 receptorexpressing CHO-K1 cells. The antibodies recognized only one protein of the expected size in the heterologous expression systems and
have been shown to be receptor subtype-specific by our colleagues
at Roche Bioscience (I. B. Ogelsby, W. G. Lachnit, G. Burnstock,
A. P. D. W. Ford, submitted for publication). Western blot analyses
with P2X5- and P2X7-specific antisera have been reported by
Gröschel-Stewart et al. (1999). Meaningful Western blot analyses of
duodenal extracts were not possible due to strong interference of E.
coli proteins (in the intestinal extracts), with the omnipresent E. coli
antibodies in all sera (immune, pre-immune and control).
Immunohistochemistry
The avidin-biotin (ABC) technique was employed according to the
protocol described by Llewellyn-Smith et al. (1992, 1993). The
sections were left at room temperature for at least 10 min. Endogenous peroxidase was blocked with 50% methanol containing
0.4% hydrogen peroxide (H2O2) for 10 min. Non-specific binding
Tissues from a total of five animals were examined. The
figures shown, however, are of adjacent sections from
the same animal and block.
Anti-smooth muscle myosin, used here as a positive
control, reacted in sections of rat duodenum with the inner circular and outer longitudinal smooth muscle layer
of the tunica muscularis, the thin muscularis mucosa lying immediately beneath the mucosal crypts, the fine
muscle strands extending from it into the villus core, and
with the vascular smooth muscle and the myosin fibrils
in the terminal web of the brush border (Drenckhahn and
Gröschel-Stewart 1980; Fig. 1a,b). In contrast, the antibody to P2X1 showed a somewhat different immunostaining pattern (Fig. 2a,b) suggestive of villus bloodand lymph-capillary network staining. P2X1 receptor immunoreactivity was clearly restricted to the villar region;
it did not extend into the crypts or beyond (Fig. 2b).
Along the crypt-villus axis, intact P2X5-immunoreactive goblet cell stems were seen in both longitudinal
(Fig. 3a) and transverse sections (Fig. 3b), with the region of such immunoreactivity extending from the base
of the villus to about 50% up. This was followed by a region of goblet cell heads that were largely P2X5-negative
and P2X7-negative. However, in the tip region, comprising about 10% of the total villus length, membrane-associated P2X7 immunoreactivity was seen. The enterocytes
did not show any P2X5 immunoreactivity along the
crypt-villus axis where exfoliation of enterocytes takes
place. At the light-microscopy level, the reaction appeared to be membrane-associated (Fig. 4a,b), giving a
honeycomb-like pattern on the apical surface. Thus P2X7
receptor immunoreactivity was clearly associated with
the region of exfoliation.
No immunoreactivity to P2X2, P2X3, P2X4 (data not
shown) or P2X6 antibodies (Fig. 5) was seen in any sections of our study of the rat duodenal villus. Other tissue
113
Fig. 1a, b Transverse section of rat duodenum: reaction with antibodies to smooth muscle myosin. a The antibody visualizes the
longitudinal and circular muscle layers, the muscularis mucosae,
the fine muscle strands in the villi, vascular smooth muscle and
myosin in the brush border (arrowhead). ×75. b A higher magnification, showing the immunoreactivity to smooth muscle myosin in
the fine muscle strands in the villi core. ×300
immunolocalization studies using these P2X antibodies,
however, have identified immunoreactivity to P2X2 (rat
hypothalamus; Xiang et al. 1998a), to P2X3 (rat trigeminal ganglion; Llewellyn-Smith and Burnstock 1998), to
both P2X2 and P2X3 (taste buds; Bo et al. 1999), and to
P2X1–6 receptors to varying degrees in the rat sensory
ganglia (Xiang et al. 1998b).
In our present study, when the primary P2X antibodies
were preabsorbed with their respective peptide antigens,
immunostaining was not observed. These results and the
previously reported western blotting analysis (Chan et al.
1998; Bo et al. 1999; Gröschel-Stewart et al. 1999) confirm the specificity of the immune reaction shown here.
Discussion
With specific antibodies to P2X receptor isoforms now
being available, a thorough screening for the receptors in
all neuronal and non-neuronal tissues by imunohistochemical methods can now be performed. The results
obtained to date confirm that P2X receptors play an important role in many physiological processes and that
their expression may be altered under pathological conditions (Abbracchio and Burnstock 1998).
In the normal rat duodenum, we have immunolocalized all seven P2X receptors, with varying intensities, in
the myenteric and submucosal plexus (to be reported
elsewhere). This present report will be restricted to the
findings in the duodenal villus.
The intestinal epithelium, a cellular monolayer, rests
on a basal lamina covering the underlying lamina propria, much of it organized into flattened leaf-like projections, the villi. The lamina propria, a connective tissue
core, contains a thin layer of smooth muscle (muscularis
114
Fig. 2a, b Transverse sections (adjacent to that of Fig. 1) of rat
duodenum: reaction with antibodies to P2X1. a Immunoreactivity
to P2X1 receptors is visualized in a fine capillary network in the
villus core. ×75. b P2X1 immunoreactivity is restricted to the villar region and does not extend into the crypts or beyond. Note a
differing pattern of reactivity to that with anti-smooth muscle
myosin (in Fig. 1b). ×430
mucosae; see Fig. 2) and supports a capillary net arising
at the villus tip from a central artery and from tiny
lymphatic vessels draining into a larger lymphatic vessel,
the so-called lacteal. Small nerve fibres also appear to be
scarcely scattered throughout the lamina propria, terminating near the villus epithelium (Madara 1991; Conigrave and Young 1996). Between the villi are well-like
depressions, the crypts, also confluently lined with epithelial cells. According to Cheng and Leblond (1974)
and Ponder et al. (1985), all cell types found in crypt and
villus arise from a small pool of continuously replenished common stem cells within the crypts. While some
cells remain in situ as stem cells, others migrate in the
direction of the crypt-villus junction while they undergo
differentiation. They continue to divide at first and eventually become the non-dividing mature villus cells. The
majority of the cells will develop into enterocytes as the
absorptive elements for nutrients, electrolytes and water.
A few will acquire mucous globules and give rise to goblet cells secreting mucins and trefoil peptides for the protection of the surface (Thim 1994; Kindon et al. 1995).
An even smaller portion acquires dense granules and
small filament bundles and differentiates into entero-endocrine cells (Cheng and Leblond 1974).
Little is known about what determines a cell to develop into a particular cell type while they begin their migration in vertical bands to the villus tip, where they will
eventually be shed. It has been shown that in late fetal
and neonatal life epithelial cells (both the absorptive and
goblet cells) project cytoplasmic processes through gaps
of the basal lamina into the lamina propria and form contacts to mesenchymal cells (Mathan et al. 1972). It is assumed that a transfer of material occurs that facilitates the
maturation of the duodenal mucosa, but it is not known
whether such signals still play a role in adult life. The ex-
115
Fig. 3a, b Transverse section of rat duodenum: reaction with antibodies to P2X5. The antibodies visualize the membranes of the tapered
stalk of villar goblet cells, both when sectioned longitudinally (a) or transversely (b). ×150
Fig. 4a, b Transverse section of rat duodenum: reaction with antibodies to P2X7. The micrographs show the villar tips. a Note the
honeycomb-like staining of the membranes, the inset showing that
it is punctate. ×200; inset x500. b Both enterocytes (arrow) and
goblet cells (arrowhead) are being extruded. ×200
clusive localization in the present study of P2X5 receptors
in the membrane of the narrow stem, where nucleus and
cell organelles are localized, suggests that nucleotides
acting via P2X5 receptors may be involved in goblet cell
maturation, possibly in collaboration with growth factors
such as epidermal growth factor (Toyoda et al. 1986).
This would be another example of this member of the
family of ATP-gated ion channels (Collo et al. 1996;
Garcia-Guzman et al. 1996) acting on peripheral tissues.
Secretion and absorption in the intestinal mucosa are
not only under endocrine and paracrine control but also
influenced by innervation through the submucosal plexus. The secretory enterocytes of the crypt are predominantly under cholinergic control (Furness and Costa
1980) and also controlled directly by vasoactive intestinal polypeptide (Costa and Furness 1983). In addition,
crypt goblet cells have been shown to discharge their
mucin granules when exposed to muscarinic cholinergic
agents, whereas the goblet cells of the villi did not respond (Phillips et al. 1984). With the demonstration in
the present study that P2X5 receptors were localized on
villar goblet cell stems, one might consider the possibility that synthesis and/or release of mucins are under purinergic control. Alternatively, it is possible that purines
are involved in the differentiation or turnover of goblet
cells. This latter proposition is supported by other studies
from this laboratory. We have previously shown
116
Fig. 5 Transverse section of rat duodenum: reaction with antibodies to P2X6. This antibody does not react with any structure in the
villar region and serves therefore as a negative control. ×50
(Gröschel-Stewart et al. 1999) that P2X5 immunoreactivity is associated largely with the cell periphery/membrane region of proliferating and differentiating (spinous
and granular) cell layers of various stratified squamous
epithelia (cornea, tongue, vagina and skin). P2X5 receptors have also been immunolocalized in various epithelia
of the urogenital tract (H. Y. Lee, M. Bardini, G. Burnstock, submitted for publication) and in primordial ovarian follicles (M. Bardini, H. Y. Lee, G. Burnstock, unpublished work). These examples give clear evidence that
P2X5 immunoreactivity is associated with metabolically
active, proliferating and differentiating cells.
When the mature villar epithelial cells arrive at the
tip of the villus, they reach the endpoint of their life
cycle and they are subjected to “programmed cell
death” (Budtz 1994). They are ejected into the lumen,
possibly by the force exerted from the upward movement of new cells. This process is clearly associated
with the appearance of P2X7 immunoreactivity. During
their upward migration, goblet cell stems eventually
loose their distinct P2X5 immunoreactivity. Only empty goblet heads are seen and at the upper extremity
their membranes acquire P2X7 immunoreactivity. The
extruding villar enterocytes at the very tip also show a
membrane-associated P2X7 immunoreactivity, result-
ing in a honeycomb pattern at the apical surface. The
same pattern is seen in the sloughed-off uterine and
cervical epithelia (M. Bardini, H. Y. Lee, G. Burnstock, unpublished work). P2X7 immunoreactivity is
strongest in the keratinized and exfoliated layers of
stratified squamous epithelia (Gröschel-Stewart et al.
1999). All these examples strongly support the view
that P2X7 receptors are associated with cell death and
apoptotic events (Surprenant et al. 1996; Collo et al.
1997; Rassendren et al. 1997).
In contrast to secretion, where dramatic changes in
transport rates are seen, the absorption processes in the
villar enterocytes take place at a relatively steady rate.
Nevertheless, adrenaline, somatostatin and other peptide
neurotransmitters can act directly on enterocytes and increase the rate of absorption (Cooke 1986; Brown and
Miller 1991; Conigrave and Young 1996). Participation
of purines and purinoceptors in epithelial transport has
been suggested, either via direct action on epithelial
cells or via a neural component (Burnstock 1979).
Although P2X receptors were not localized on the ascending absorptive enterocytes themselves, we found
P2X1 receptor immunoreactivity associated with the
capillary network of the lamina propria. P2X1 is usually
seen in the membranes of smooth muscle cells in arteries, bladder, ureter and vas deferens. However, the
staining pattern seen here does not resemble that of
smooth muscle in the villus core but more closely resembled the previously reported visualization of the villus blood- and lymph-capillary network by antibodies to
non-muscle myosin (Gröschel-Stewart et al. 1985). A
double-staining experiment using fluoresceine-labelledantibodies to P2X1 and smooth muscle myosin proved
that the staining patterns are indeed different (data not
shown). The P2X1 staining, restricted to the villus core
and not connecting to the muscularis mucosae, closely
resembles the pattern previously seen after the application of antibodies selective for non-muscle myosin, reacting with the villar core capillary network (GröschelStewart et al. 1985). Although the presence of P2X receptors on endothelial cells is still being discussed, it is
possible that ATP, via P2X1 receptors, either directly or
indirectly influences the absorptive capacity of the
lymph and blood capillaries and thus the transport of absorbed nutrients in to the body circulation. This would
also explain why P2X1 receptor immunoreactivity terminates at the base of the villus proper.
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