Department of Medicine, Division of Gastroenterology and Hepatology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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ABSTRACT |
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Recent investigations have suggested carbon monoxide (CO) as a putative messenger molecule. Although several studies have implicated the heme oxygenase (HO) pathway, responsible for the endogenous production of CO, in the neuromodulatory control of the internal anal sphincter (IAS), its exact role is not known. Nitric oxide, produced by neuronal nitric oxide synthase (nNOS) of myenteric neurons, is an important inhibitory neural messenger molecule mediating nonadrenergic noncholinergic (NANC) relaxation of the IAS. The present studies were undertaken to investigate in detail the presence and coexistence of heme oxygenase-2 (HO-2) with nNOS in the opossum anorectum. In perfusion-fixed, frozen-sectioned tissue, HO-2 immunoreactive (IR) and nNOS IR nerves were identified using immunocytochemistry. Ganglia containing HO-2 IR neuronal cell bodies were present in the myenteric and submucosal plexuses throughout the entire anorectum. Colocalization of HO-2 IR and nNOS IR was nearly 100% in the IAS and decreased proximally from the anal verge. In the rectum, colocalization of HO-2 IR and nNOS IR was ~70%. Additional confocal microscopy studies using c-Kit staining demonstrated the localization of HO-2 IR and nNOS IR in interstitial cells of Cajal (ICC) of the anorectum. From the high rate of colocalization of HO-2 IR and nNOS IR in the IAS as well as the localization of HO-2 IR and nNOS IR in ICC in conjunction with earlier studies of the HO pathway, we speculate an interaction between HO and NOS pathways in the NANC inhibitory neurotransmission of the IAS and rectum.
nonadrenergic noncholinergic; interstitial cells of Cajal; internal anal sphincter; inhibitory neurotransmission
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INTRODUCTION |
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CARBON MONOXIDE (CO) has recently attracted much attention as a possible neuronal messenger molecule. The heme oxygenase (HO) pathway is primarily responsible for the endogenous production of CO. HO cleaves heme producing CO and biliverdin and heme-releasing iron. To date, three isoforms of HO have been characterized, HO-1, HO-2, and HO-3. These isoforms are distinct in their regulation and expression. HO-1 is a stress protein and is induced by a wide variety of stimuli such as metal ions, hypoxia, and bacterial and viral toxins (15). The spleen characteristically has HO-1 as the predominant form of the isozyme, and it has also been localized in bone marrow, liver, and intestinal mucosa. HO-2 is the constitutive isoform and is more prevalent in peripheral neural tissues, the brain, testes, and endothelial cells of the arteries (15). HO-3 has recently been found in spleen, liver, thymus, prostate, kidney, brain, and testes (16).
Only a few studies have examined the presence of HO-2, HO activity, and CO effects in the gastrointestinal tract. HO-2 has been localized primarily in the enteric neurons of different regions of the gastrointestinal tract, the parietal and gastrin-containing cells of the gastric mucosa, and the interstitial cells of Cajal (ICC) networks of the mouse small intestine (7, 11, 17, 19, 20, 28).
Nitric oxide (NO), produced by neuronal nitric oxide synthase (nNOS), is an established neural messenger. NOS and HO bear many similarities and have been colocalized in the murine ileum (30). In addition, mice with gene deletions respectively targeted for HO-2 and nNOS exhibited profound changes in the gastrointestinal motility (30). Specifically, nonadrenergic noncholinergic (NANC) relaxation was found to be reduced dramatically in HO-2 and nNOS knockout mice compared with normal wild-type mice (30).
NANC nerves intrinsic to the enteric nervous system mediate relaxation of the internal anal sphincter (IAS) smooth muscle. Histochemical, immunocytochemical, and physiological studies have displayed the multiplicity of neural messengers involved in NANC relaxation of the IAS (3, 14, 18, 21, 22, 26). CO directly relaxes the IAS and increases intracellular concentrations of cGMP, and an HO inhibitor, zinc protoporphyrin IX, inhibits NANC relaxation of the IAS (22). However, the exact role of the HO pathway in the inhibitory neurotransmission and the exact localization of HO in the IAS is not known.
Both morphological and physiological investigations have implicated ICC in gastrointestinal electrical rhythmicity and neurotransmission (4, 12, 23). Numerous studies have characterized ICC types in various regions of the gastrointestinal tract in several distinct species (1, 10, 17). Recent studies in humans have shown the presence of ICC in the IAS and rectum myenteric and submucosal plexuses (9). However, in these studies, the presence of HO-2 and nNOS in the ICC has not been examined. Interestingly, a specific type of ICC, intramuscular ICC, has been proposed to be involved in NO-dependent neurotransmission in the lower esophageal sphincter (LES) and pyloric sphincter (27). With the advent of a marker for ICC, antibodies against the protooncogene c-kit, morphological studies have targeted the localization of molecules involved in neurotransmission, such as HO-2 and nNOS, in ICC (1, 17, 23, 29). However, the localization of HO-2 and nNOS in the ICC of anorectum has yet to be demonstrated.
Recent studies by Ny et al. (20) have shown the presence of HO-2 and its colocalization with nNOS in different regions of the gastrointestinal tract, including the sphincteric regions. However, the specific details of the presence of HO-2 and its colocalization with nNOS in the myenteric as well as the submucosal plexuses and the regional variation of their distribution scanning the entire anorectum have not been examined. Also, this study did not specifically examine the localization of HO-2 and nNOS in ICC. To date, the bulk of the physiological data regarding the neural regulation of the IAS, especially the HO and NOS pathways, have been obtained in a experimental animal model, the opossum. The presence of HO and its copresence with NOS, however, have not been examined in this animal model.
Therefore, the aim of the present study was to examine systematically the localization of HO-2 and its colocalization with nNOS in the myenteric and submucosal plexuses in the IAS and their regional distribution throughout the anorectum of the opossum. Furthermore, specific studies were designed to examine the localization of HO-2 and nNOS in the ICC of the anorectum.
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MATERIALS AND METHODS |
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Tissue preparation. Adult opossums were deeply anesthetized with intraperitoneal pentobarbital sodium (40 mg/kg) and then transcardially perfused with heparinized PBS solution (0.01 M) at pH 7.4 at room temperature. This was followed by perfusion with ice-cold fixative (4% paraformaldehyde and 0.2% picric acid in PBS, pH 7.4). The details of the procedure have been given before (14). Animals were handled according to procedures approved by the Institutional Laboratory Animal Use and Care Committee (Thomas Jefferson University). The anorectum was opened with a single longitudinal cut, rinsed with PBS, laid flat (but not stretched), and pinned to a rubber pad. The tissue was postfixed for 2 h, followed by cold 20% sucrose in PBS overnight at 4°C. The IAS, defined as the region of thickened muscular wall, and rectum, beginning where the thickened wall ends, were frozen over dry ice and set into blocks measuring 1 cm by 2 cm (longitudinal direction) in OCT (Miles, Elkhart, IN). In some studies, the anorectum was procured as described above. The tissue was cut into three blocks of 2 cm (longitudinal direction) by 1 cm in OCT and frozen over dry ice with the distal and proximal ends carefully marked. An approximate length of 6 cm ensured the inclusion of the entire IAS, the transitional area between the IAS and rectum, and a distal portion of the rectum. The tissue was frozen-sectioned (20 µm) in the cross-sectional plane proceeding from the distal end to the proximal end. The sections were collected on chrome-alum-coated glass slides (coating to improve adhesion to the slide) and collected in sets of every fourth serial section. Cell counts were performed on every fourth section.
HO-2 and nNOS immunoreactivity. Immunocytochemistry was performed by the indirect immunofluorescence method as described in our previously published studies (14). Sections were incubated in a mixture of 1:1,000 HO-2 antiserum raised in rabbit (Stressgen, Victoria, BC) and 1:600 nNOS antiserum raised in mice (Transduction Labs, Lexington, KY) diluted in PBS containing 0.5% BSA and 0.2% Triton X-100 overnight at room temperature in a humid chamber. Additional sections were incubated in 1:800 HO-1 antiserum raised in rabbit (Stressgen). The sections were then rinsed in PBS (three times for 5 min) and incubated in a mixture of secondary antibodies raised in donkey (Jackson Laboratories, West Grove, PA). HO-2 was labeled with secondary antibody conjugated to Texas red diluted 1:200 in a solution of 2% normal donkey serum and 0.3% Triton X-100 in PBS, whereas nNOS was labeled with secondary antibody conjugated to FITC diluted 1:100 in the same solution. The slides were incubated for 60 min at room temperature, rinsed in PBS, air dried, and coverslipped with Vectashield (Vector Labs, Burlingame, CA). The sections were observed with fluorescent microscopy using the appropriate filters and photographed. Some sections were immunostained as described above but with the primary antibodies removed (used as controls).
A neuron was only considered to be HO-2 immunoreactive (IR) or nNOS IR when several specific criteria were met. The majority of neurons were found clustered in ganglia throughout the plexuses. Only neurons with clear, full-size profiles of cell bodies were counted. Often, there were obstructions, such as fibers, overlapping with ganglia, making it difficult to determine their immunoreactivity. Neurons that were not distinct, due to obstruction, were not counted. The technique ensured a high degree of confidence in our cell counts (14).c-Kit IR and confocal microscopy.
For immunohistochemical studies examining the distribution of ICC in
the anorectum, a rat monoclonal antibody raised against c-Kit protein (GIBCO BRL, Gaithersburg, MD), an
established marker for ICC (29), was used. Freshly dissected tissues
were thoroughly cleaned with PBS and then cryoprotected in graded
sucrose solutions (5, 10, 15, and 20% in PBS). Tissues were next set
into 2 cm by 2 cm blocks over liquid nitrogen. Sections were cut as
outlined above. Sections were fixed in cold acetone at 20°C
for 2 min and then thoroughly washed in PBS. Nonspecific antibody
binding was blocked by 60-min incubation in 10% normal goat serum
(NGS) in PBS in a humid chamber at room temperature.
Immunocytochemistry was performed by the indirect immunofluorescence
method as described in HO-2 and nNOS immunoreactivity. Sections
were incubated either in a mixture of 5 µg/ml c-Kit antiserum and
1:1,000 HO-2 antiserum diluted in PBS containing 0.5% BSA, 2% NGS,
and 0.2% Triton X-100 overnight at room temperature in a humid chamber
or in c-Kit antiserum (5 µg/ml) and 1:500 anti-nNOS polyclonal
antibody raised in rabbit (Transduction Labs) prepared in the same
solution. The sections were then rinsed and incubated in secondary
antibodies raised in goat (Vector Laboratories). c-Kit was labeled with
secondary antibody conjugated to Texas red diluted 1:200 in a solution
of 2% NGS in PBS, whereas HO-2 and nNOS were labeled with secondary antibodies conjugated to FITC for 60 min at room temperature, rinsed in
PBS, air dried, and coverslipped with antifade medium (Slow-Fade;
Molecular Probes, Eugene, OR). Some sections were immunostained as
above but with primary antibodies removed (used as controls).
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RESULTS |
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Neurons were found clustered into ganglia throughout the myenteric and submucosal plexuses of the anorectum. Ganglia were smaller and more evenly spaced in the rectum than in the IAS. Single neurons were occasionally observed, more often in the rectum than in the IAS.
Distribution of HO IR.
HO-2 IR was found throughout the myenteric and submucosal plexuses of
the anorectum (Figs.
1-3).
HO-1 IR was seldom present in any of these plexuses. Because of this,
further detailed analyses of HO-1 IR in the present studies were not
carried out. HO-2 IR was mostly present in the neuronal cell bodies.
However, occasional staining of nerve fiber bundles and fine nerve
fibers extending into the circular muscle was also found. Also, single
neurons in the circular muscle exhibited HO-2 IR. The fluorescence of HO-2 IR nerve cell bodies was variable. Although in the majority of
cases the fluorescence was intense, in some cases it was less intense.
HO-2 IR was also found in the epithelium as well as endothelial linings
of arteries. Interestingly, the epithelium and endothelium linings of
the veins were found to be devoid of HO-2 IR, and HO-2 IR
was virtually nonexistent in the smooth muscle cells. These results are
summarized in Table 1.
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Colocalization of HO-2 and nNOS IR. Distribution of nNOS IR was similar to that of HO-2 with certain notable differences. nNOS IR was only observed in the epithelium in endocrine-like cells. The majority of nerve fibers and specifically fiber bundles exhibited a strong fluorescence for nNOS.
In the IAS, HO-2 IR and nNOS IR colocalized 98% of the time (228/233 neurons counted) in the myenteric plexus and 96% of the time (165/171 neurons counted) in the submucosal plexus. In the rectum, HO-2 IR and nNOS IR were colocalized ~70% of the time in both the myenteric plexus (323/467 neurons counted) and submucosal plexus (229/328 neurons counted) (Fig. 1). To further examine the regional variation in colocalization of HO-2 IR and nNOS IR within the anorectum, data were recorded from every fourth section from a set of serial sections from the anal verge up to 52 mm proximally. A total of 2,131 neurons were counted, of which 1,695 colocalized HO-2 IR and nNOS IR. The high percentage of colocalization in the myenteric plexus fell from almost 100% in the IAS to 56% proximally in the rectum (determined as 52 mm above the anal verge) (Fig. 1). This readily apparent trend of decreasing colocalization in the myenteric plexuses was less apparent in the submucosal plexus (Fig. 3). We found the region of highest nerve density to be the transition region between the IAS and rectum and the distal rectum. The localization of these neurons may correspond to that of the NANC inhibitory neurotransmission in the IAS. Although the transition from squamous to cuboidal epithelium was apparent more distally, at 16 mm from the anal verge, the true rectum was distinguished as the end of the thickened muscular wall and began proximally at 24 mm. The change in epithelium was used to mark a transition zone spanning 6 mm until the rectum began at 24 mm. Very few neurons were found in the most distal 6 mm of the anal canal. Both HO-2 and nNOS appeared in nonneuronal cells that morphologically fit the description of ICC (25). ICC were characterized by a fusiform cell body and dendritic processes. This suggests a possible nonneuronal role for HO-2 and nNOS in the opossum anorectum. This is consistent with other studies that examined HO-2 IR and nNOS distribution throughout the rat, canine, and feline gastrointestinal tracts (7, 8, 17, 20). Furthermore, we carried out studies that were not undertaken before. We used a combination of antibodies specific for c-Kit, HO-2, and nNOS to examine the presence and colocalization of HO-2 and nNOS IR in the ICC of the anorectum. c-Kit IR was observed throughout the anorectum. Control studies eliminating primary antibodies showed no labeling. Consistent with other studies, two cell types exhibited c-Kit IR: ICC and mast cells (9). These cells could be distinguished on the basis of their morphology. Mast cells are larger than ICC and are round with a round central nucleus. ICC are smaller fusiform cells with an oval nucleus. ICC also contain varying numbers of processes. ICC were often found to be associated with neural elements, such as ganglia and nerve fibers. However, clusters of ICC were found in septa separating muscle fiber bundles. From our thin section studies, the ICC present among muscle fibers were more interspersed within the IAS than within the rectum. ICC would also interconnect in networks; this was more readily observable in the rectum than in the IAS. ICC were more readily observable in the myenteric than in the submucosal plexuses. However, abundant c-Kit labeling was found in the submucosa. Most of the c-Kit-IR cells were easily identifiable as ICC, as explained above. In addition to the myenteric and submucosal plexuses, HO-2 IR and nNOS IR were also found to be present within the ICC (Figs. 4-7). The colocalization of c-Kit IR with HO-2 IR and nNOS IR further suggests an interaction between HO and NOS pathways in the ICC for the inhibitory neurotransmission of the IAS.
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DISCUSSION |
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This is the first report that describes the distribution of HO-2 and its colocalization with nNOS in both the myenteric and submucosal plexuses and that describes the localization of HO-2 and nNOS in ICC of the anorectum. In neurons in the IAS, HO-2 IR and nNOS IR colocalized nearly 100% of the time. In the rectum, the percent colocalization drops to nearly 70% and decreases further proximally. Also, the percent colocalization varies in the rectum between the myenteric and submucosal plexuses. In the submucosal plexus, although there was a general decrease as one proceeded proximally in the rectum, it was not as defined as in the myenteric plexus. This suggests a higher level of functional interactions at the level of motor inhibitory neurotransmission between NO and HO-2 in the IAS than in the rectum.
The level of colocalization of HO-2 and nNOS in the enteric plexuses in the opossum IAS was found to be considerably higher than reported before in other parts of the gastrointestinal tract (20). The reason for these differences may be related to species differences, the experimental design, and the large number of neurons (~2,000) counted in the present study. In our earlier studies in the opossum (22), we have demonstrated the presence of an actual interaction between the HO and NOS pathways, since NANC relaxation of the IAS was found to be suppressed by an HO inhibitor. The primary mediator of the NANC relaxation of the IAS is NO generated via the NOS pathway.
In addition, the studies showed the equally high level of colocalization of HO-2 and nNOS in the submucosal plexuses of the IAS. This high level of colocalization (~100%) of these pathways in the IAS has not been shown before. The colocalization of HO-2 and nNOS in the submucosal plexus in the rectum appears to be relatively less compared with that in the myenteric plexuses. The nature of interaction between the HO and NOS pathways in the motor inhibitory neurotransmission and at the sensory level in the anorectum remains to be determined.
The immunocytochemical studies lend further support to the possibility of CO having a functional role in the NANC relaxation of the IAS that is mediated via the NOS pathway. Our laboratory has already shown direct relaxation of the IAS smooth muscle in response to CO via an increase in cGMP and an attenuation of the NANC relaxation by the HO inhibitor (22). Similar responses of CO were later verified in the feline and porcine LES (19, 28). Furthermore, CO has been shown to modulate potassium current and membrane potentials of isolated circular smooth muscle cells of human and canine jejunum (6, 7). In the feline LES, an increase in the HO activity corresponded to the NANC relaxation of the circular smooth muscle, but the HO inhibitors (zinc protoporphyrin and tin protoporphyrin) failed to affect the NANC relaxation of the smooth muscle (19). The data suggest that HO inhibitors may exert a wide range of species-related effects in the same gastrointestinal sphincteric region, facilitatory modulation of the NOS pathway in one (2) and inhibitory modulation in the other (5, 22).
Our laboratory has shown previously that an HO inhibitor activates NOS in the rabbit IAS (2) and suppresses that in the opossum (22). Furthermore, studies of transgenic mice with the HO-2 or nNOS gene deleted suggest a complementary role for CO and NO in NANC relaxation of murine ileum, with each being responsible for approximately one-half (30). In the same study, HO-2 and nNOS were colocalized 60-70% of the time in myenteric ganglia. In contrast, in rat endothelial cells, HO-2 and NOS have been shown to have a compensatory relationship (24).
In the present study, in contrast to HO-2 IR, in general, HO-1 IR was found not to be present in the anorectum. HO-1 IR, according to other studies, was relatively more visible compared with that in the opossum anorectum, in different regions of the canine and feline gastrointestinal tract. Furthermore, in those studies, HO IR and nNOS IR have been shown to be colocalized to some degree in different regions of the feline and canine alimentary tracts including the sphincteric regions (20). It is possible that the differences of HO-1 IR in the opossum anorectum vs. feline and canine gut are due to species differences.
Both HO-2 and nNOS appeared in nonneuronal cells that morphologically and anatomically fit into the description of ICC. This suggests a possible nonneuronal role for HO-2 and nNOS in the opossum anorectum. This is consistent with other studies in which HO-2 IR and nNOS distribution were examined throughout the rat, canine, and feline gastrointestinal tracts (7, 8, 17, 20). Interestingly, abnormal ICC have been specifically associated with Hirschsprung's disease (9, 13). The exact anatomic location of ICC in relation to myenteric neurons in myenteric plexuses and IAS smooth muscle cells has not been ascertained. However, on the basis of earlier studies in different systems, it is speculated that a major role of ICC in the anorectum may be to amplify the NANC inhibitory neurotransmission, which is primarily NOS mediated. The presence of nNOS IR in the ICC of the anorectum supports that speculation and implies that NO release from ICC may act at the myenteric inhibitory neurons, which may cause further release of NO from ICC before its action at the smooth muscle cells. In addition, it is possible that NO released from ICC may act at the myenteric neurons to potentiate or otherwise modulate the inhibitory neurotransmission. The exact roles of ICC in the genesis of slow waves and in the electromechanical coordination or rhythmicity in the anorectum remain to be determined.
The variations in the densities of ICC in different areas of the anorectal region suggest specific functional differences (9). Our studies demonstrating differences in the density of ICC, lower in the IAS than in the rectum, may support that speculation. The exact significance of these findings remains to be determined. It is possible that, in whole mount preparations of the IAS, some ICC may be obscured that are buried deep in the smooth muscle. The use of confocal microscopy in the present study may have facilitated the visibility of such cells.
It is well known that ICC are of different types and may have multiple roles in gastrointestinal motility (9, 23). A thorough characterization of ICC types in the anorectum, and specifically in the IAS, would help in further delineating the roles of ICC in this region. The exact identification and quantification of different classes of ICC in different regions of the anorectum would require confocal microscopic examination of whole mounts and detailed electron microscopic studies of the IAS and rectum. The present studies demonstrating the localization of HO-2 and nNOS in the ICC of anorectum suggest a role for ICC and of NOS and HO pathways in the inhibitory neurotransmission of the anorectum.
We conclude that HO-2 IR, but not HO-1 IR, is present in the enteric plexuses of the anorectum. There is a high degree of colocalization between HO-2 IR and nNOS IR throughout the anorectum with some regional variations. In the anorectum, the colocalization is highest in the IAS. The studies also show the presence of HO-2 IR and nNOS IR in the ICC. The findings suggest a modulatory role for the HO pathway in the NOS-mediated NANC inhibitory neurotransmission of the IAS. We speculate the involvement of HO and NOS pathways at both the levels of the enteric nervous system and the ICC. The exact site and nature of the interaction between the two pathways at the synaptic and ICC levels remain to be determined.
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ACKNOWLEDGEMENTS |
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We thank Priya Hingorani and Natalie Innocent for assistance in the preparation and interpretation of confocal immunofluorescent images.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-35385 and an institutional grant from Thomas Jefferson University.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. Rattan, Professor of Medicine and Physiology; 901 College, Dept. of Medicine, Division of Gastroenterology and Hepatology, 1025 Walnut St., Philadelphia, PA 19107 (E-mail: satish.rattan{at}mail.tju.edu).
Received 23 March 1999; accepted in final form 30 September 1999.
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