1 Gastrointestinal Diseases Research Unit, Queens University, Kingston K7L 5G2; and 2 Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5
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ABSTRACT |
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The calcium-binding protein neuronal calcium sensor 1 (NCS-1) is involved in modulation of neurotransmitter release in the peripheral and central nervous systems. Since intestinal inflammation impairs neurotransmitter release, we evaluated the expression of NCS-1 in the normal rat colon and in dinitrobenzene sulfonic acid (DNBS)-induced colitis. Immunocytochemistry and Western blots showed high levels of NCS-1 in the myenteric plexus and in axons in the smooth muscle layers; 23 ± 2% of myenteric neurons were NCS-1 positive, with staining restricted to the largest neurons. NCS-1-positive axons decreased to 13.3 ± 0.4% of total axons by day 2 and dropped further to 7.0 ± 0.1% by day 4, returning to control levels by day 16. Dual-label Western blot analysis showed that the expression of NCS-1 relative to PGP 9.5 decreased by 50% on day 4 but returned to control by day 16. The selective loss of NCS-1 during colitis may underlie the altered neural function seen in the inflamed intestine.
frequenin; enteric nervous system; dinitrobenzene sulfonic acid
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INTRODUCTION |
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THE RELEASE OF NEUROTRANSMITTER from the synaptic terminal is a complex process, requiring calcium-binding proteins to respond to increases in intracellular calcium by undergoing conformational changes that then allow interactions with target proteins. The neuronal calcium sensor (NCS) family of calcium-binding proteins has been shown to bind calcium with high affinity through characteristic EF-hand motifs (3). NCS-1 [termed frequenin in invertebrate systems (27)] has parallel actions to calmodulin in promoting the activity of enzymes and ion channels required for synaptic efficacy, suggesting an important role for NCS-1 in neurotransmitter release from synaptic terminals. In particular, NCS-1 can activate 3',5'-cyclic nucleotide phosphodiesterase and the phosphatase calcineurin as well as potentiate nitric oxide synthase activity (22). NCS-1 can also participate in phosphoinositol signaling, with evidence for interaction with phosphatidylinositol 4-kinase in MDCK cells and yeast (10, 26).
An important role for NCS-1 in synaptic neurotransmission was confirmed by electrophysiological studies in Drosophila and in neuroblastoma-myocyte cocultures, in which overexpression caused frequency-dependent facilitation of transmitter release at the neuromuscular junction (5, 16). Enhanced synaptic efficacy was also measured in Xenopus spinal neurons after introduction of exogenous frequenin by early blastomere injection (18).
NCS-1 is widely distributed within the mammalian nervous system, with expression in neuronal cell bodies throughout the adult rat brain and spinal cord, in some nerve terminals of the rat central nervous system, and in presynaptic structures at the neuromuscular junction (16, 19). Combined with evidence for widespread localization in neurons of several species (1, 16, 17), this suggests a broad significance for NCS-1 in neuronal function. However, the presence and function of NCS-1 within the mammalian enteric nervous system (ENS) is unknown, despite the extremely large number of neurons and their complex intrinsic sensory reflex mechanisms (e.g., see Refs. 13 and 14).
Although virtually nothing is known about the events of synaptic vesicle exocytosis in the ENS, it is clear that the suppression of neurotransmitter release from ENS neurons is a characteristic of intestinal inflammation in several different animal models (reviewed in Ref. 6). Typically, inflammation decreased the stimulated release of ACh from preparations containing the myenteric plexus and smooth muscle (7). These changes were also present in adjacent, noninflamed areas of the gut (12) and could be mimicked by application of exogenous interleukin (IL)-1 to control preparations (15), suggesting that ENS function is sensitively affected by the appearance of proinflammatory mediators.
The earlier experiments showing suppression of neurotransmitter release in the inflamed intestine were controlled for any effects of structural damage (7). Our subsequent studies showed that suppression of synthesis of neurotransmitter was not responsible for the decreased release of ACh, because we observed both increased precursor uptake and marked upregulation of the activity of choline acetyltransferase activity in the inflamed intestine (8). Therefore, we hypothesized that inflammation might target the presence or amount of synaptic vesicle proteins and, in particular, that alterations to calcium-sensor proteins such as NCS-1 might be critical in leading to impaired function and, hence, reduced neurotransmitter release.
Therefore, we have used immunocytochemistry and Western blotting to characterize the pattern of expression of NCS-1 in the ENS of the normal rat colon. Using the well-established model of chemically induced colitis in the rat, we determined the effects of intestinal inflammation on NCS-1 distribution and localization.
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METHODS |
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Animal use and induction of intestinal inflammation. Experiments were performed on adult (225-300 g) male Sprague-Dawley rats (Charles River, PQ, Canada) housed in pairs in microfilter-isolated cages with free access to food and water. Food was removed from the rats 24 h before induction of colitis. All experimental procedures were approved by the local University Animal Care Committee.
For experimental inflammation of the colon, rats were lightly anesthetized with halothane, and 29 mg dinitrobenzene sulfonic acid (DNBS; ICN) dissolved in 250 µl of 50% ethanol was introduced into the rectum 8 cm proximal to the anus by using a PE-50 catheter (21). Control animals were not treated, because previous studies have shown that treatment with either 50% ethanol or 0.9% saline results in no significant alterations (21). Animals were killed by cervical dislocation under halothane anesthesia at various times after the initiation of colitis. The inflamed region of the mid descending colon was identified, removed, and fixed in 10% neutral buffered formalin for 24 h, followed by dehydration in ethanol and embedding in paraffin. The mid descending colon was removed from control animals and processed as above. Cross sections (4 µm) were stained with hematoxylin and eosin for routine examination or processed for immunocytochemistry as described below. For whole mount preparations, the appropriate region was opened along the mesenteric border and pinned mucosal side down in Hanks' balanced salt solution with 10 mM HEPES (pH 7.0). The smooth muscle layer containing the myenteric plexus (SM/MP) was gently removed intact, fixed in neutral buffered formalin for 3 h, permeabilized in phosphate-buffered saline (PBS) containing 1% Triton X-100, and processed for immunocytochemistry.Immunocytochemistry. Paraffin sections were blocked for 1 h in 0.2% Triton X-100 in PBS-Tween-20 containing 1% goat serum. Sections were labeled with affinity-purified antibodies raised in rabbits against NCS-1 (1:500; Drs. J. Roder and A. Jeromin, Mt. Sinai Hospital, Toronto, ON, Canada) in antibody-diluting fluid (DAKO) overnight at room temperature. This was followed by a 2-h incubation in goat anti-rabbit secondary antibody conjugated to Alexa 546 (1:500; Molecular Probes) followed by visualization using fluorescence microscopy (Olympus BX-60). In some cases, the secondary antibody was conjugated to Alexa 488 (1:500; Molecular Probes).
In some experiments, previously stained tissues were stripped of bound antibodies for subsequent reprobing by incubation in a solution containing 0.2% H2SO4 and 0.1% potassium permanganate in dH2O for 10 min (23, 24). This was necessary because all of the primary antibodies were raised in rabbits, precluding the use of colabeling with two antibodies simultaneously. Removal of initial antibody staining was verified by viewing with a fluorescent microscope. After being washed in PBS for 30 min, the tissues were then incubated with rabbit antibodies to either PGP 9.5 (1:1,000; Ultraclone), tyrosine hydroxylase (TH, 1:500; Chemicon), or glial fibrillary acidic protein (GFAP, 1:500; DAKO) by using the protocol described above. To evaluate the distribution of NCS-1 within the cell bodies of the myenteric plexus, 20 nonadjacent ganglia from whole mount preparations were analyzed for the number of neurons with positive staining for NCS-1. Data was expressed as the average of the percentage of the total neurons present in each ganglion. The number of NCS-1-positive axons within the circular smooth muscle (CSM) layer was determined in cross sections of the colon, with manual counting of stained axons in 4-12 nonadjacent microscope fields (×40 objective; total field area 0.09 mm2). The tissue was then relabeled with an antibody to PGP 9.5, as described above. The number of PGP 9.5-positive axons was then counted in the same manner to determine the total number of axons. The number of NCS-1-positive fibers was expressed as a percentage of the total.Western blot analysis. After protein determination of tissue lysates, samples containing equal amounts of protein were resolved by 12% SDS-PAGE and transferred to a polyvinylidine difluoride membrane. The membrane was then incubated in 5% nonfat milk in Tris-buffered saline (TBS) containing 0.2% Tween-20 (TBS-T) and then with anti-NCS-1 diluted in antibody-diluting fluid (1:2,000) for 2.5 h. The membrane was washed in TBS buffer, incubated for 1 h with goat anti-rabbit IgG conjugated with alkaline phosphatase (1:2,000; Sigma) in TBS-T. The membrane was then incubated in a substrate solution (SigmaFast; Sigma). The blots were scanned and imported into the Image Pro program (Media Cybernetics) to measure the integral optical density (IOD) of individual bands. This method was repeated two or three times for each sample analyzed. Of the several homogenates of SM/MP obtained from control rats, one specified sample was included in each blot to allow values from different blots to be compared with each other.
Statistics. Data were analyzed by using single-factor analysis of variance, with P < 0.05 considered to be statistically significant.
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RESULTS |
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NCS-1 is expressed in control colon.
Immunocytochemistry using an antibody to NCS-1 on cross sections of rat
colon showed strong and consistent staining of the ENS. NCS-1 was
expressed in neuronal cell bodies of both the myenteric and submucosal
plexuses, as well as in axon bundles distributed within the
longitudinal and CSM layers and the mucosa (Fig.
1, A and B). This
immunoreactivity was verified as NCS-1 by Western blot analysis (Fig.
1C), where a single 22-kDa band was obtained in tissue
homogenates from the control rat colon and rat brain, corresponding to
the molecular size for NCS-1 as reported elsewhere (3).
This was detected in homogenates of the SM/MP, in the tissue layer
containing the submucosal plexus, and in lesser amounts in the mucosa.
No signal was detected when primary antibody was omitted from the
protocol or when a mismatched secondary antibody made against mouse or
goat was used in place of the anti-rabbit IgG (data not shown).
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Expression of NCS-1 in colitis. Intrarectal administration of DNBS in ethanol caused transmural inflammation of the distal colon, as described previously (21). Macroscopically, a 2- to 4-cm length of the mid descending colon was dilated and overtly inflamed, with serosal adhesions. Histological examination of cross sections from days 2 and 4 after DNBS showed extensive immune cell infiltration and edema, with large areas of mucosal damage. By day 16, the appearance was largely normal, with an intact mucosa, although increased numbers of immune cells remained detectable and the muscle wall was much thickened.
NCS-1 continued to be expressed in neuronal cell bodies and axons of the rat colon at days 2 and 4 after administration of DNBS. However, there was a dramatic decrease in the number of stained axons within the smooth muscle layers (Fig. 3A), although the intensity of labeling of neuronal cell bodies was similar to that observed in control tissue. To study the changes in the number of axons expressing NCS-1 during colitis, quantitative measurements of NCS-1-positive axons per field were obtained in the randomly selected areas of the CSM (Fig. 3B) . This showed a nearly 50% decrease in number, from 4.7 ± 0.7 axons/field in control tissue to 2.1 ± 0.6 axons/field (P < 0.05) on day 2 after DNBS. This value showed significant further decrease by day 4 after DNBS, when only 0.5 ± 0.3 positive axons per field were present (n = 4; P < 0.05).
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Western blot analysis of inflamed tissue.
Since the number of NCS-1 axons was decreased in colitis, Western blots
were used to evaluate the effect of inflammation on NCS-1 expression in
the SM/MP, which contained both the cell bodies and axons of the
myenteric plexus. Figure 4A
shows a Western blot containing samples from cohort animals, in which
equal amounts of tissue from the SM/MP of cohort control and day
4 post-DNBS rats were probed for NCS-1 expression. In each case,
there was a clear decrease in NCS-1 expression in inflamed tissue
compared with control. Videodensitometry showed that the average IOD
for the NCS-1 bands was 0.8 ± 0.05 (n = 3) in
control tissues, and that this was significantly reduced to 0.29 ± 0.02 (n = 3) by day 4 after DNBS (Fig.
4B; P < 0.05). This decrease was reversed by day 16, when the value was similar to control.
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DISCUSSION |
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In isolated systems, the molecular properties of NCS-1 include extremely high calcium sensitivity and the ability to substitute for, or even synergistically enhance, the actions of calmodulin (22). The broad general importance of calcium regulation in neural function suggested that NCS-1 could have important roles through the entire spectrum of events leading from electrical signaling through the exocytosis of neurotransmitters. Therefore, we have investigated the presence and distribution of NCS-1 in the ENS, where complex neuronal interactions regulate largely autonomous intestinal function.
We have shown that NCS-1 is widely present within myenteric neurons and axons within the smooth muscle layers of the rat colon. Characteristically, NCS-1 was found in a subset of the neuronal cell bodies, with positive staining of at least some neurons in each ganglion and showing a narrow range of distribution among animals. In addition, the relatively large average size of positively staining cell bodies suggested that these may be intrinsic sensory neurons (i.e., Dogiel type II), a class of neurons that may be particularly reliant on the precise regulation of intracellular calcium levels for subtle modulation of electrical signaling. However, an immunocytochemical analysis is required to investigate whether NCS-1 is an additional factor of use in the chemical coding of neurons within the ENS (9).
The stereotypical appearance of NCS-1 within the rat colon was extremely sensitive to DNBS-induced colitis, and there was nearly complete loss from identified axons by day 4. Since NCS-1 immunoreactivity remained detectable within the adjacent neuronal cell bodies in the same sections, this was unlikely to be an artifact. Furthermore, dual-label immunocytochemistry showed that axonal structures remained present and detectable with the PGP 9.5 antibody, although negative for NCS-1. We interpret this as evidence for the selective loss or removal of NCS-1 from the myenteric axonal extensions during colitis. Western blot analysis confirmed this, since evaluation of the expression of NCS-1 relative to PGP 9.5 (as a representative of a neuron-specific protein) showed a similar sharp drop and subsequent recovery. This indicated the selective loss of NCS-1 within the neuronal protein pool, similar to that seen in the proportion of NCS-1-positive axons.
The time course of decreased expression of NCS-1 was similar to the impairment of neurotransmitter release seen in enteritis; both in chemically induced colitis and in Trichinella-induced inflammation of the rat jejunum, inflammation caused suppression of the stimulated release of ACh, and this was largely reversed by day 23 after infection (7, 12). It is possible that the near-complete loss of this sensitive calcium-sensor protein could be responsible for impaired function of these neurons, indirectly supported by evidence that increased expression of NCS-1 led to the facilitation of neurotransmitter release (20). However, the direct evidence is lacking to prove that loss of NCS-1, or similar calcium-sensor proteins in other subpopulations of enteric neurons, is responsible for impaired neurotransmitter release in the intestinal inflammation.
Although it is not completely clear how intestinal inflammation causes impaired neurotransmitter release, the application of exogenous IL-1 on control tissues could mimic this effect and showed a requirement for protein synthesis before the onset of suppression of neurotransmitter release (15). Leukemia inhibitory factor was identified as a key intermediate, because neutralizing antibodies to leukemia inhibitory factor could block the IL-1-induced suppression of ACh release (25). Although this sequence is compatible with the time course of decreased NCS-1 expression seen in the present study, further experiments are necessary to prove the link between IL-1 and modulation of expression of NCS-1.
The above evidence proves that enteric neurons can respond either directly or indirectly to proinflammatory mediators by alteration of protein expression. An additional mechanism allowing far more rapid modulation of function also exists, because electrophysiological experiments show that exogenous application of IL-1 or IL-6 at physiological levels suppresses neurotransmitter release within seconds of application to guinea pig submucosal neurons (28).
The loss of NCS-1 expression in colitis was apparently completely reversed by day 16, when the relative abundance of NCS-1-positive axons was again similar to control. However, our recent work in this model emphasizes the extensive changes that have occurred by this time (21). Significant levels of neuronal death occur early, followed by axonal proliferation and smooth muscle cell hyperplasia. These latter events occurred in proportion, so that a constant level of innervation density was maintained. Therefore, restoration of the relative abundance of NCS-1 levels actually represents a large increase in absolute amount and occurs in the presence of major structural adaptation and remodeling. That these processes may fall short of restoration of the normal environment is suggested by evidence in this and similar animal models, such as persistence of increased smooth muscle mass, maintained upregulation of choline acetyltransferase activity, and the failure of complete restoration of neurotransmitter release to control levels (2, 7, 8).
Overall, evidence from rat models of intestinal inflammation suggests the hypothesis that NCS-1, and possibly other synaptic vesicle proteins, are sensitive targets of the proinflammatory mediators present in the inflamed intestinal wall. Decreased expression of function-related proteins such as these may parallel the onset of structural changes that jointly allow for axonal proliferation and the preservation of homeostasis.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. G. Blennerhassett, Gastrointestinal Diseases Research Unit, Queens University, Hotel Dieu Hospital, 166 Brock St., Kingston, Ontario, Canada K7L 5G2 (E-mail: mblen{at}meds.queensu.ca).
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. Section 1734 solely to indicate this fact.
First published February 20, 2002;10.1152/ajpgi.00320.2001
Received 20 July 2001; accepted in final form 4 February 2002.
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