Intestinal inflammation modulates expression of the synaptic vesicle protein neuronal calcium sensor-1

S. Lourenssen1, A. Jeromin2, J. Roder2, and M. G. Blennerhassett1

1 Gastrointestinal Diseases Research Unit, Queens University, Kingston K7L 5G2; and 2 Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 1.   Immunocytochemistry and Western blotting demonstrating that the synaptic vesicle protein neuronal calcium sensor 1 (NCS-1) is abundant within the innervation of the control rat colon. A: fluorescence photomicrograph of a cross section of the rat colon showing positive staining (e.g., arrow) of nerve cell bodies within the myenteric plexus (MP) and axons in the adjacent smooth muscle (SM) layers (LM, longitudinal muscle; CM, circular smooth muscle). B: fluorescence photomicrograph of the section in A after stripping and reprobing for the panneuronal marker PGP 9.5, showing that elements with positive staining for NCS-1 in A (e.g., arrow) were a subset of those positive for PGP 9.5 and confirming that NCS-1 is a neural protein within the enteric nervous system. Arrowheads indicate additional axons stained with PGP 9.5 but not NCS-1. Bar = 50 µm. C: representative Western blot showing that antibodies to NCS-1 detect a single 22-kDa protein in homogenates of the rat colon and brain. Each lane contains 80 µg of protein from the SM/MP, submucosal plexus (SMP), mucosa, or whole brain.

NCS-1 staining of the myenteric plexus was localized to a proportion of the myenteric neurons, where staining was generally strong when present. Analysis of whole mounts labeled with anti-NCS-1 confirmed this appearance, showing the presence of relatively uniform staining throughout the neuronal perikarya of a proportion of the myenteric neurons within each ganglion (Fig. 2). In whole mounts and in cross sections, NCS-1 staining was uniformly distributed along the lengths of both the large bundles of axons connecting adjacent ganglia as well as of small axons within the smooth muscle layers (Figs. 1 and 2).


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 2.   The presence and distribution of NCS-1 within the large myenteric neurons of the rat colon. A: fluorescence micrograph of a whole mount preparation of the myenteric plexus after immunocytochemistry for NCS-1, showing the characteristic selective labeling of a subset of neuronal cell bodies, with extensive labeling of axons both within the ganglion and in the internodal strands. Bar = 50 µm. B: graph showing the relative proportions of NCS-1-positive cell bodies within myenteric ganglia. Typically, 20-30% of neurons within each ganglion were NCS-1 positive. Inset: comparison of the average maximum length of NCS-1-positive (+ve) and negative (-ve) neurons, showing that NCS-1 staining was associated with large neuronal size.

To determine the proportion of myenteric plexus neurons that expressed NCS-1, whole mount preparations were labeled with anti-NCS-1 and the number of positive cell bodies were counted. This analysis showed that 22.6 ± 1.8% (n = 66 ganglia from 3 animals) of myenteric neurons expressed this protein. Interestingly, the distribution of neurons expressing NCS-1 among the myenteric plexus ganglia was characteristically variable (Fig. 2B): only one or two NCS-1-positive neurons were detected in some ganglia, whereas almost 40% of neurons were labeled with anti-NCS-1 in other ganglia.

The labeled neuronal cell bodies appeared characteristically larger than unstained ones within the same ganglia, and this was evaluated by comparison of the mean area in digital images (Fig. 2B, inset). This showed that NCS-1-positive neurons were typically twice the size of adjacent unstained neurons when compared by average area [364 ± 15 vs. 163 ± 7 µm2 (n = 3 animals; 22 neurons per animal)] or by average maximum diameter (28 ± 0.5 vs. 17 ± 0.2 µm).

We used tissue cross sections to examine the distribution of NCS-1 within axons in the CSM layer. The intensity of NCS-1 labeling was uniform along the length of the axon bundles, indicating that NCS-1 was localized throughout the axons and was not confined to areas of synaptic vesicle accumulation. Analysis of cross sections of the control colon showed an average of 4.7 ± 0.7 (n = 4 animals) axon bundles per microscope field in the CSM layer. The diameter of the NCS-1-positive elements ranged between 2.3 and 7.2 µm [average 4.4 ± 0.4 µm (n = 4)]. The average length of these axon bundles in the plane of the tissue cross section was 53 ± 7 µm (n = 4).

Since NCS-1 was not detected in all neuronal cell bodies, it was likely that NCS-1 was expressed in only a proportion of the axonal bundles innervating the CSM. To study this, sections of control colon were first labeled with anti-NCS-1, analyzed, and then reprobed with an antibody to the panneuronal marker PGP 9.5 to reveal all neuronal elements (see Fig. 1, A and B). All NCS-1-labeled elements were also stained with PGP 9.5, confirming their neural nature. NCS-1-labeled axons were indeed a subset of the total number of axons in the CSM and comprised 28 ± 0.3% (n = 4 animals) of the total.

Since the proportion of NCS-1-positive axon bundles in the CSM layer is greater than that of the NCS-1-positive cell bodies in the myenteric plexus, we thought that some of these axon bundles might be of extrinsic origin. To test this, we used antibodies to TH to detect if some of the fibers expressing NCS-1 were also TH positive. Since TH is an enzyme involved in norepinephrine synthesis in sympathetic neurons that is not present in rat intrinsic enteric neurons, TH presence in NCS-1-positive axons would confirm an extrinsic origin. Positive staining for TH was found principally in axons that terminated onto cell bodies of the myenteric plexus, with labeling of some axons in the smooth muscle layers. In both cases, TH immunoreactivity showed a typically punctate appearance, in contrast to the uniform distribution of NCS-1, which suggested localization to areas of neurotransmitter accumulation. Dual-label immunocytochemistry showed the presence of punctate labeling for TH in 33 ± 6.1% (n = 3) of the NCS-1-stained elements. Therefore, some of the NCS-1-positive axons in the CSM may originate from extrinsic, sympathetic fibers.

The ENS contains both enteric neurons and glial cells, which have a common origin in cells of neural crest derivation (4). To verify that expression of NCS-1 was limited to neurons and their extensions, we used an antibody to the marker protein GFAP and compared this with NCS-1 staining in sequentially stained sections. No overlap of staining by NCS-1 with GFAP-positive cells or their extensions was seen, confirming that expression of NCS-1 expression was confined to neurons and not present in glial cells (data not shown).

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 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).


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 3.   Inflammation of the rat colon causes marked reduction of NCS-1 expression. A: photomicrograph showing typical appearance of NCS-1 immunocytochemistry on day 2 after dinitrobenzene sulfonic acid (DNBS)-induced colitis. NCS-1 labeling of axons within the smooth muscle was greatly reduced compared with control (compare with Fig. 1B), whereas staining within the myenteric plexus remained strong. An arrow identifies the middle portion of a rare NCS-1-positive axonal fiber. B: graph showing the decrease in the number of NCS-1 profiles per field on days 2 and 4 after DNBS (*, significantly different from control) and subsequent return by day 16 to a level similar to control. C: graph showing that NCS-1 was lost from identifiable axons during colitis and then reexpressed by day 16. Sections were sequentially stained and photographed for NCS-1 and PGP 9.5 to determine the proportion of NCS-1-positive axons within the total population, as determined by staining for PGP 9.5.

The sharp decrease in NCS-1-positive axonal fibers within the CSM during colitis might result from either the loss of neuronal structures or from the loss of NCS-1 expression within existing axons. To distinguish between these possibilities, we measured the number of NCS-1-positive axons in the CSM layer and expressed this as a proportion of the number of PGP 9.5-positive axons, as determined before for control animals (Fig. 3C). This showed a significant decrease to 13.3 ± 0.4% by day 2 after DNBS, less than half of the control value of 28 ± 0.3%. This was further decreased on day 4 of inflammation, when the number of NCS-1-positive elements (0.5 ± 0.3 axons/field) was only 7.0 ± 0.1% of the PGP 9.5-positive axons. This value is equivalent to 25% of the control number (P < 0.05) and confirms that NCS-1 expression was lost from axons that remained present in the CSM during colitis.

Our previous work has shown that the appearance of the colon was largely restored to normal by day 16 after DNBS (21). Although significant smooth muscle hyperplasia occurs in colitis (11), the density of PGP 9.5-positive fibers in the CSM on day 16 was not different from control, indicating that significant axonal proliferation occurred in proportion to newly arising smooth muscle cells (21). Since these rapid and extensive changes might affect the expression of NCS-1-positive axons following colitis, the number of NCS-1-positive profiles per field in the CSM was determined in animals at day 16 after DNBS. This value (6.0 ± 1.2 axons/field; P = 0.3) was not different from control (Fig. 3B). Similarly, the ratio of NCS-1 to PGP 9.5-positive fibers was similar to that measured in control tissue (29.2 ± 0.4 vs. 28 ± 0.3%; P < 0.05) by day 16 after DNBS (Fig. 3C). Overall, these data show that the large decreases in both the density and proportion of NCS-1-positive axons were fully reversed following colitis.

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.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4.   The amount of NCS-1 protein decreases during DNBS-induced colitis. A: representative Western blot of the SM/MP layer from 3 control rats and 3 animals at day 4 after DNBS colitis, showing the uniform decrease in NCS-1 protein in the inflamed animals. B: graph showing the relative intensity of staining [integral optical density (IOD)] for NCS-1 in immunoblots. The IOD of NCS-1 was significantly reduced by day 4 after DNBS relative to control, with recovery to a level similar to control by day 16.

Since this could be affected by the loss of NCS-1-positive neurons from the myenteric plexus, and the ensuing axonal proliferation [occurring early in DNBS colitis (21)], a dual-label Western blot analysis was performed using antibodies to both NCS-1 and PGP 9.5 (Fig. 5). Initial control studies showed that the average IOD of the NCS-1 band from control SM/MP was similar to that of NCS-1 bands produced on blots colabeled with PGP 9.5 (P > 0.05), indicating that dual labeling did not interfere with NCS-1 labeling. Thus the amount of NCS-1 could be expressed relative to the amount of PGP 9.5 as a marker reflecting change within the myenteric plexus. Analysis of the results of the dual-labeled Western blots is shown in Fig. 5B, which illustrates that the ratio of the IODs of NCS-1 to PGP 9.5 dropped by 50% on day 4 after DNBS (0.17 ± 0.04 vs. 0.08 ± 0.01; n = 3; P < 0.05). By day 16 after DNBS, this ratio was similar to control levels (P > 0.05). We interpret the sharp drop in this ratio during colitis, and its subsequent return toward normal, as evidence for the selective impairment of expression of NCS-1 in enteric neurons during intestinal inflammation.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 5.   Dual-label Western blotting suggests that NCS-1 is selectively reduced within the neuronal protein pool. A: representative Western blot of SM/MP from two control animals showing dual labeling with antibodies to NCS-1 and PGP 9.5. B: graph showing the ratio of the IOD values of the anti-NCS-1-labeled bands relative to the anti-PGP 9.5-labeled bands from tissue taken from the control colon at days 4 and 16 after inflammation (n = 4, 3, and 4 animals, respectively). Each sample was probed at least 3 times, and the IODs were averaged. There was a significant decrease in the amount of NCS-1 protein relative to the panneuronal marker PGP 9.5 on day 4 of inflammation relative to control (P < 0.05), with subsequent return to control level by day 16 after DNBS.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Angaut-Petit, D, Toth P, Rogero O, Faille L, Tejedor FJ, and Ferrus A. Enhanced neurotransmitter release is associated with reduction of neuronal branching in a Drosophila mutant overexpressing frequenin. Eur J Neurosci 10: 423-434, 1998[Medline].

2.   Blennerhassett, MG, Vignjevic P, Vermillion DL, and Collins SM. Inflammation causes hyperplasia and hypertrophy in smooth muscle of rat small intestine. Am J Physiol Gastrointest Liver Physiol 262: G1041-G1046, 1992[Abstract/Free Full Text].

3.   Burgoyne, RD, and Weiss JL. The neuronal calcium sensor family of Ca2+-binding proteins. Biochem J 353: 1-12, 2001[ISI][Medline].

4.   Chalazonitis, A, Rothman TP, Chen J, Lamballe F, Barbacid M, and Gershon MD. Neurotrophin-3 induces neural crest-derived cells from fetal rat gut to develop in vitro as neurons or glia. J Neurosci 14: 6571-6584, 1994[Abstract].

5.   Chen, XL, Zhong ZG, Yokoyama S, Bark C, Meister B, Berggren PO, Roder J, Higashida H, and Jeromin A. Overexpression of rat neuronal calcium sensor-1 in rodent NG108-15 cells enhances synapse formation and transmission. J Physiol 532: 649-659, 2001[Abstract/Free Full Text].

6.   Collins, SM. The immunomodulation of enteric neuromuscular function: implications for motility and inflammatory disorders. Gastroenterology 111: 1683-1699, 1996[ISI][Medline].

7.   Collins, SM, Blennerhassett PA, Blennerhassett MG, and Vermillion DL. Impaired acetylcholine release from the myenteric plexus of Trichinella-infected rats. Am J Physiol Gastrointest Liver Physiol 257: G898-G903, 1989[Abstract/Free Full Text].

8.   Davis, KA, Masella J, and Blennerhassett MG. Acetylcholine metabolism in the inflamed rat intestine. Exp Neurol 152: 251-258, 1998[ISI][Medline].

9.   Furness, JB. Types of neurons in the enteric nervous system. J Auton Nerv Syst 81: 87-96, 2000[ISI][Medline].

10.   Hendricks, KB, Wang BQ, Schnieders EA, and Thorner J. Yeast homologue of neuronal frequenin is a regulator of phosphatidylinositol-4-OH kinase. Nat Cell Biol 1: 234-241, 1999[ISI][Medline].

11.   Hogaboam, CM, Jacobson K, Collins SM, and Blennerhassett MG. The selective beneficial effects of nitric oxide inhibition in experimental colitis. Am J Physiol Gastrointest Liver Physiol 268: G673-G684, 1995[Abstract/Free Full Text].

12.   Jacobson, K, McHugh K, and Collins SM. Experimental colitis alters myenteric nerve function at inflamed and noninflamed sites in the rat. Gastroenterology 109: 718-722, 1995[ISI][Medline].

13.   Lomax, AE, and Furness JB. Neurochemical classification of enteric neurons in the guinea-pig distal colon. Cell Tissue Res 302: 59-72, 2000[ISI][Medline].

14.   Lomax, AE, Sharkey KA, Bertrand PP, Low AM, Bornstein JC, and Furness JB. Correlation of morphology, electrophysiology and chemistry of neurons in the myenteric plexus of the guinea-pig distal colon. J Auton Nerv Syst 76: 45-61, 1999[ISI][Medline].

15.   Main, C, Blennerhassett P, and Collins SM. Human recombinant interleukin 1 beta suppresses acetylcholine release from rat myenteric plexus. Gastroenterology 104: 1648-1654, 1993[ISI][Medline].

16.   Martone, ME, Edelmann VM, Ellisman MH, and Nef P. Cellular and subcellular distribution of the calcium-binding protein NCS-1 in the central nervous system of the rat. Cell Tissue Res 295: 395-407, 1999[ISI][Medline].

17.   Olafsson, P, Soares HD, Herzog KH, Wang T, Morgan JI, and Lu B. The Ca2+ binding protein, frequenin is a nervous system-specific protein in mouse preferentially localized in neurites. Brain Res Mol Brain Res 44: 73-82, 1997[ISI][Medline].

18.   Olafsson, P, Wang T, and Lu B. Molecular cloning and functional characterization of the Xenopus Ca(2+)-binding protein frequenin. Proc Natl Acad Sci USA 92: 8001-8005, 1995[Abstract].

19.   Paterlini, M, Revilla V, Grant AL, and Wisden W. Expression of the neuronal calcium sensor protein family in the rat brain. Neuroscience 99: 205-216, 2000[ISI][Medline].

20.   Pongs, O, Lindemeier J, Zhu XR, Theil T, Engelkamp D, Krah-Jentgens I, Lambrecht HG, Koch KW, Schwemer J, and Rivosecchi R. Frequenin---a novel calcium-binding protein that modulates synaptic efficacy in the Drosophila nervous system. Neuron 11: 15-28, 1993[ISI][Medline].

21.   Sanovic, S, Lamb DP, and Blennerhassett MG. Damage to the enteric nervous system in experimental colitis. Am J Pathol 155: 1051-1057, 1999[Abstract/Free Full Text].

22.   Schaad, NC, De Castro E, Nef S, Hegi S, Hinrichsen R, Martone ME, Ellisman MH, Sikkink R, Rusnak F, Sygush J, and Nef P. Direct modulation of calmodulin targets by the neuronal calcium sensor NCS-1. Proc Natl Acad Sci USA 93: 9253-9258, 1996[Abstract/Free Full Text].

23.   Staines, WA, Meister B, Melander T, Nagy JI, and Hokfelt T. Three-color immunofluorescence histochemistry allowing triple labeling within a single section. J Histochem Cytochem 36: 145-151, 1988[Abstract].

24.   Tramu, G, Pillez A, and Leonardelli J. An efficient method of antibody elution for the successive or simultaneous localization of two antigens by immunocytochemistry. J Histochem Cytochem 26: 322-324, 1978[Abstract].

25.   Van Assche, G, and Collins SM. Leukemia inhibitory factor mediates cytokine-induced suppression of myenteric neurotransmitter release from rat intestine. Gastroenterology 111: 674-681, 1996[ISI][Medline].

26.   Weisz, OA, Gibson GA, Leung SM, Roder J, and Jeromin A. Overexpression of frequenin, a modulator of phosphatidylinositol 4-kinase, inhibits biosynthetic delivery of an apical protein in polarized madin-darby canine kidney cells. J Biol Chem 275: 24341-24347, 2000[Abstract/Free Full Text].

27.   Werle, MJ, Roder J, and Jeromin A. Expression of frequenin at the frog (Rana) neuromuscular junction, muscle spindle and nerve. Neurosci Lett 284: 33-36, 2000[ISI][Medline].

28.   Xia, Y, Hu HZ, Liu S, Ren J, Zafirov DH, and Wood JD. IL-1beta and IL-6 excite neurons and suppress nicotinic and noradrenergic neurotransmission in guinea pig enteric nervous system. J Clin Invest 103: 1309-1316, 1999[Abstract/Free Full Text].


Am J Physiol Gastrointest Liver Physiol 282(6):G1097-G1104
0193-1857/02 $5.00 Copyright © 2002 the American Physiological Society