Gastrointestinal Diseases Research Unit, Queens University, Hotel Dieu Hospital, Kingston, Ontario K7L 5G2, Canada
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ABSTRACT |
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The loss of intrinsic neurons is an early event in inflammation of the
rat intestine that precedes the growth of intestinal smooth muscle
cells (ISMC). To study this relationship, we cocultured ISMC and
myenteric plexus neurons from the rat small intestine and examined the
effect of scorpion venom, a selective neurotoxin, on ISMC growth. By 5 days after neuronal ablation, ISMC number increased to 141 ± 13%
(n = 6) and the uptake of [3H]thymidine
in response to mitogenic stimulation was nearly doubled. Atropine
caused a dose-dependent increase in [3H]thymidine uptake
in cocultures, suggesting the involvement of neural stimulation of
cholinergic receptors in regulation of ISMC growth. In contrast,
coculture of ISMC with sympathetic neurons increased
[3H]thymidine uptake by 45-80%, which was sensitive
to propranolol (30 µM) and was lost when the neurons were
separated from ISMC by a permeable filter. Western blotting showed that
coculture with myenteric neurons increased -smooth muscle-specific
actin nearly threefold to a level close to ISMC in vivo. Therefore, factors derived from enteric neurons maintain the phenotype of ISMC
through suppression of the growth response, whereas catecholamines released by neurons extrinsic to the intestine may stimulate their growth. Thus inflammation-induced damage to intestinal innervation may
initiate or modulate ISMC hyperplasia.
actin; immunocytochemistry; sympathetic neurons; tissue culture; Western blotting
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INTRODUCTION |
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INFLAMMATION AFFECTS ALL of the cellular systems within the intestine, altering their functions as well as their ability to deliver the integrated responses necessary for normal motility. However, these changes usually become reversed as the markers of inflammation subside. For example, animal models of intestinal inflammation have shown the development of accelerated intestinal transit and hyperreactivity of the smooth muscle, which returned to baseline values when inflammation was no longer present (reviewed in Ref. 9).
Although this suggests that the effect of inflammation on the control mechanisms of intestinal motility is transient and can be met with adequate repair or replacement of cellular components, we have recently found that some aspects of intestinal structure undergo significant, essentially irreversible changes that require functional adaptation for preservation of normal intestinal motility. For example, a significant loss of myenteric and submucosal neurons was found to occur during the first 48 h following induction of colitis in the rat, with no further change observed in the subsequent 8- to 10-day period of overt inflammation (26). In earlier work in this model, as well as in experimental inflammation of the rat jejunum, we had observed that marked hyperplasia of the intestinal smooth muscle cells (ISMC) occurred through days 4-6 following induction of inflammation (4, 18). Although mitosis of the smooth muscle was normally virtually undetectable, up to 6% of the cell population were mitotic during inflammation, and this gave rise to an essentially permanent increase in the smooth muscle cell number.
Of further significance to motility, examination of the effect of
growth of smooth muscle on its differentiated nature showed a
significant increase in cell size as well as alterations in amount and
proportion of mRNA and protein for -smooth muscle-specific actin
(
-SM actin) (3). Although a cycle of dedifferentiation, growth, and resumption of contractile phenotype is normal for repair of
smooth muscle (21), an altered outcome in the case of
inflammation-induced hyperplasia suggests that changes occur at the
cellular level of smooth muscle, which may then affect the contractile
properties of the tissue.
These findings suggest that the impact of inflammation on the enteric nervous system (ENS) is an underestimated, early challenge to intestinal function that is followed closely by large increases in the number of smooth muscle cells, which require innervation for appropriate contractile responses. These changes to both the neuronal and smooth muscle cell populations could contribute to the altered motility seen during inflammation, and these irreversible or slowly reversing changes may contribute to the frequent reports of persistence of symptoms following acute inflammatory episodes (e.g., see Ref. 16).
Elsewhere in the nervous system, the close neuron-target cell relationship is essential for mutual survival and function. This suggested to us that disruption of normal innervation of intestinal smooth muscle could be implicated in the subsequent hyperplasia. Supporting evidence comes from the observations that the neural damage precedes the onset of smooth muscle mitosis, as well as restoration of the normal density of innervation of smooth muscle thereafter (26).
Specifically, we hypothesized that innervation of ISMC may directly or indirectly influence their growth. In the absence of evidence in the literature, we developed a tissue culture model to pursue this, involving the coculture of rat ISMC with neurons from the myenteric plexus. Since extrinsic innervation of the intestine might contrast with the effects of enteric innervation, we also examined the consequences of coculture of ISMC with neurons from the sympathetic nervous system. The strong and contrasting outcomes of these studies represent evidence for a new and significant aspect of the nerve-target cell relationship within the intestine.
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METHODS |
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Tissue culture of intestinal smooth muscle and myenteric neurons. To obtain cultures of ISMC and neurons from the ENS, the longitudinal and circular smooth muscle layer and the enclosed myenteric plexus were removed from the small intestine of 3- to 4-wk-old male Sprague-Dawley rats (Charles River, Montreal, PQ, Canada) and dissociated using 0.25% trypsin II (Sigma, St. Louis, MO) in HEPES-buffered Hanks' saline (pH 7.35). Cell suspensions were plated onto 48-well culture plates previously coated with Matrigel (Collaborative Research). Medium (DMEM; GIBCO) containing 10% fetal calf serum (FCS) and 2.5% rat serum (RS) was then added and replaced at 48-h intervals. These were called "cocultures" to reflect the predominance of neurons and ISMC, although other cell types such as glial cells could also be present in low numbers. For subsequent use in coculture with sympathetic neurons, primary cultures of ISMC were established and maintained in DMEM plus 10% FCS alone, in which ENS neurons were undetectable after 5-7 days.
In some cultures, ENS neurons were selectively removed by treatment with the neurotoxin scorpion venom (Sigma) at 24 h. Scorpion venom contains selective agonists of the fast Na+ channel found on neurons (25), and in initial experiments, we found that a single treatment caused a dose-dependent neurotoxicity without affecting the glial or smooth muscle cells. Subsequently, we used scorpion venom at 30 µg/ml, about two times higher than the dose required for removal of all neurons in pilot experiments, and we followed this with immunocytochemistry of cohort cultures to confirm the absence of neurons.Coculture of ISMC with sympathetic neurons. Sympathetic neurons were established in vitro according to previously published techniques (2). Briefly, the superior cervical ganglia (SCG) from neonatal rats were dissociated and cultured on Matrigel-coated multiwell plates in medium containing 50 ng/ml 2.5 S nerve growth factor (NGF). Accessory cells were removed by an initial treatment with the antimitotic cytosine arabinoside (2.5 µM; Sigma), which resulted in a complex network of pure neurons by 2 days and thereafter.
ISMC were obtained by dissociation of 14- to 21-day-old primary cultures. These were added to 4- to 6-day-old cultures of SCG neurons or maintained in pure control on similarly treated wells. NGF was added to all wells at 48-h intervals. In coculture, the ISMC adhered and grew underneath the neurons, which responded by forming extensive contacts through the growth and rearrangement of neurites. Ganglion-like plexuses developed on top of the smooth muscle by 2-3 days, with extensive neurite contact with the ISMC. The cultures were then synchronized and tested for growth response using [3H]thymidine uptake, with the level of NGF maintained by addition of concentrated aliquots when replacement of medium was not carried out.Immunocytochemistry and histochemistry. Immunocytochemistry with the pan-neuronal marker PGP 9.5 (Ultraclone, Isle of Wight, UK) was used to study the presence of neurons within cultures of ISMC. Briefly, cultures were fixed with neutral buffered formalin, washed, incubated overnight at room temperature with PGP 9.5 (1/1,000 in phosphate-buffered saline with 0.2% Triton X-100), followed by a biotinylated secondary anti-rabbit antibody (1/300; DAKO) and visualized with Cy3-labeled streptavidin (1/500; Jackson Labs), each for 1 h at room temperature. Staining was observed with an Olympus BX-60 microscope, and images were digitally captured (ImagePro Plus; Media Cybernetics).
Immunocytochemistry with a mouse monoclonal antibody toGrowth assays. The growth of ISMC in vitro was evaluated by direct counting of cell number or by [3H]thymidine incorporation (reflecting the entry into S phase of mitosis). Results are reported as the average of individual experiments, and each experiment was the average from triplicate wells.
For determination of cell number, cultures were dissociated with 0.15% trypsin in HEPES-buffered Hanks' saline, and cell number was determined with a hemocytometer. To assess [3H]thymidine incorporation, a standard protocol was used involving the synchronization of mitotic cycle before testing of the growth response of ISMC, either in pure culture or in cocultures. All cultures were washed, exposed to fresh medium containing serum as appropriate, and changed to DMEM without serum 24 h later. After further incubation for 72 h, all media was replaced with either medium alone (baseline condition) or medium containing mitogenic stimuli. These were FCS, RS, or 10 ng/ml platelet-derived growth factor (PDGF; UBI). At 20 h following this, [3H]thymidine was added for 4 h, followed by routine processing for scintillation counting.Western blotting.
The presence and relative abundance of -SM actin was studied using
Western blotting and videodensitometry as previously described (3). Briefly, ISMC in culture or freshly dissected smooth
muscle tissue were enzymatically dissociated and ISMC number was
determined by hemocytometer counting in triplicates. Cell suspensions
were centrifuged to a pellet and resuspended in lysis buffer containing protease inhibitors. Equal volumes of 2× sample buffer were added to
the tissue lysates, and the samples were boiled for 5 min, subsequently
resolved by SDS-PAGE, transferred to polyvinylidene difluoride
membranes, blocked in 5% BSA, and incubated overnight at 4°C with an
antibody to
-SM actin (1A4) at 1:1,000 in Tris-buffered saline (TBS) containing 0.2% Tween 20 (TBS-T). The blots were washed
in TBS, incubated for 2 h at room temperature with biotinylated goat anti-mouse IgG (DAKO) at 1:3,000 in TBS-T, washed again, and
incubated for 2 h with horseradish peroxidase-conjugated
streptavidin antibody (1:5,000; DAKO). The blots were washed again and
immersed in a substrate solution of 50 mM Tris containing 0.06%
diaminobenzidine and 0.0125% H2O2 (vol/vol).
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RESULTS |
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ENS neurons and ISMC in coculture.
Cultures of ENS neurons and ISMC were established from the jejunum or
colon from 2- to 4-wk-old rats. Initially, ISMC formed a relatively
uniform layer 1-2 cells thick that showed regular and coordinated
contraction. The cell shape was typically bipolar and spindle- or
ribbon-shaped and was uniform throughout the cultures. By 3-4 days
in vitro, spontaneous contraction was infrequent and ISMC were arranged
in multilayered regions with relatively sparse intervening areas (Fig.
1A). Local retraction of the
cellular sheet began to develop, and after 7 days, retraction of the
entire cell mass from the culture surface began to occur.
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ENS innervation regulates the growth response of ISMC.
Initially, direct determination of cell number was used to evaluate
ISMC growth, and this was found to increase by two- to threefold over a
6-day period. To study the effect of loss of innervation on growth of
ISMC, some cultures were treated with scorpion venom (30 µg/ml), a
selective toxic agonist that targets the Na+ channels
present on neurons. Immunocytochemistry showed that ENS neurons were
undetectable after 18 h, without direct damage to ISMC. ISMC
number was determined in cultures exposed to scorpion venom 6 days
earlier, and this was compared with ISMC number in untreated cohort
cultures. Figure 2 shows the results of a
typical experiment, in which the treatment with scorpion venom and the resulting loss of enteric neurons led to an increased ISMC number compared with controls. On average, treatment of cocultures of ISMC
from the jejunum caused a 41 ± 13% increase in ISMC
number (n = 6 experiments; P < 0.05).
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Sympathetic innervation increases ISMC growth response.
The sympathetic nervous system has a trophic influence on vascular
smooth muscle, and the neural mechanism is thought to involve local or
circulating levels of catecholamines (5). Sympathetic innervation of the intestine is inhibitory, causing relaxation of
smooth muscle largely by -adrenergic receptor-mediated input to ENS
neurons (15). To justify examination of a possible
influence of sympathetic innervation on rat ISMC growth, we looked for
evidence for direct innervation of ISMC using histochemistry for
catecholamines in whole mounts of the smooth muscle layers of rat
jejunum. As expected, this showed dense innervation of the myenteric
plexus neurons but also revealed the spread of nerves within the smooth muscle (Fig. 5A). These
progressively decreased in caliber as they branched, and single axons
with highly fluorescent varicosities were seen to terminate among the
ISMC (Fig. 5A). Therefore, we examined the possibility that
sympathetic innervation might directly influence ISMC growth response.
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ENS innervation maintains ISMC phenotype.
Prolonged mitosis of vascular smooth muscle cells in vitro and in vivo
is associated with alterations in intracellular protein content and an
increased emphasis on protein synthesis (8). Therefore,
inhibition of ISMC growth by ENS neurons in vitro might preserve the
differentiated contractile phenotype. To test this, we examined the
effects of coculture with ENS neurons on the -SM actin content of ISMC.
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DISCUSSION |
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Recent work in animal models of intestinal inflammation showed
that damage to the ENS was an early event, preceding the onset of
hyperplasia of smooth muscle in both the circular and longitudinal muscle layers (26). We suspected a causal relationship,
proposing that ENS-derived factors normally regulate the proliferative
state of smooth muscle, and investigated this through development of a
tissue culture model. In this model, myenteric plexus neurons survived
dissociation from the tissue well and rapidly extended dense arrays of
neurites among the neighboring smooth muscle cells. The smooth muscle
cells were uniform in appearance, and all showed at least some
expression of -SM actin following immunocytochemistry with the 1A4
antibody. The initial ratio of 1:8 of neurons among the smooth muscle
cells allowed good opportunity for interactions and the possible
development of growth control.
To study the effect of neuronal coculture on smooth muscle growth, we used scorpion venom to cause the selective ablation of neurons and compared the growth of ISMC in these cultures with that in untreated parallel but otherwise identical cultures. After 5 days, the culture wells treated with scorpion venom contained significantly more ISMC than the untreated controls. The possibility that neuronal coculture led to suppression of the response of ISMC to growth factors was investigated through growth assays involving [3H]thymidine incorporation, showing that the presence of enteric neurons was associated with a significant suppression of the ISMC response to serum mitogens.
Our investigation of the possible mechanisms involved the testing of the role of cholinergic neurotransmitter release in regulation of ISMC growth. Hexamethonium had no effect over the dose range used, whereas atropine increased the growth response of ISMC in cocultures. This is interpreted as evidence that suppression of nicotinic cholinergic receptors, involved in interneuronal interactions, did not affect neurally mediated growth control and provided support for a role for direct neuron-smooth muscle interactions acting via muscarinic receptors on the ISMC. Although daily application of carbachol to pure cultures of ISMC had no effect, this may not adequately mimic the local release of acetylcholine into the cellular microenvironment.
Clearly, the complex array of neuropeptides that are present in the ENS are expected to be present in vitro as well, requiring a more extensive examination of the effect of both excitatory and inhibitory factors. For example, ATP has recently been described both as a neurotransmitter in guinea pig enteric neurons (1) and as a growth factor for vascular smooth muscle (12). In addition, vasoactive intestinal polypeptide inhibited the growth response of rabbit colonic smooth muscle (29), whereas neuropeptide Y stimulated growth in human vascular smooth muscle cells (13).
Examination of the consequences of coculture of ISMC with pure cultures of rat sympathetic neurons showed significant enhancement of the ISMC growth response, the opposite effect from that seen with enteric neurons. The close proximity of these neurons among the ISMC was required, since their separation by a permeable filter removed the stimulatory effect. Under these culture conditions, sympathetic neurons remain adrenergic (30), which suggests that neurally released catecholamines stimulate ISMC growth. In support of this, both the acute and chronic addition of propranolol to ISMC in cocultures caused significant inhibition of the growth response, showing the sensitivity of ISMC to catecholamines. However, a similar response was seen among ISMC in pure culture when exposed to propranolol, which prevents the clearer identification of a neural mechanism for this effect. Although the presence and significance of direct adrenergic innervation of the rodent ISMC has been debated (15, 27, 31), recent functional evidence has shown that sympathetic nerves innervate adrenoceptors of various types in the intestinal muscle layers (20). Although adrenergic innervation of ISMC has been difficult to demonstrate in standard sections (27), this was readily seen in whole mounts of rat jejunum and thus may reflect technical differences.
Elsewhere, a trophic influence of the sympathetic nervous system on vascular smooth muscle is well established, and sympathetic hyperinnervation is thought to be a major determinant of intimal hyperplasia and increased blood pressure in the spontaneously hypertensive rat model (17). Catecholamines released by these neurons are held responsible for this effect and may act directly to cause increased smooth muscle growth. For example, supersensitivity to catecholamines without change in receptor number is indicated in hyperplasia of rabbit aortic smooth muscle in vivo (22). An indirect effect is also possible, since noradrenaline increased the number of receptors for PDGF in rat aortic smooth muscle in vitro (6). Studies in vitro have shown that sympathetic innervation acts via adrenergic neurotransmitters to cause hypertrophy of the cardiac myocyte (19) as well as regulation of ion channel physiology (32).
Our evidence suggests that neurally mediated release of neurotransmitters was responsible for growth control in vitro, rather than a nonspecific effect of contact with neural membrane. This was shown by the loss of neural growth control following the application of receptor antagonists and, most strikingly, by the opposite effects of coculture of ISMC with ENS vs. sympathetic neurons.
Neurally mediated inhibition or stimulation of smooth muscle growth in the intestine may also be a specific phenomenon that is linked to the neuronal phenotype. The outcome of a net increase in smooth muscle cell number during inflammation may potentially reflect an imbalance among the intrinsic and extrinsic neural influences on the ISMC. For example, the loss of enteric neurons requires sprouting from survivors into a new target field, which may take substantially longer than the regeneration of damaged sympathetic axons whose extrinsic cell bodies are intact.
There is extensive remodeling of innervation of the intestine, due in part to transient or permanent damage affecting both intrinsic and extrinsic innervation. For example, inflammation has been shown to impair the release of acetylcholine (10) and noradrenaline (28) from the muscle wall of the rat jejunum. In jejunitis, we have shown earlier that inflammation caused a permanent upregulation of choline acetyltransferase (11), evidence for increased synthesis of acetylcholine and potentially reflecting the response of intrinsic cholinergic enteric neurons to the increased target population of smooth muscle. As evidence of axonal proliferation, we have shown that the density of innervation of smooth muscle is maintained following inflammation-induced hyperplasia in the rat colon (26).
Smooth muscle cells are highly differentiated, which is evident from the large proportion of contractile filaments within the cell, and must undergo a process of dedifferentiation, referred to as "phenotypic modulation" for mitosis and an increase in cell number to occur (8, 23). In general, smooth muscle proliferation is associated with a decrease of contractile proteins and an increase in protein synthesis. Although these changes are normally reversed, smooth muscle in disease states such as atherosclerosis continue to show increased growth responsiveness, altered lipid metabolism, increased matrix production, and loss of contractile proteins (24). The dedifferentiation of smooth muscle into proliferative, synthetic myoblasts and their subsequent resumption of a myogenic program is necessary for normal homeostasis as well as pathogenesis.
Cultured gastrointestinal smooth muscle cells display a coordinated
program of gene expression reflecting this process (7). Factors that regulate the extent of cell division may influence the
outcome of cell division in vivo, leading to significant alterations in
phenotype that are possibly only slowly reversed and may contribute to
altered motility. For example, we found that smooth muscle cells in the
inflamed intestine were substantially larger, with altered -SM actin
content (3). The inverse relationship between differentiation and cell division led us to examine the effect of
innervation in vitro on the
-SM actin content of ISMC in vitro, using similar techniques as before (3). Although
examination of total protein content as a measure of cell size showed
no significant changes with coculture, semiquantitative Western
blotting and video densitometry provided clear evidence for greatly
increased actin content in ISMC cocultured with ENS neurons. This is
interpreted as evidence that the innervation of ISMC has a critical
role in maintenance of the contractile phenotype through suppression of proliferation.
Overall, the available evidence suggests strongly that neurons are effective regulators of intestinal smooth muscle growth and the maintenance of their differentiated state. This emphasizes the importance of increasing our knowledge of the effects of both acute and chronic inflammation of the intestine on its innervation.
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ACKNOWLEDGEMENTS |
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This work was supported by the Canadian Association of Gastroenterology and the Medical Research Council of Canada.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. G. Blennerhassett, Gastrointestinal Diseases Research Unit, Queens Univ., Hotel Dieu Hospital, 166 Brock St., Kingston, ON K7L 5G2, Canada.
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.
Received 13 September 1999; accepted in final form 14 March 2000.
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