1 Dipartimento Farmaco-Biologico, Università della Calabria, 87036 Arcavacata di Rende (CS); 2 Dipartimento di Anatomia, Istologia, e Medicina Legale, Università di Firenze, 50134 Florence; and 3 Dipartimento di Biologia cellulare e dello Sviluppo, Università di Palermo, 90128 Palermo, Italy
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The aim of the present study
was to evaluate whether alterations in the distribution and/or function
of nitric oxide synthase (NOS) could be involved in the development of
the spontaneous mechanical tone observed in colon from dystrophic
(mdx) mice. By recording the intraluminal pressure of
isolated colon from normal mice, we showed that
N-nitro- L-arginine methyl
ester (L-NAME) increased the tone, even in the presence of
tetrodotoxin. The effect was prevented by L-arginine, nifedipine, or Ca2+-free solution. In colon from
mdx mice, L-NAME was ineffective. Immunohistochemistry revealed that the presence and distribution of
neuronal (nNOS), endothelial, and inducible NOS isoforms in smooth
muscle cells and neurons of colon from mdx mice were the same as in controls. However, the expression of myogenic nNOS was
markedly reduced in mdx mice. We conclude that there is a myogenic NOS in mouse colon that can tonically produce nitric oxide to
limit influx of Ca2+ through L-type voltage-dependent
channels and modulate the mechanical tone. This mechanism appears to be
defective in mdx mice.
nitric oxide synthase; nitric oxide; Duchenne muscular dystrophy; intestinal smooth muscle; spontaneous tone; immunohistochemistry
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
DYSTROPHIN IS A LARGE PROTEIN (17) localized at the inner face of the cell membrane (35) in skeletal, cardiac, and smooth muscles (7) and in brain (19, 33) and enteric neurons (34). Dystrophin deficiency occurs in the X-linked hereditary disease Duchenne muscular dystrophy (DMD) (17). An animal model for study of DMD is provided by mdx mice, a strain lacking dystrophin because of an X-linked mutation (6).
The roles of dystrophin are not completely established. It has been suggested that dystrophin acts as a transsarcolemmal linker between the subsarcolemmal cytoskeleton and the extracellular matrix (11, 12) because it is associated, by its COOH-terminal domain, with a complex of sarcolemmal glycoproteins (13) and, by its NH2-terminal region, with cytoskeletal proteins (14). Furthermore, dystrophin might also be implicated in other functions. For instance, dystrophin anchors nitric oxide synthase (NOS) at the inner surface of the sarcolemma of skeletal fibers (5), and in DMD patients and mdx mice the sarcolemma is devoid of NOS (5, 9). These changes have stimulated speculation that NOS-related defects may contribute to the pathophysiology of DMD.
In DMD, although skeletal muscle failure is the most prominent manifestation, gastrointestinal disorders such as gastric dilation and intestinal pseudoobstruction have been also reported (3, 18). Similarly, functional alterations of gastric and colonic mechanical activity have been observed in mdx mice (2, 20, 22). These changes in gastrointestinal motility have been attributed to an impairment of nitric oxide (NO) function (1-2, 22, 31). In particular, we found that proximal colon from dystrophic mice, in contrast to control animals, developed in vitro an extra spontaneous tone caused by increased Ca2+ influx through L-type voltage-dependent channels (23) and that circular muscle from mdx colon had a more depolarized membrane potential than that observed in control animals (31). This finding could account for the sustained influx of Ca2+ in mdx colon.
In digestive smooth muscle that maintains resting tone, such as the lower esophageal sphincter (LES), the contractile state depends on the intracellular Ca2+ level. The increase in Ca2+ concentration would activate a myogenic Ca2+-dependent NOS responsible for ongoing production of NO able to limit Ca2+ entry and to restrict contraction (26, 27). Therefore, the alterations of the resting tone reported in mdx mice colon could depend on changes in NO production by a myogenic NOS. However, data concerning cellular and subcellular distribution of NOS isoforms in the gastrointestinal tract of dystrophic mice are lacking.
Three different NOS isoforms have been described; two are Ca2+ dependent, constitutively expressed, and distinct in neuronal (nNOS) and endothelial (eNOS) forms, and one is Ca2+ independent, induced by several stimuli, and called "inducible" (iNOS). In the gastrointestinal tract, Ca2+-dependent NOS isoforms have been found not only in nonadrenergic, noncholinergic neurons (4) but also in the smooth cells of different species (8, 24, 26, 32). Recent data have shown that smooth muscle cells of colon express both the Ca2+-dependent and the Ca2+-independent NOS isoforms with different subcellular distributions (Ref. 10; unpublished results).
Our working hypothesis was that the lack of dystrophin leads to alterations of the distribution and/or function of NOS in mdx mouse colon. In this view, in vitro mechanical functional studies were performed to clarify whether the development of the extra tone observed in mdx mice was due to changes in NO production. In parallel, immunohistochemical investigations were carried out using antibodies to nNOS, eNOS, and iNOS isoforms to assess possible differences in their presence and distribution in colon from mdx mice.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experiments were authorized by the Ministero della Sanità (Rome, Italy). Adult (12-18 mo old) male control (C57BL/10SnJ) and dystrophic (mdx mutants; C57BL/ 10Sn-Dmd/J) mice were used. The animals were killed by cervical dislocation, and the abdomen was opened immediately. The colon was removed just distal to the cecum.
In Vitro Functional Studies
Recording of mechanical activity. The contents of the excised colonic segments were gently flushed out with Krebs solution with the following composition (mM): 119 NaCl, 4.5 KCl, 2.5 MgSO4, 25 NaHCO3, 1.2 KH2PO4, 2.5 CaCl2, and 11.1 glucose. Colonic segments were mounted horizontally in a custom-designed organ bath continuously perfused with oxygenated (95% O2-5% CO2) and heated (37°C) Krebs solution. The distal end of each segment was tied around the mouth of a J tube that was connected to a pressure transducer (Statham model P23XL). The ligated proximal end was secured with a silk thread to an isometric force transducer (Grass FT03) to preload the preparations of 0.5 g. The preparations were allowed to equilibrate for at least 30 min. Mechanical activity was detected as changes of intraluminal pressure, which are mainly generated by the circular muscle, and was recorded on an ink-writer polygraph (Grass model 7D).
Experimental protocol.
Experiments using the NOS inhibitor
N-nitro-L-arginine methyl ester
(L-NAME) were designed to determine its effects on
mechanical tone both in control and in mdx colonic muscle.
L-NAME (100 µM) was introduced into the Krebs reservoir
and superfused for 30 min. The effects of perfusion with
L-NAME were determined both in control conditions and after
pretreatment for 30 min with L-arginine (L-Arg,
1 mM; substrate for NOS production of NO and competitor for the same
NOS site of action as L-NAME), TTX (1 µM; Na+
channel blocker), or nifedipine (1 µM; L-type
Ca2+ channel blocker) or in the presence of
Ca2+-free solution. For these experiments, the Krebs
solution was prepared with the same composition described above
except that CaCl2 was omitted and 100 µM EGTA was added.
The experiments using the photosensitive nifedipine were conducted in a
darkened laboratory.
Drugs. The following drugs were used: L-NAME, L-Arg hydrochloride, TTX, nifedipine, and EGTA (all purchased from Sigma, St. Louis, MO). All drugs were dissolved in distilled water except nifedipine, which was dissolved in 70% ethanol. Experiments using the solvent alone showed that it had no effects on the tissue.
Data analysis and statistical tests. Spontaneous mechanical activity was evaluated as mechanical tone. All data are expressed as means ± SE; n indicates the number of experiments and is equivalent to the number of experimental animals. Statistical analysis was performed by means of Student's t-test or analysis of variance when appropriate. A probability value of <0.05 was regarded as significant.
Morphological Studies
Immunohistochemistry.
After excision, specimens of colon from normal and dystrophic mice (3 animals in each group) were cleaned of any digestive material and fixed
in 4% paraformaldehyde in 0.1 M PBS, pH 7.4, for 6 h at 4°C.
The specimens were then placed in 30% sucrose in PBS overnight at
4°C, and the following day they were embedded in OCT compound (Miles,
Elkhart, IN) and frozen at 80°C. Cryosections (14-µm thick) were
obtained from each specimen. After being washed in PBS containing 3%
normal goat serum and 0.5% Triton X-100, all the sections were
incubated with anti-nNOS polyclonal and monoclonal antibodies, with
anti-iNOS and anti-eNOS polyclonal antibodies, and, to label neurons,
with neuron-specific enolase (NSE) polyclonal antibody. All antibodies
were incubated for 24 h at 4°C. Sources and working dilutions of
the antibodies are reported in Table 1.
For double labeling, the sections were simultaneously incubated with
monoclonal nNOS antiserum and polyclonal NSE antiserum. To check the
specificity of the immunostaining, negative controls were performed by
omitting the primary antibodies, replacing them with a nonimmune rabbit
or mouse serum, or, for eNOS antibody, adding the eNOS
(599) blocking peptide. To avoid aspecific
binding by the monoclonal anti-nNOS antibody to mouse tissues, the
sections were pretreated with unlabeled goat anti-mouse whole IgG
molecules (Sigma) diluted 1:50 in PBS and applied to the sections for
15 min, followed by rinsing in PBS. At the end of incubation, the sections received three 10-min washes in PBS. After the final wash, the polyclonal primary antisera were revealed by using
fluorescein Cy2-conjugated AffiniPure F(ab')2 fragment goat
anti-rabbit IgG (H+L; Jackson Immuno-Research, West Grove, PA)
secondary antibody, diluted 1:100 for 2 h at room temperature.
nNOS monoclonal antibody was detected by incubating the sections in the
presence of rhodamine tetramethylrhodamine isothiocyanate-conjugated
AffiniPure rabbit anti-mouse IgG, F(ab')2 fragment specific
(Jackson Immuno-Research) secondary antibody diluted 1:50 for 2 h
at room temperature. The sections were then mounted in an aqueous
medium (Gel Mount; Biomeda, Foster City, CA), and the immunoreaction
products were observed under an epifluorescence Zeiss Axioskop
microscope and photographed.
|
Quantitative analysis. Ten serial cryosections (14-µm thick) were obtained from each specimen and alternatively placed on two different slides. Three series were collected for each animal. One slide of each series was incubated with the NSE antibody and the other with the polyclonal nNOS, eNOS, and iNOS antibodies, respectively. Immunoreactive (IR) neurons were counted under a ×100 objective in the five sets of twin sections obtained for each specimen and the NSE-nNOS double-labeled sections (5 sections for each animal). Percentages (means ± SE) of nNOS/iNOS/eNOS-IR neurons were calculated from total NSE-IR neurons. Statistical analysis was performed by means of Student's t-test. A probability value of <0.05 was regarded as significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In Vitro Functional Studies
As previously described (22, 23), colonic segments from both control and mdx mice showed spontaneous mechanical activity. Once mounted in the organ bath, colon from mdx mice, in contrast to that from control mice, developed an extra spontaneous tone, detectable as an increase in the recording baseline, which reached a stable level (3-4 cmH2O) within 20 min.Perfusion with the NO synthase inhibitor L-NAME (100 µM)
consistently caused a significant increase in tone in normal colon, whereas it had very little or no effect on the tone of mdx
colon (Fig. 1). In any case, the tone
values reached in the presence of L-NAME in control and in
mdx colon were not significantly different (P > 0.05; n = 6). The effect of
pretreatment with L-Arg (1 mM), a NOS substrate, was also
studied. In control animals, L-Arg addition markedly
reduced the mechanical tone. Moreover, it prevented the increase in
tone induced by L-NAME, consistent with the conclusion that
the actions of L-NAME result from the inhibition of NOS. In
colon from mdx mice, L-Arg (1 mM) decreased the
mechanical tone nonsignificantly (Fig. 1).
|
TTX (1 µM) increased the mechanical tone of colonic segments from
both control animals and mdx mice, indicating that the same tonic neural inhibition influences the mechanical tone in both tissues.
In fact, even when the values of the tone reached were significantly
different in the two tissues, there was no significant difference
(P > 0.05; n = 5) in the increase of
tone induced by TTX in control (+2.1 ± 0.7 cmH2O) and
mdx colon (+1.7 ± 0.8 cmH2O). In control
animals, the addition of L-NAME (100 µM) in the presence of TTX gave rise to a further increase in the mechanical tone, whereas
L-NAME was once again without effect in mdx
colon (Fig. 2).
|
To verify whether membrane Ca2+ channels of the smooth
muscle cells are involved in the increase in tone induced by
L-NAME in normal animals, the effects of the NOS inhibitor
were studied in the presence of nifedipine or Ca2+-free
solution. In colon from control animals pretreatment with nifedipine (1 µM) or Ca2+-free solution, which failed to affect the
mechanical tone by themselves (23), prevented the
L-NAME-induced effect on the tone. In contrast, when either
nifedipine (1 µM) or Ca2+-free solution was added after
L-NAME, each reduced mechanical tone (Fig.
3).
|
Morphological Studies
Smooth muscle cells.
In control mice, nNOS-IR was detected on the smooth muscle cells of
longitudinal and circular muscle layers only with the monoclonal
antibody. IR was intense and appeared as a continuous, peripheral ring
(Fig. 4, A and B).
In mdx mice most of the smooth muscle cells of both layers
were unlabeled, and a few cells showed nNOS-IR but the labeling was
faint and interrupted (Fig. 4, C and D). eNOS-
and iNOS-IR were detected on smooth muscle cells of both muscle layers
both in control and in mdx mice. In a manner similar to the
controls, in mdx mice the eNOS-IR was intense and detected
on granular structures mainly located at the cell periphery and
matching the typical mitochondrial distribution (Fig.
5A). Both in control and
mdx mice, no eNOS labeling was present after incubation of
the primary antibody with its blocking peptide. iNOS-IR was faint and
evenly distributed within the cytoplasm (Fig. 5B).
|
|
Neuronal cells.
In mdx mice, nNOS-IR obtained with both antibodies,
eNOS-IR, and iNOS-IR showed labeling intensity and intracellular
distribution similar to those in controls. In particular, the nNOS-IRs
were evenly distributed within the perikaryon and nerve fibers (Fig. 6A), the eNOS-IR was on
granular structures scattered throughout the perikaryon (Fig.
6B), and the iNOS-IR was faint and evenly distributed in the
perikaryon (Fig. 6C). Quantitative analysis demonstrated
that almost half of the NSE-IR neurons were labeled either with
monoclonal (49.70 ± 6.6% controls; 45.40 ± 5.1%
mdx) or polyclonal (48.60 ± 4.7% controls; 44.50 ± 5.6% mdx) nNOS antibody, with no significant difference
between the two groups of animals. As in controls, 100% of the neurons
were eNOS- and iNOS-IR.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present functional and immunohistochemical findings demonstrate that in mouse colon a Ca2+-dependent myogenic NOS can tonically produce NO, without neural input, for maintainance of muscular tone and that its reduced expression in the colon of dystrophic mice can be responsible for the development of the spontaneous tone increment.
Recent data demonstrated the development in mdx colon of an extra mechanical tone, which was independent of alterations in the tonic neural inhibition and caused by an increased influx of Ca2+ through voltage-dependent Ca2+ channels (22, 23). In the present study, the use of the NOS inhibitor L-NAME indicated that an ongoing production of NO modulating the mechanical behavior of colonic muscle existed in control animals. Continuous suppression of colonic smooth muscle activity by NO is a common occurrence, and many studies have provided evidence for this phenomenon (16, 21). The observation that L-NAME was ineffective in mdx colon indicates that, in this tissue, the endogenous production of NO is not in a concentration sufficient to maintain a certain degree of suppression of the mechanical tone. The hypothesis concerning an impairment of the NO function has been already advanced to explain the different mechanical and electrical behavior observed in colon from mdx mice (1, 22, 31). The results obtained in the latter studies favor the existence of a defective nitrergic neurotransmission in mdx colon. In addition, the present study shows the presence in normal colon of a nonneural NOS, likely myogenic, whose defect could contribute to the abnormal development of the mechanical tone in colon from mdx mice. The observation that in controls L-NAME produced a further increase in mechanical tone in the presence of TTX also suggests an extraneural origin of NO. The specificity of the effect of L-NAME in control mice was confirmed by the ability of L-Arg to compete for the active NOS site and to inhibit L-NAME actions. Indeed, L-Arg reduced mechanical tone, indicating that endogenous L-Arg was not adequate to saturate the tonically active NOS and that there is submaximal activity of NOS. The observation that L-Arg failed to appreciably modify the mechanical tone of colon from mdx mice might indicate a defect of NOS because, even in the presence of abundant substrate, the endogenous production of NO is inadequate.
In normal colon the effects on tone induced by L-NAME were prevented or reduced by nifedipine or Ca2+-free solution, indicating that they required Ca2+ influx through a voltage-dependent L-type Ca2+ channel. Therefore, we suggest that when NO production is pharmacologically blocked with L-NAME in control animals, or defective as in mdx mice, there is an increase in Ca2+ influx caused by an enhancement of the opening probability of voltage-sensitive L-type Ca2+-channels, which leads to the tone development. Our previous findings (31), obtained with intracellular recordings in normal colon, demonstrated that block of NOS activity by applied L-NAME caused cell membrane depolarization. This depolarization probably enhances the opening probability of voltage-dependent L-type Ca2+ channels on the plasma membrane and results in Ca2+ influx.
Because one source for TTX-insensitive NO release could be the smooth
muscle cells (8, 10, 26, 32), immunohistochemical studies
were performed to verify possible differences between control and
mdx colon in the distribution at the muscular or neuronal level of the different NOS isoforms. In controls, as previously described (Ref. 10; unpublished results), both smooth
muscle cells and neurons of mouse colon express
Ca2+-dependent and -independent NOS isoforms with different
subcellular distribution. In agreement with Salapatek et al.
(26), who reported the presence in the esophageal smooth
muscle cells of a constitutive, membrane-bound myogenic NOS using the
NADPH-diaphorase method, we could observe, using monoclonal nNOS
antibody, that murine colonic smooth muscle cells also express a
constitutive, membrane-bound NOS. This NOS isoform presumably is the
product of one of the splice variants of nNOS mRNA found in
preparations of intestinal muscle coat (15, 32) and, more
precisely, one of the membrane-associated nNOS proteins (nNOS). We
previously demonstrated (10) the presence of another
constitutive myogenic NOS, eNOS, whose subcellular distribution matched
that of mitochondria. In the colon of mdx mice the eNOS was
unchanged. Conversely, few cells were nNOS immunolabeled in these
animals, and the labeling was very faint; therefore, only
membrane-bound NOS is affected in mdx mice.
Interestingly, the subcellular distribution of nNOS labeling along the cell contour of smooth muscle cells was similar to that reported for skeletal muscle fibers using the same antibody (5). The demonstration that sarcolemmal nNOS labeling is absent in skeletal muscle of mdx mice and DMD patients has raised the possibility that nNOS in these muscles is membrane bound through a link to dystrophin and that the absence of nNOS-IR in these pathological conditions is due to the lack of dystrophin. It is likely that nNOS is also linked to dystrophin in colonic smooth muscle cells and that the loss of this protein would affect the expression of nNOS.
In mdx mice, the number of neuronal cells labeled by eNOS, iNOS, and the two nNOS antibodies and the subcellular distribution of the three NOS isoforms were the same as in control animals (unpublished results). Therefore, in contrast to smooth muscle cells, there is an apparent sparing of nNOS in the neurons from mdx mouse colon. This sparing might depend on the fact that nNOS in the neurons is not linked to dystrophin. The observation that nNOS expression is not changed in the neurons of mdx mouse colon might appear to contrast with our previous studies (22, 31) suggesting a defective nitrergic neurotransmission in mdx colon. However, it cannot be excluded that the neural nitrergic impairment of NO depends on changes in nNOS activity. In addition, because it is well known that the interstitial cells of Cajal (ICC) play a crucial role in nerve-to-muscle signal transmission in the gastrointestinal tract (28), it could be hypothesized that ICC abnormalities lead to the observed alterations of neuromuscular transmission, as suggested for other intestinal motility dysfunctions (29).
We presume that in mouse colon a myogenic Ca2+-dependent NOS, through NO production, limits Ca2+ channel opening probability and modulates the mechanical tone. A similar mechanism has been shown for canine LES, in which high intracellular Ca2+ levels maintained by continual Ca2+ influx through L-type Ca2+ channels continuously activate a myogenic NOS, resulting in an ongoing release of NO that limits the contractile state (25-27). This mechanism of tonic inhibition mediated by myogenic NOS appears to be defective in mdx mouse colon.
In conclusion, there is a Ca2+-dependent membrane-bound nNOS in the smooth muscle cells of mouse colon that is continuously active under physiological conditions and modulates smooth muscle contractile state. The decreased expression of this nNOS in the colonic smooth muscle cells of mdx mice, presumably consequent to the deficiency in dystrophin, contributes to the reported abnormal mechanical behavior.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank F. Bonvissuto for technical assistance.
![]() |
FOOTNOTES |
---|
This work was supported by a grant (no. 1134) from Comitato Telethon Fondazione Organizzazioni non lucrative de utilitá sociale Italy.
Address for reprint requests and other correspondence: F. Mulè, Dipartimento di Biologia cellulare e dello Sviluppo, Laboratorio di Fisiologia generale, Università di Palermo, Viale delle Scienze, 90128 Palermo, Italy (E-mail: fmule{at}unipa.it).
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.
Received 20 February 2001; accepted in final form 11 July 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Azzena, GB,
and
Mancinelli R.
Nitric oxide regenerates the normal colonic peristaltic activity in mdx dystrophic mouse.
Neurosci Lett
282:
105-108,
2000[ISI][Medline].
2.
Baccari, MC,
Romagnani P,
and
Calamai F.
Impaired nitrergic relaxations in the gastric fundus of dystrophic (mdx) mice.
Neurosci Lett
261:
9-12,
1999[ISI][Medline].
3.
Barohn, RJ,
Levine EJ,
Olson JO,
and
Mendell JR.
Gastric hypomotility in Duchenne's muscular dystrophy.
N Engl J Med
319:
15-18,
1988[Abstract].
4.
Bredt, DS,
Hwang PM,
and
Snyder SH.
Localization of nitric oxide synthase indicating a neural role for nitric oxide.
Nature
347:
768-770,
1990[ISI][Medline].
5.
Brenman, JE,
Chao DS,
Xia H,
Aldape K,
and
Bredt DS.
Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy.
Cell
82:
743-752,
1995[ISI][Medline].
6.
Bulfield, G,
Siller WG,
Wight PA,
and
Moore KJ.
X chromosome-linked muscular dystrophy (mdx) in the mouse.
Proc Natl Acad Sci USA
81:
1189-1192,
1984[Abstract].
7.
Byers, TJ,
Kunkel LM,
and
Watkins SC.
The subcellular distribution of dystrophin in mouse skeletal, cardiac, and smooth muscle.
J Cell Biol
115:
411-421,
1991[Abstract].
8.
Chakder, S,
and
Rattan S.
Evidence for VIP-induced increase in NO production in myenteric neurons of opossum internal anal sphincter.
Am J Physiol Gastrointest Liver Physiol
270:
G492-G497,
1996
9.
Chang, WJ,
Iannacone ST,
Lau KS,
Masters BSS,
McCabe TJ,
McMillan K,
Padre RC,
Spencer MJ,
Tidball JG,
and
Stull JT.
Neuronal nitric oxide synthase and dystrophin-deficient muscular dystrophy.
Proc Natl Acad Sci USA
93:
9142-9147,
1996
10.
Corsani, L,
Vannucchi MG,
and
Faussone-Pellegrini MS.
Nitric oxide synthase (NOS) distribution in the muscle coat of mouse colon (Abstract).
Ital J Anat Embryol
105, Suppl1:
54,
2000.
11.
Ervasti, GM,
and
Campbell KP.
Membrane organization of the dystrophin-glycoprotein complex.
Cell
66:
1121-1131,
1991[ISI][Medline].
12.
Ervasti, GM,
and
Campbell KP.
Dystrophin and the membrane skeleton.
Curr Opin Cell Biol
5:
82-87,
1993[Medline].
13.
Ervasti, GM,
Ohlendieck K,
Kahl SD,
Gaver MG,
and
Campbell KP.
Deficiency of a glycoprotein component of the dystrophin complex in dystrophic muscle.
Nature
345:
315-319,
1990[ISI][Medline].
14.
Hemmings, L,
Kuhlman PA,
and
Critchley DR.
Analysis of the actin-binding domain of alpha-actinin by mutagenesis and demonstration that dystrophin contains a functionally homologous domain.
J Cell Biol
116:
1369-1380,
1992[Abstract].
15.
Huber, A,
Saur D,
Kurjak M,
Schusdziarra V,
and
Allescher HD.
Characterization and splice variants of neuronal nitric oxide synthase in rat small intestine.
Am J Physiol Gastrointest Liver Physiol
275:
G1146-G1156,
1998
16.
Keef, KD,
Murray DC,
Sanders KM,
and
Smith TK.
Basal release of nitric oxide induces an oscillatory motor pattern in canine colon.
J Physiol (Lond)
499:
773-786,
1997[Abstract].
17.
Koenig, M,
Monaco AP,
and
Kunkel LM.
The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein.
Cell
53:
219-228,
1988[ISI][Medline].
18.
Leon, SH,
Schuffler MD,
Kettler M,
and
Rohrmann CA.
Chronic intestinal pseudo-obstruction as a complication of Duchenne's muscular dystrophy.
Gastroenterology
90:
455-459,
1986[ISI][Medline].
19.
Lidov, HGW,
Byers TJ,
and
Kunkel LM.
The distribution of dystrophin in the murine central nervous system: an immunocytochemical study.
Neuroscience
54:
167-187,
1993[ISI][Medline].
20.
Mancinelli, R,
Tonali P,
Servidei S,
and
Azzena GB.
Analysis of peristaltic reflex in young mdx dystrophic mice.
Neurosci Lett
192:
57-60,
1995[ISI][Medline].
21.
Mulè, F,
D'Angelo S,
Amato A,
Contino I,
and
Serio R.
Modulation by nitric oxide of spontaneous mechanical activity in rat proximal colon.
J Auton Pharmacol
18:
1-6,
1998[ISI][Medline].
22.
Mulè, F,
D'Angelo S,
Tabacchi G,
Amato A,
and
Serio R.
Mechanical activity of small and large intestine in normal and mdx mice: a comparative analysis.
Neurogastroenterol Motil
11:
133-139,
1999[ISI][Medline].
23.
Mulè, F,
and
Serio R.
Increased calcium influx is responsible for the sustained mechanical tone in colon from dystrophic (mdx) mice.
Gastroenterology.
120:
1430-1437,
2001[ISI][Medline].
24.
Murthy, KS,
and
Makhlouf GM.
Vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide-dependent activation of membrane-bound NO synthase in smooth muscle mediated by pertussis toxin-sensitive Gi1-2.
J Biol Chem
269:
15977-15980,
1994
25.
Salapatek, AMF,
Lam A,
and
Daniel EE.
Calcium source diversity in canine lower esophageal sphincter muscle.
J Pharmacol Exp Ther
287:
98-106,
1998
26.
Salapatek, AMF,
Wang Y-F,
Mao Y-K,
Lam A,
and
Daniel EE.
Myogenic nitric oxide synthase activity in canine lower oesophageal sphincter: morphological and functional evidence.
Br J Pharmacol
123:
1055-1064,
1998[Abstract].
27.
Salapatek, AMF,
Wang YF,
Mao Y-K,
Mori M,
and
Daniel EE.
Myogenic NOS in canine lower oesophageal sphincter: enzyme activation, substrate recycling, and product actions.
Am J Physiol Cell Physiol
274:
C1145-C1157,
1998
28.
Sanders, KM.
A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract.
Gastroenterology
111:
492-515,
1996[ISI][Medline].
29.
Sanders, KM,
Ördög T,
Koh SD,
Torihashi S,
and
Ward SM.
Development and plasticity of interstial cells of Cajal.
Neurogastroenterol Motil
11:
311-338,
1999[ISI][Medline].
30.
Saur, D,
Paehge H,
Schusdziarra V,
and
Allescher HD.
Distinct expression of splice variants of neuronal nitric oxide synthase in the human gastrointestinal tract.
Gastroenterology
118:
849-858,
2000[ISI][Medline].
31.
Serio, R,
Bonvissuto F,
and
Mulè F.
Altered electrical activity in colonic smooth muscle cells from dystrophic (mdx) mice.
Neurogastroenterol Motil.
13:
169-175,
2001[ISI][Medline].
32.
Teng, BQ,
Murthy KS,
Kuemmerle JF,
Grider JR,
Sase K,
Michel T,
and
Makhlouf GM.
Expression of endothelial nitric oxide synthase in human and rabbit gastrointestinal smooth muscle cells.
Am J Physiol Gastrointest Liver Physiol
275:
G342-G351,
1998
33.
Uchino, M,
Teramoto H,
Naoe H,
Yoshioka K,
Miike T,
and
Ando M.
Localisation and characterisation of dystrophin in the central nervous system of controls and patients with Duchenne muscular dystrophy.
J Neurol Neurosurg Psychiatry
57:
426-429,
1994[Abstract].
34.
Vannucchi, MG,
Corsani L,
Giovannini MG,
and
Faussone-Pellegrini MS.
Expression of dystrophin in the mouse myenteric neurones.
Neurosci Lett
300:
120-124,
2001[ISI][Medline].
35.
Zubrzycka-Gaarn, EE,
Bulman DE,
Karpati G,
Burghes AH,
Belfall B,
Klamut HJ,
Talbot J,
Hodges RS,
Ray PN,
and
Worton RG.
The Duchenne muscular dystrophy gene product is localised in sarcolemma of human skeletal muscle.
Nature
333:
466-469,
1988[ISI][Medline].
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |