1Department of Physiology and Cell Biology and 2Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada 89557
Submitted 26 September 2002 ; accepted in final form 15 April 2003
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ABSTRACT |
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muscarinic receptor; enteric nervous system; gastrointestinal motility
In the small intestine, ICC-IM are concentrated in the region of the deep muscular plexus (DMP) (6). ICC-DMP also form close associations with varicosities of excitatory and inhibitory enteric motoneurons (11, 37, 40, 46) ICCDMP express neurokinin 1 (NK1) receptors (16, 23, 34, 38), and recent immunocytochemical studies show synaptic-like contacts between SP-containing varicosities and ICC-DMP (40). Exposure of small intestinal muscles to SP caused NK1-receptor internalization in ICC-DMP (19). Taken together, these data suggest that ICC-DMP could be an important site of excitatory innervation in the small intestine, but direct tests demonstrating functional innervation of ICC-DMP by excitatory neurons have not been performed. ICC-DMP are not lost in animals with mutations in the proteins responsible for Kit signaling (21, 42), and no animal model has been identified in which ICC-DMP are selectively removed. Therefore, other techniques are needed to evaluate the role of ICCDMP in neurotransmission.
Stimulation of GI muscles with excitatory neuro-transmitters results in a number of changes in post-junctional cells, some of which might be monitored with antibodies to specific elements of second-messenger cascades. We reasoned that because some isozymes of PKC are activated and translocated by muscarinic stimulation, it may be possible to test whether ICC-DMP are innervated by excitatory motoneurons using an immunohistochemical approach. In doing these experiments, we were also able to obtain evidence of muscarinic responses in neurons within enteric ganglia.
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METHODS |
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The entire gastrointestinal tract from 0.5 cm above the lower esophageal sphincter to 1 cm above the internal sphincter was removed and placed in Krebs-Ringer buffer (KRB). For functional immunohistochemical experiments, 3- to 4-cm strips of small intestine were removed from a region 1 cm above the ileocecal sphincter. The mucosa was removed by sharp dissection, and the remaining strips of tunica muscularis (2 x 1 cm) were pinned to the base of a dish partially filled with Sylgard elastomer (Dow Corning, Midland, MI) with the mucosal side of the circular muscle layer facing upward. Tissues were cut into small strips (5 x 10 mm) and pinned to elastomer panels. Parallel platinum electrodes were placed on either side of the muscle strips. Tissues were subsequently immersed in an organ bath and allowed to equilibrate in oxygenated KRB (97% O2-3% CO2) at 37 ± 0.5°C for 60-90 min before experiments were initiated. Neural responses were elicited by square wave pulses of electrical field stimulation (EFS; 0.5-ms duration, 5 Hz, train duration of 60 s, 15 V) using a Grass S48 stimulator (Quincy, MA).
Functional immunohistochemistry. Immediately after EFS or 20 min
after exposure to agonists, the Sylgard elastomer panels with strips of
tunica muscularis attached were rapidly transferred to ice-cold
acetone or paraformaldehyde (4% wt/vol made up in 0.1 M sodium phosphate
buffer with 0.9% NaCl; PBS, pH 7.4). After 10 min fixation at 4°C, the
muscle strips were removed from the Sylgard panels and washed with 0.05 M PBS
(pH 7.4) for 1 h with several changes of the solution. Nonspecific antibody
binding was reduced by incubation of the tissues in BSA (1%, Sigma, St. Louis,
MO) for 1 h at room temperature. To demonstrate colocalization of Kit and
vimentinlike immunoreactivities, acetone-fixed muscles were incubated
consecutively with primary antibodies to both Kit (ACK2, rat monoclonal
antiserum, 5 µg/ml, GIBCO-BRL, Gaithersburg, MD) and vimentin (goat
polyclonal antiserum, 1:200, Polysciences, Warrington, PA). To show the
immunoreactivities of PKC- and vimentin, primary antibodies to both
PKC-
(rabbit polyclonal antiserum, 1:150, Boehringer Mannheim, Mannheim,
Germany) and vimentin were used consecutively. All antibody incubations were
carried out for 48 h at 4°C. For secondary antibodies, FITC-coupled rabbit
anti-rat (for ACK2), goat anti-rabbit (for PKC-
), and Texas
red-conjugated rabbit anti-goat (for vimentin) IgG were used, respectively.
All secondary antibodies were obtained from Vector Laboratories (Burlingame,
CA; dilution 1:100), and incubations were carried outfor1hat room temperature.
Control tissues were prepared by either omitting the primary antibodies from
the incubation solutions or using preabsorbed anti-PKC-
primary antibody
[i.e., the PKC-
antibody was preincubated with PKC-
peptide
(Boehringer Mannheim) for 12 h before use]. All the antisera were diluted with
0.3% Triton X-100 in 0.05 M PBS (pH 7.4).
Because the anti-Kit antibody performed poorly unless tissues were fixed
with acetone, we used vimentinlike immunoreactivity (vimentin-LI) to identify
ICC, as previously described
(40). It was necessary to use
paraformaldehyde fixation to obtain adequate labeling with the anti-PKC-
antibody. Therefore, we verified that Kit-like immunoreactivity (Kit-LI) and
vimentin-LI were colocalized in the same population of cells within the murine
small intestine, and then we used vimentin antibody (which worked well in
tissues fixed with paraformaldehyde or acetone) to identify ICC in other
experiments.
Tissues were examined with a Bio-Rad MRC 600 confocal microscope (Hercules, CA) with an excitation wavelength appropriate for FITC (488 nm) or Texas Red (595 nm). Confocal micrographs shown are digital composites of Z-series scans of 10-20 optical sections through a depth of 6-20 µm. Final images were constructed with Bio-Rad "Comos" software. n Represents the number of tissues from separate animals examined under each experimental condition.
Solutions and drugs. Muscles were maintained in KRB (37.5 ±
0.5°C; pH 7.3-7.4) containing (in mM): 137.4 Na+, 5.9
K+, 2.5 Ca2+, 1.2 Mg, 134 Cl-, 15.5
HCO3-, 1.2 H2PO4-, and
11.5 dextrose and bubbled with 97% O2-3% CO2. Solutions
of ACh, SP, phorbol ester, atropine sulfate, TTX, guanethidine sulfate, sodium
nitroprusside, and N-nitro-L-arginine
(L-NNA) or N
-nitro-L-arginine
methyl ester (L-NAME; Sigma) were dissolved in distilled water at
10-2 to 10-4 M and diluted in KRB to the stated final
concentrations.
Immunoblotting. A 6- to 8-cm segment of small intestine beginning
1 cm above the ileocecal sphincter was used for Western blot analysis. The
mucosa was removed by sharp dissection, and the remaining strips of tunica
muscularis (2 x 1 cm) were pinned to Sylgard with the mucosal side
of the circular muscle layer facing upward. Tissues were immersed in organ
baths and allowed to equilibrate, as described in Tissue preparation,
before experiments were initiated. Unstimulated muscles (control) and muscles
stimulated with EFS or ACh were removed after stimulation, excess Krebs was
rapidly removed, and muscles were dropped into liquid nitrogen. Extracts were
obtained by mechanical grinding of the tissue in ice-cold extraction buffer
containing (in mM): 20 Tris, 50 EGTA, 50 Na2EDTA, 1 leupeptin, and
1 4-(2-aminoethyl)-benzene sulfonyl fluoride (AEBSF) hydrochloride to
homogenize the tissues. The ratio of buffer to tissue was 20 µl/mg wet wt
of tissue. The homogenized tissue mixture was centrifuged at 100,000
g for 40 min at 4°C (Beckman model L5-75 Ultracentrifuge, RPM 30,
000). After centrifugation, the supernatant was taken as the cytosolic
extract. Membrane-associated extracts were obtained by grinding pellets in
ice-cold extraction buffer (SDS, Tris, EGTA, EDTA, leupeptin, NaF, Na
orthovanadate, 20 µl buffer/mg wet wt of tissue) to dissolve the
particulate fraction. These extracts were centrifuged at 10,000 g for
5-10 min at 4°C. Three parts of each extract were added to one part of SDS
buffer, and the mixture was incubated for 20 min at 37°C before
centrifugation at 10,000 g for 5 min. Electrophoresis of protein was
carried out with 25 µl of supernatant loaded per lane for 60 min at 200 V
at room temperature. After protein separation on 10% SDS-polyacrylamide gel
(0.75-mm spacers), the Western blot was transferred onto nitrocellulose
membrane (Genie electroblotter, run at 24 V, 4°C, 2 h). The sample was
blocked with 0.5% gelatin in TNT buffer (10 mM Tris, 150 mM NaCl, 0.05%
Tween-20) for 1 h and incubated overnight with the same primary PKC-
antibody (1:2,000) used for immunohistochemistry. Goat anti-rabbit alkaline
phosphatase (1:5,000, 1.5-h incubation) was used as a secondary antibody. Both
antibodies were diluted in 0.05% gelatin in TNT. Alkaline phosphatase reaction
was performed with 5-bromo-chloro-3-indolyl phosphate p-toluidine
salt (BCIP) and nitro blue tetrazolium (NBT).
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RESULTS |
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EFS (0.5-ms pulses at 5 Hz for 60 s; n = 6) produced translocation
of PKC--LI in myenteric neurons from a central cytoplasmic distribution
to a peripheral distribution along the edges of cells
(Fig. 1, D and
F). The exact number of myenteric neurons that displayed
translocation of PKC-
-LI was difficult to access due to the distribution
of immunoreactivity within cells and overlap between neurons. After EFS, nerve
fibers within interganglionic tracts were more intensely labeled with
PKC-
-LI (Fig.
1D); however, evidence for translocation within nerve
fibers could not be resolved. EFS also resulted in detectable levels of
PKC-
-LI in a second population of cells at the level of the DMP
(Fig. 1, D and
F). The level of resolution at the light microscope level
could not discern whether PKC-
-LI was cytoplasmic or membrane associated
within the thin process of cells in the DMP. Double-labeling experiments with
vimentin antibody showed that cells in which PKC-
-LI appeared after EFS
were vimentin immunopositive (Fig. 1,
E and F; n = 8).
We tested whether the translocation of PKC--LI in myenteric neurons
and the appearance of PKC-
-LI in cells within the DMP was dependent on
the activation of intrinsic neurons by performing experiments in the presence
of TTX (0.3 µM). After incubation of TTX for 20 min to inhibit action
potential-dependent responses, EFS did not produce translocation of
PKC-
-LI from the cytoplasmic domain to the peripheral regions in
myenteric neurons, and PKC-
-LI was not resolved in cells at the level of
the DMP (Fig. 1,
G-I; n = 6).
To determine the nature of the cells with vimentin-LI at the level of the
DMP, we performed double-labeling experiments with vimentin and Kit antibodies
(markers for ICC in the GI tract)
(40). Double-labeling
experiments revealed a 100% colocalization of vimentin-LI and Kit-LI at the
level of the DMP, confirming that the cells in which PKC--LI was
resolved after EFS were ICC-DMP (Fig. 2,
A-C).
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Addition of ACh (10 µM, 20 min) mimicked the actions of nerve
stimulation on the translocation of PKC--LI from the cytoplasmic region
to the periphery of myenteric neurons. We also found that ACh increased the
resolution of PKC-
-LI in nerve fibers within interganglionic tracts and
caused the appearance of resolvable PKC-
-LI in ICC-DMP
(Fig. 3,
A-C). Similar to the response to EFS,
PKC-
-LI in ICC-DMP could not be resolved to any particular region within
the cells (n = 6).
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Preincubation of tissues in atropine (1 µM) for 20 min completely
inhibited the translocation of PKC--LI in myenteric neurons and the
detection of PKC-
-LI in ICC-DMP by exogenous ACh
(Fig. 3, D-F)
and to EFS (Figs. 3,
G-I; n = 6 each).
During nerve stimulation, multiple neurotransmitters, including ACh and
excitatory neuropeptides such as SP, may be released from excitatory motor
nerves and have actions resulting in the translocation of PKC within a variety
of cell types. The experiments testing atropine suggest that the majority of
the PKC response is due to stimulation of muscarinic receptors. However, we
also tested the effects of SP on PKC--LI in myenteric neurons and
ICC-DMP of the murine small intestine. Addition of SP (1 µM for 20 min) did
not produce a translocation of PKC-
-LI in myenteric neurons, and after
SP stimulation, we did not resolve PKC-
-LI observed in any other
population of cells within the tunica muscularis
(Fig. 4, A-C;
n = 6).
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Phorbol esters have been used to induce translocation of PKC isozymes in a
variety of tissues (1,
2). We examined the actions of
-phorbol ester on the translocation of PKC-
-LI in myenteric
neurons and development of PKC-
-LI in ICC-DMP. Preincubation of tissues
in phorbol ester (10 µM for 20 min) caused a dramatic shift in the
localization of PKC-
-LI in myenteric neurons in a manner similar to that
observed with nerve stimulation and exposure to exogenous ACh.
Immunoreactivity was detected along the periphery of myenteric neurons and
within nerve fibers located within interglangionic tracts. ICC-DMP were
positive for PKC-
-LI following the treatment of tissues with phorbol
ester (Fig. 4, D-F;
n = 6).
As a negative control we examined the effects of the NO donor sodium
nitroprusside (SNP) on translocation of PKC--LI in myenteric neurons and
ICC-DMP. SNP (100 µM) was added to the organ bath for 20 min before
fixation of the tissues. SNP did not produce a translocation of PKC-
-LI
in myenteric neurons, and PKC-
-LI was not observed in ICC-DMP following
exposure to SNP (Fig. 4,
G-I; n = 6).
To examine the specificity of the antibody used in immunohistochemistry
studies, we examined the soluble and particulate fractions of tissues prepared
for Western blot analysis. Under control conditions, immunoblots revealed a
positive band with a molecular weight of 83.3 kDa in the soluble fraction
(Fig. 5), which is the known
molecular weight of PKC-. No detectable band was observed in the
particulate fraction at a similar molecular weight. After nerve stimulation,
using the same parameters used for immunohistochemisty, the density of the
immunoblot in the soluble fraction decreased and a concurrent increase in
density of the immunoblot of the particular cellular fraction was observed at
a similar molecular weight of 83.3 kDa
(Fig. 5B; n =
4). Similar to nerve stimulation, ACh applied in an identical manner to that
performed in the immunohistochemical studies caused a similar reduction in the
soluble immunoblot band and an increase in density of the immunoblot band of
the particulate fraction (Fig.
5C; n = 4).
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DISCUSSION |
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Smooth muscles express a variety of PKC isozymes, and the functions of
these kinases are of great importance in cell activities such as contraction,
motility, and gene expression
(20,
22,
33,
39). We chose to concentrate
on PKC-, because previous studies have shown that this isozyme can be
activated and translocated in response to a variety of physiological agonists.
The specific PKC isozymes that respond to a given agonist vary between cell
types (17), and PKC-
has
been reported to translocate in response to muscarinic stimulation
(33) or to show no response
(22,
33). In the present study, we
used an antibody that did not resolve PKC-
-LI in either smooth muscle
cells or ICC under resting conditions. Muscarinic stimulation resulted in
PKC-
-LI in ICC-DMP, but immunoreactivity was never resolved in smooth
muscle cells. This observation suggests that either the levels of PKC-
were too low to be resolved in smooth muscle cells with the antibody we used
or muscarinic stimulation does not activate translocation of this isozyme in
intestinal smooth muscle cells. The absence of smooth muscle PKC-
-LI was
beneficial for our experiments, because we were able to observe a strong
signal-to-noise ratio in ICC-DMP fluorescence after muscarinic stimulation.
The results suggest that ICC-DMP receive motor input from cholinergic,
excitatory motoneurons. As a result of the lack of immunoreactivity in smooth
muscle cells, however, we were unable to determine whether cholinergic neurons
also innervate and activate smooth muscle cells (i.e., parallel innervation of
both cell types occurs).
It is interesting that ICC-DMP lacked PKC--LI in unstimulated
tissues, and significant immunoreactivity appeared after muscarinic
stimulation. These observations suggest that the immunoreactivity of
PKC-
in ICC-DMP was increased by activation or translocation. It is
possible that the epitope for the antibody was shielded when PKC-
was
unstimulated. Conformational changes during activation or translocation may
have unmasked the epitope and increased the affinity for the antibody. At the
present time, little has been reported about the conformational changes that
occur during PKC-
translocation; however, activation and translocation
of other isoforms of PKC as well as translocation of other cytosolic proteins,
e.g., p47 (phox), to the plasma membrane result in conformational changes that
could affect antibody affinities
(4,
35). Western blots
demonstrated the presence of PKC-
in tissue extracts before and after
cholinergic stimulation (see Fig.
5). In unstimulated tissues, immunoreactivity was primarily in the
soluble fraction, and stimulation with exogenous ACh or by activation of
intrinsic neurons led to translocation of PKC-
-LI to the particulate
fraction. These data support the notion that the PKC-
epitope was
present in unstimulated tissues, and the lack of PKC-
-LI in unstimulated
ICC-DMP was due to relative unavailability of the epitope.
In addition to ICC-DMP, changes in PKC- immunoreactivity were also
noted in a subpopulation of enteric neurons. Immunoreactivity was concentrated
in the central regions of cells in resting neurons, and a clear translocation
to a more peripheral compartment occurred after stimulation. PKC-
responses were blocked by TTX, suggesting that neuroneuronal transmission was
necessary, and inhibition by atropine suggested that translocation was
initiated by muscarinic receptor occupation. Enteric neurons have been
reported to express muscarinic receptors
(5) and to respond to
cholinergic stimulation via muscarinic mechanisms
(8). In the guinea pig antrum,
for example, fast nicotinic responses to applied ACh were followed by slow
depolarizations mediated by muscarinic receptors in 32% of neurons studied
(36). Others
(18) have concluded that
transmission from sensory neurons in the ascending and descending reflex
pathways in the guinea pig ileum is mediated by ACh acting at both nicotinic
and muscarinic receptors. Despite these studies, the importance of muscarinic
inputs in enteric neural integration is still not well understood. The present
study provides a new technique to evaluate the activation of muscarinic
responses during GI reflexes.
Although some investigations have linked effects of SP to activation of PKC
(9) and specifically to
translocation of PKC-
(31), others
(13) have shown that
muscarinic stimulation can induce translocation of PKC, whereas SP was
ineffective. Similar observations were made in the present study, and we found
that muscarinic stimulation but not stimulation by SP caused translocation of
PKC-
in myenteric neurons and resolution of PKC-
-LI in ICC-DMP. If
the actions of SP are mediated via a PKC mechanism in these cells, then other
isoforms of PKC must be linked to the neurokinin receptors expressed by
neurons and ICC-DMP.
In summary, ICC-DMP of the small intestine are innervated by excitatory
motoneurons. Funtional innervation is supported by the observation that
stimulation of enteric motoneurons increased PKC--LI in ICC-DMP. Neural
responses were mimicked by exogenous ACh and blocked by atropine, suggesting
that cholinergic neurons, which form close associations with ICC-DMP
(40), release ACh and activate
PKC-
via a muscarinic receptor-linked mechanism. We also found
translocation of PKC-
-LI in enteric neurons, suggesting muscarinic
neurotransmission results from field stimulation in a subpopulation of enteric
nerves. PKC immunoreactivity is a useful means of detecting the use of
muscarinic pathways during GI reflexes.
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DISCLOSURES |
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FOOTNOTES |
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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.
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REFERENCES |
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