PKC-{epsilon} translocation in enteric neurons and interstitial cells of Cajal in response to muscarinic stimulation

Xuan-Yu Wang,1 Sean M. Ward,1 William T. Gerthoffer,2 and Kenton M. Sanders1

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


    ABSTRACT
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Interstitial cells of Cajal in the deep muscular plexus (ICC-DMP) of the small intestine express excitatory neurotransmitter receptors. We tested whether ICC-DMP are functionally innervated by cholinergic neurons in the murine intestine. Muscles were stimulated by intrinsic nerves and ACh and processed for immunohistochemistry to determine these effects on PKC-{epsilon} activation. Under control conditions, PKC-{epsilon}-like immunoreactivy (PKC-{epsilon}-LI) was only observed in myenteric neurons within the tunica muscularis. Electrical field stimulation or ACh caused translocation of neural PKC-{epsilon}-LI from the cytosol to a peripheral compartment. After stimulation, PKC-{epsilon}-LI was found in spindle-shaped cells in the DMP. These cells were identified as ICC-DMP by Kit-LI and vimentin-LI. PKC-{epsilon}-LI in ICC-DMP and translocation of PKC{epsilon}-LI in neurons were blocked by tetrodotoxin or atropine, suggesting that these responses were due to activation of muscarinic receptors. Western blots also confirmed translocation of PKC-{epsilon}-LI. In conclusion, PKC-{epsilon} translocation is linked to muscarinic receptor activation in ICC-DMP and a subpopulation of myenteric neurons. These studies demonstrate that ICC-DMP are functionally innervated by excitatory motoneurons.

muscarinic receptor; enteric nervous system; gastrointestinal motility


INTRAMUSCULAR ICC (ICC-IM) in gastrointestinal (GI) muscles lie in close association with varicose nerve terminals of enteric motoneurons (3, 10, 12, 14, 15, 26-29, 40). These morphological observations have led many investigators to suggest that ICC may be involved in mediating neural inputs to the GI tract. Isolated ICC have been shown to respond to enteric neurotransmitters including nitric oxide (NO) and substance P (SP) (34, 38), and the use of specific antibodies to cGMP has shown that ICC are specific targets for NO-dependent neurotransmission (32, 45). Recent physiological experiments have supported the role for ICC in enteric neurotransmission by showing that GI muscles lacking ICC-IM have greatly reduced post-junctional responses to nerve stimulation (3, 7, 41, 44).

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.


    METHODS
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Tissue preparation. Twenty-eight BALB-c mice between the ages of 30 and 50 days were used for these studies. Mice were anesthetized with isoflurane (Baxter, Deerfield, IL) before cervical dislocation and exsanguination by decapitation. The Institutional Animal Use and Care Committee at the University of Nevada approved the use and treatment of animals.

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-{epsilon} and vimentin, primary antibodies to both PKC-{epsilon} (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-{epsilon}), 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-{epsilon} primary antibody [i.e., the PKC-{epsilon} antibody was preincubated with PKC-{epsilon} 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-{epsilon} 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{omega}-nitro-L-arginine (L-NNA) or N{omega}-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-{epsilon} 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).


    RESULTS
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 METHODS
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 DISCUSSION
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 REFERENCES
 
PKC-{epsilon}-like immunoreactivity (PKC-{epsilon}-LI) in enteric nerves and in cells within the tunica muscularis was characterized before and after stimulation with EFS and various agonists. Preliminary experiments showed that whole mount preparations produced the best resolution of the changes in PKC-{epsilon}-LI that occurred in response to the stimuli chosen for these studies. Under control conditions PKC-{epsilon}-LI was localized to the cytoplasm of a subpopulation of myenteric neurons (Fig. 1, A and C). An average of 10.7 ± 0.3 neurons per ganglia was immunopositive for PKC-{epsilon}-LI (120 myenteric ganglia from 6 animals). Interganglionic nerve tracts and nerve fibers were slightly immunoreactive for PKC-{epsilon}. Smooth muscle cells within the circular and longitudinal muscle layers and ICC did not display PKC-{epsilon}-LI (Fig. 1, B and C) under control conditions (n = 6).



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Fig. 1. PKC-{epsilon}-like immunoreactivity (PKC-{epsilon}-LI) and vimentinlike immunoreactivity (vimentin-LI) in small intestinal muscles before and after electrical field stimulation (EFS). A and B: staining for PKC-{epsilon} and vimentin, respectively. PKC-{epsilon}-LI was found throughout the cytoplasm of a subpopulation of myenteric neurons (arrows). Some cells were immunonegative (arrowheads). Interganglionic nerve fibers were weakly stained (*). PKC-{epsilon}-LI was not resolved in other cells within the tunica muscularis in unstimulated tissues. Vimentin-LI was found in spindle-shaped cells at the level of the deep muscular plexus. C: digital composition of the images in A and B. D-F: tissues after EFS (0.5-ms duration; 5 Hz for 60 s). PKC-{epsilon}-LI was translocated in myenteric neurons to more peripheral regions of the cell soma (arrows). Interganglionic fiber tracts were more immunopositive after EFS (*). PKC-{epsilon}-LI was also observed in spindle-shaped cells at the level of the deep muscular plexus (arrowheads). E: the spindle-shaped cells were immunopositive for vimentin (arrowheads). F: digital composition of the images in D and E. Yellow pixels represent regions in which PKC-{epsilon}-LI and vimentin-LI are colocalized (i.e., in the spindle-shaped cells at the level of the deep muscular plexus; arrowheads). G-I: preincubation of muscles with TTX (0.3 µM) block the translocation of PKC-{epsilon}-LI in myenteric neurons (arrows) and development of PKC-{epsilon}-LI in interganglionic nerve tracts (*) and cells in the deep muscular plexus in response to EFS. Scale bar in I is applicable to all panels.

 

EFS (0.5-ms pulses at 5 Hz for 60 s; n = 6) produced translocation of PKC-{epsilon}-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-{epsilon}-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-{epsilon}-LI (Fig. 1D); however, evidence for translocation within nerve fibers could not be resolved. EFS also resulted in detectable levels of PKC-{epsilon}-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-{epsilon}-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-{epsilon}-LI appeared after EFS were vimentin immunopositive (Fig. 1, E and F; n = 8).

We tested whether the translocation of PKC-{epsilon}-LI in myenteric neurons and the appearance of PKC-{epsilon}-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-{epsilon}-LI from the cytoplasmic domain to the peripheral regions in myenteric neurons, and PKC-{epsilon}-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-{epsilon}-LI was resolved after EFS were ICC-DMP (Fig. 2, A-C).



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Fig. 2. Interstitial cells in the deep muscular plexus. A: labeling of deep muscular plexus interstitial cells of Cajal (ICC-DMP) with Kit antibody. B: cells with the same morphology are labeled with vimentin antibody. C: digital composition of the images in A and B and demonstrates that the vimentin-positive, spindle-shaped cells are ICC-DMP. All cells that were Kit positive were also vimentin positive. Scale bar in C is applicable to all panels.

 

Addition of ACh (10 µM, 20 min) mimicked the actions of nerve stimulation on the translocation of PKC-{epsilon}-LI from the cytoplasmic region to the periphery of myenteric neurons. We also found that ACh increased the resolution of PKC-{epsilon}-LI in nerve fibers within interganglionic tracts and caused the appearance of resolvable PKC-{epsilon}-LI in ICC-DMP (Fig. 3, A-C). Similar to the response to EFS, PKC-{epsilon}-LI in ICC-DMP could not be resolved to any particular region within the cells (n = 6).



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Fig. 3. Changes in PKC-{epsilon}-LI induced by ACh. A-C: the effects of exogenous ACh on PKC-{epsilon}-LI in intestinal muscle. A shows that application of ACh mimics the effects of EFS (see Fig. 1). PKC-{epsilon}-LI is translocated to the peripheral margins of myenteric neurons (arrows), and immunoreactivity was detected in interganglionic connectives (*). Spindle-shaped cells at the level of the deep muscular plexus also contain PKC-{epsilon}-LI after ACh exposure (arrowheads). B shows that spindle-shaped cells contain vimentin-LI (arrowheads). C shows a digital composite of the images in A and B. Yellow pixels represent colocalization of PKC-{epsilon}-LI and vimentin-LI in ICC-DMP (arrowheads). D-F demonstrate that atropine pretreatment of muscles blocked the effects of ACh on PKC-{epsilon}-LI translocation in myenteric neurons (arrows) and appearance of PKC-{epsilon}-LI in interganlionic connectives (*) and ICC-DMP (arrowheads). G-I demonstrate that atropine pretreatment blocked the effects of EFS on PKC-{epsilon}-LI translocation in myenteric neurons (arrows) and appearance of PKC-{epsilon}-LI in interganlionic connectives (*) and ICC-DMP (arrowheads). Scale bar in I is applicable to all panels.

 

Preincubation of tissues in atropine (1 µM) for 20 min completely inhibited the translocation of PKC-{epsilon}-LI in myenteric neurons and the detection of PKC-{epsilon}-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-{epsilon}-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-{epsilon}-LI in myenteric neurons, and after SP stimulation, we did not resolve PKC-{epsilon}-LI observed in any other population of cells within the tunica muscularis (Fig. 4, A-C; n = 6).



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Fig. 4. Effects of substance P, phorbol ester, and sodium nitroprusside (SNP) on PKC-{epsilon}-LI. A-C show the effects of exogenous substance P (SP; 1 µM) on PKC-{epsilon}-LI in intestinal muscle. A shows that application of SP did not affect PKC-{epsilon}-LI or mimic the effects of EFS (see Fig. 1). PKC-{epsilon}-LI was found in cytoplasmic regions of myenteric neurons (arrows), and immunoreactivity was detected only faintly in interganglionic connectives (*). PKC-{epsilon}-LI was not detected in other cells in the muscle layer after exposure to SP. B shows spindle-shaped cells labeled with vimentin-LI (arrowheads). C shows a digital composite of the images in A and B and demonstrates that ICC-DMP (arrowheads) were immunonegative for PKC-{epsilon}-LI after SP. D-F show the effects of phorbol ester (10 µM) on PKC-{epsilon}-LI in intestinal muscle. D shows that application of phorbol ester mimics the effects of EFS and ACh (see Figs. 1 and 3). PKC-{epsilon}-LI is translocated to the peripheral margins of myenteric neurons (arrows), and immunoreactivity was detected in interganglionic connectives (*). Spindle-shaped cells at the level of the deep muscular plexus also contain PKC-{epsilon}-LI after phorbol ester (arrowheads). E shows that spindle-shaped cells contain vimentin-LI (arrowheads). F shows a digital composite of the images in D and E. G-I show the effects of sodium nitroprusside (SNP; 100 µM) on PKC-{epsilon}-LI in intestinal muscle. G shows that application of SNP did not affect PKC-{epsilon}-LI or mimic the effects of EFS (see Fig. 1). PKC-{epsilon}-LI was found in cytoplasmic regions of myenteric neurons (arrows), and immunoreactivity was detected only faintly in interganglionic connectives (*). PKC-{epsilon}-LI was not detected in other cells in the muscle layer after exposure to SNP. H shows spindle-shaped cells labeled with vimentin-LI (arrowheads). I shows a digital composite of the images in G and H and demonstrates that ICC-DMP were immunonegative for PKC-{epsilon}-LI after SNP. Scale bar in I is applicable to all panels.

 

Phorbol esters have been used to induce translocation of PKC isozymes in a variety of tissues (1, 2). We examined the actions of {alpha}-phorbol ester on the translocation of PKC-{epsilon}-LI in myenteric neurons and development of PKC-{epsilon}-LI in ICC-DMP. Preincubation of tissues in phorbol ester (10 µM for 20 min) caused a dramatic shift in the localization of PKC-{epsilon}-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-{epsilon}-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-{epsilon}-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-{epsilon}-LI in myenteric neurons, and PKC-{epsilon}-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-{epsilon}. 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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Fig. 5. Immunoblots of PKC-{epsilon}-LI in extracts of intestinal muscles. A shows PKC-{epsilon}-LI in the soluble fraction (S) and little or no immunoreactivity in the particulate fraction (P) under control conditions. B and C show a shift in PKC-{epsilon} immunoreactivity from the soluble to the particulate fraction after EFS and ACh stimulation, respectively.

 


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This study shows changes in PKC-{epsilon} immunoreactivity in murine small intestinal muscles in response to cholinergic stimulation and activation of intrinsic neurons. Both ICC-DMP and enteric neurons responded to muscarinic stimulation with changes in PKC-{epsilon}-LI or in the cellular localization of PKC-{epsilon}-LI. The data are consistent with morphological studies that have suggested that ICC-DMP are innervated by enteric excitatory motoneurons (40), and ICC-DMP express receptors for excitatory neurotransmitters (16, 19, 23, 34, 38). We have recently shown that when ICC-IM are lost from the gastric fundus of mutant animals that express a functionally compromised form of c-kit (W/WV mutants) or its natural ligand stem cell factor (Sl/Sld mutants), excitatory cholinergic neural inputs are significantly reduced (3, 41). These studies suggested that the close synaptic relationship between ICC-IM and enteric excitatory motor terminals are necessary for cholinergic neurotransmission. Similar synaptic contacts between excitatory motoneurons and smooth muscle cells are far less common (3, 41). ICCDMP of the murine small intestine survive in W/WV mutants (21), so the type of experiments performed on fundus muscles was not possible. Therefore, we used PKC-{epsilon} immunoreactivity as a novel means to test whether ICC-DMP receive cholinergic input. ICC-DMP were identified by vimentin-LI and Kit-LI. Kit has been shown to be a specific marker for ICC in the small intestine (40). When considered with previous morphological studies, the present study suggests that ICCDMP serve a role in the small intestine similar to ICC-IM in the stomach: these cells are a prominent site of excitatory neurotransmission in GI muscles.

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-{epsilon}, 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-{epsilon} 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-{epsilon}-LI in either smooth muscle cells or ICC under resting conditions. Muscarinic stimulation resulted in PKC-{epsilon}-LI in ICC-DMP, but immunoreactivity was never resolved in smooth muscle cells. This observation suggests that either the levels of PKC-{epsilon} 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-{epsilon}-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-{epsilon}-LI in unstimulated tissues, and significant immunoreactivity appeared after muscarinic stimulation. These observations suggest that the immunoreactivity of PKC-{epsilon} in ICC-DMP was increased by activation or translocation. It is possible that the epitope for the antibody was shielded when PKC-{epsilon} 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-{epsilon} 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-{epsilon} 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-{epsilon}-LI to the particulate fraction. These data support the notion that the PKC-{epsilon} epitope was present in unstimulated tissues, and the lack of PKC-{epsilon}-LI in unstimulated ICC-DMP was due to relative unavailability of the epitope.

In addition to ICC-DMP, changes in PKC-{epsilon} 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-{epsilon} 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-{epsilon} (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-{epsilon} in myenteric neurons and resolution of PKC-{epsilon}-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-{epsilon}-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-{epsilon} via a muscarinic receptor-linked mechanism. We also found translocation of PKC-{epsilon}-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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This project was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-40569. Immunohistochemical experiments were supported by the morphology core of NIDDK Grant DK-41315.


    FOOTNOTES
 

Address for reprint requests and other correspondence: K. M. Sanders, Dept. of Physiology and Cell Biology, Univ. of Nevada School of Medicine, Reno, NV 89557 (E-mail: kent{at}physio.unr.edu).

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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