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Inflammation and Inflammatory Agents Activate Protein Kinase C ε Translocation and Excite Guinea-Pig Submucosal Neurons

2007; Elsevier BV; Volume: 133; Issue: 4 Linguagem: Inglês

10.1053/j.gastro.2007.07.002

1528-0012

Daniel P. Poole, Hayato Matsuyama, Trung Van Nguyen, Emily M. Eriksson, Christopher J. Fowler, John B. Furness,

Ion Channels and Receptors

Background & Aims: Properties of enteric neurons are transformed by inflammation and protein kinase C (PKC) isoforms are involved both in long-term changes in enteric neurons, and in transducing the effects of substances released during inflammation. We investigated roles of PKCε in submucosal neurons by studying translocation in response to inflammatory mediators, effects on neuron excitability, and the changes in PKCε distribution in a trinitrobenzene sulphonate model of ileitis. Methods: Immunohistochemical detection and analysis of association with membrane and cytosolic fractions, and Western blot analysis of cytosolic and particulate fractions were used to quantify translocation. Electrophysiology methods were used to measure effects on neuron excitability. Results: All submucosal neurons were immunoreactive for the novel PKC, PKCε, and direct PKC activators, phorbol 12,13-dibutyrate, ingenol 3,20-dibenzoate, and the PKCε-specific activator, transactivator of transduction-ψε receptor for activated C kinase, all caused PKCε translocation from cytoplasm to surfaces of the neurons. Electrophysiologic studies showed that the stimulant of novel PKCs, ingenol (1 μmol/L), increased excitability of all neurons. Stimulation of protease-activated receptors caused PKCε translocation selectively in vasoactive intestinal peptide secretomotor neurons, whereas a neurokinin 3 tachykinin receptor agonist caused translocation in neuropeptide Y and calretinin neurons. In all cases translocation was reduced significantly by a PKCε-specific translocation inhibitor peptide. Increased PKCε at the plasma membrane occurred in all neurons 6–7 days after an inflammatory stimulus. Conclusions: Major targets for PKCε include ion channels near the plasma membrane. PKCε is likely to have a significant role in controlling the excitability of submucosal neurons and is probably an intermediate in causing hyperexcitability after inflammation. Background & Aims: Properties of enteric neurons are transformed by inflammation and protein kinase C (PKC) isoforms are involved both in long-term changes in enteric neurons, and in transducing the effects of substances released during inflammation. We investigated roles of PKCε in submucosal neurons by studying translocation in response to inflammatory mediators, effects on neuron excitability, and the changes in PKCε distribution in a trinitrobenzene sulphonate model of ileitis. Methods: Immunohistochemical detection and analysis of association with membrane and cytosolic fractions, and Western blot analysis of cytosolic and particulate fractions were used to quantify translocation. Electrophysiology methods were used to measure effects on neuron excitability. Results: All submucosal neurons were immunoreactive for the novel PKC, PKCε, and direct PKC activators, phorbol 12,13-dibutyrate, ingenol 3,20-dibenzoate, and the PKCε-specific activator, transactivator of transduction-ψε receptor for activated C kinase, all caused PKCε translocation from cytoplasm to surfaces of the neurons. Electrophysiologic studies showed that the stimulant of novel PKCs, ingenol (1 μmol/L), increased excitability of all neurons. Stimulation of protease-activated receptors caused PKCε translocation selectively in vasoactive intestinal peptide secretomotor neurons, whereas a neurokinin 3 tachykinin receptor agonist caused translocation in neuropeptide Y and calretinin neurons. In all cases translocation was reduced significantly by a PKCε-specific translocation inhibitor peptide. Increased PKCε at the plasma membrane occurred in all neurons 6–7 days after an inflammatory stimulus. Conclusions: Major targets for PKCε include ion channels near the plasma membrane. PKCε is likely to have a significant role in controlling the excitability of submucosal neurons and is probably an intermediate in causing hyperexcitability after inflammation. Neurons within submucosal ganglia play key roles in controlling the movement of fluid between body compartments and the gut lumen1Furness J.B. The enteric nervous system.in: Blackwell, Oxford, U.K2006: 270Google Scholar and derangements of this control by toxins can cause life-threatening loss of water and electrolytes.2Lundgren O. Enteric nerves and diarrhoea.Pharmacol Toxicol. 2002; 90: 109-120Google Scholar, 3Field M. Intestinal ion transport and the pathophysiology of diarrhea.J Clin Invest. 2003; 111: 931-943Google Scholar Inflammation also affects the excitability of submucosal neurons and compromises the regulation of fluid movement.4Lomax A.E. Mawe G.M. Sharkey K.A. Synaptic facilitation and enhanced neuronal excitability in the submucosal plexus during experimental colitis in guinea-pig.J Physiol. 2005; 564: 863-875Google Scholar, 5Lomax A.E. Linden D.R. Mawe G.M. et al.Effects of gastrointestinal inflammation on enteroendocrine cells and enteric neural reflex circuits.Autonom Neurosci. 2006; 126: 250-257Google Scholar Among the factors that are released during intestinal inflammation are proteases from mast cells and tachykinins. Proteases stimulate protease activated receptors (PARs) that occur on submucosal neurons.6Reed D.E. Barajas-Lopez C. Cottrell G. et al.Mast cell tryptase and proteinase-activated receptor 2 induce hyperexcitability of guinea-pig submucosal neurons.J Physiol (Lond). 2003; 547: 531-542Google Scholar Tachykinins released within the mucosa, which act on submucosal neurons, also contribute to the inflammatory response.7Koon H.W. Pothoulakis C. Immunomodulatory properties of substance P.Ann N Y Acad Sci. 2006; 1088: 23-40Google Scholar In myenteric ganglia, activation of protein kinase C (PKC) causes substantial increases in neuron excitability.8Nguyen T.V. Poole D.P. Harvey J.R. et al.Investigation of PKC isoform-specific translocation and targeting of the current of the late afterhyperpolarising potential of myenteric AH neurons.Eur J Neurosci. 2005; 21: 905-913Google Scholar Both PARs and tachykinin receptors are linked to PKC, but how PAR stimulation, tachykinin receptor activation, and inflammation affect PKCs in submucosal neurons is uninvestigated, even though a link between PKC and the consequences of inflammation in the intestine has been shown.9Di Mari J.F. Mifflin R.C. Powell D.W. The role of protein kinase C in gastrointestinal function and disease.Gastroenterology. 2005; 128: 2131-2146Abstract Full Text Full Text PDF Scopus (22) Google Scholar Among the isoforms of PKC, the novel PKC, PKCε, is expressed by all submucosal neurons.10Poole D.P. Hunne B. Robbins H.L. et al.Protein kinase C isoforms in the enteric nervous system.Histochem Cell Biol. 2003; 120: 151-161Google Scholar PKCs translocate to surface and organelle membranes after activation and the redistribution of PKCs is an indication of enzyme activation.11Dorn II, G.W. Mochly-Rosen D. Intracellular transport mechanisms of signal transducers.Annu Rev Physiol. 2002; 64: 407-429Google Scholar Relocation of activated PKCs targets PKC catalytic activity close to potential substrates and provides a mechanism through which substrate specificity can be conferred.11Dorn II, G.W. Mochly-Rosen D. Intracellular transport mechanisms of signal transducers.Annu Rev Physiol. 2002; 64: 407-429Google Scholar Thus, by tracking the translocation of PKCε, it is possible to trace the links between receptor activation and cell signaling.12Hucho T.B. Dina O.A. Levine J.D. Epac mediates a cAMP-to-PKC signalling in inflammatory pain: an isolectin B4(+) neuron-specific mechanism.J Neurosci. 2005; 25: 6119-6126Google Scholar In the current study, we have used this strategy to investigate the role of PKCε in mediating the effects of inflammation and of inflammatory mediators in submucosal neurons. Guinea pigs of either sex (180–250 g) were stunned by a blow to the head and killed by severing their carotid arteries and spinal cords. All procedures conformed to National Health and Medical Research Council of Australia guidelines and were approved by the University of Melbourne Animal Experimentation Ethics Committee. Every effort was made to minimize the number of animals used. The distal ileum was removed and placed in physiologic saline containing nicardipine (1 μmol/L; Sigma-Aldrich, Sydney, Australia) to inhibit tissue contraction. Segments were opened along the mesenteric attachment and pinned flat, mucosa-down, under moderate tension in oxygenated physiologic saline for 30 minutes before experiments were commenced. After equilibration, the preparations were exposed to drug treatments before overnight fixation (2% formaldehyde plus 0.2% picric acid in 0.1 mol/L sodium phosphate buffer, pH 7.2, at 4°C) for immunohistochemistry or cell lysis solution for protein extraction. Immunohistochemical methods were as described previously.10Poole D.P. Hunne B. Robbins H.L. et al.Protein kinase C isoforms in the enteric nervous system.Histochem Cell Biol. 2003; 120: 151-161Google Scholar Briefly, whole-mount preparations were incubated in 10% normal sheep serum plus Triton X-100 in phosphate-buffered saline (PBS) for 30 minutes before incubation with primary antibodies for 48 hours at 4°C (Table 1). The whole mounts then were washed in PBS before incubation with secondary antibodies. Labeled secondary antibodies were from Amersham (Melbourne, Australia), Jackson Immunoresearch (West Grove, PA), and Molecular Probes (Eugene, OR). No PKCε-IR was detected when primary antibodies were omitted. Preparations were analyzed by confocal microscopy on a Bio-Rad MRC1024 (Bio-Rad, Melbourne, Australia) confocal scanning laser system. Images were captured using the 63× objective and were processed using CorelDraw (Corel Corporation, Dublin, Ireland) and Corel Photo-Paint software (Corel Corporation).Table 1Antibodies and Labels Used, With Concentrations and SuppliersTargets and labeled secondary antibodiesHost speciesImmunohistochemistryWestern blotImmunoprecipitationSourceCalretininGoat1:500SwantNeuNMouse1:500Chemicon InternationalNPYSheep1:400E221039Furness J.B. Costa M. Gibbins I.L. et al.Neurochemically similar myenteric and submucous neurons directly traced to the mucosa of the small intestine.Cell Tissue Res. 1985; 241: 155-163Google ScholarPKCεMouse1:5001:5002 μL/mgBD BiosciencesPKCεRabbit1:2001:5002 μL/mgUpstate BiotechnologyPhosphorylated serine PKC substrate siteRabbit1:4001:5002 μL/mgCell Signalling TechnologyVIPMouse1:500V3140Accili E.A. Dhatt N. Buchan A.M.J. Neural somatostatin, vasoactive intestinal polypeptide and substance P in canine and human jejunum.Neurosci Lett. 1995; 185: 37-40Google ScholarNOTE. Locations of manufacturers listed in Table 1: BD Biosciences, North Ryde, Australia; Chemicon International, Temecula, CA; Cell Signalling Technology, Beverley, MA; Swant, Bellinzona, Switzerland; Upstate Biotechnology, Charlottesville, VA. Open table in a new tab NOTE. Locations of manufacturers listed in Table 1: BD Biosciences, North Ryde, Australia; Chemicon International, Temecula, CA; Cell Signalling Technology, Beverley, MA; Swant, Bellinzona, Switzerland; Upstate Biotechnology, Charlottesville, VA. Preparations for protein extraction were placed into ice-cold celLytic (Sigma-Aldrich) mammalian tissue extraction reagent containing a protease inhibitor cocktail and phenylmethylsulfonyl fluoride (5 μmol/L), all from Sigma. Samples were minced before sonication and prepared as previously described.8Nguyen T.V. Poole D.P. Harvey J.R. et al.Investigation of PKC isoform-specific translocation and targeting of the current of the late afterhyperpolarising potential of myenteric AH neurons.Eur J Neurosci. 2005; 21: 905-913Google Scholar Protein samples were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (20 μg total protein, 10% resolving gel) followed by electrophoretic transfer to polyvinylidene difluoride membranes (Hybond-P; Amersham) as described previously.10Poole D.P. Hunne B. Robbins H.L. et al.Protein kinase C isoforms in the enteric nervous system.Histochem Cell Biol. 2003; 120: 151-161Google Scholar Blots were visualized using enhanced chemiluminescence (Amersham). For immunoprecipitation, submucosal lysates (1 mg) were precleared of immunoglobulins and other protein A interacting proteins by agitation with 5 μL of protein A–sepharose beads (75% slurry; Amersham) for 1 hour at 4°C. Beads were removed by centrifugation (10,500g, 2 min). Anti-PKCε antibodies (2 μL) were added to the supernatant and incubated at 4°C with agitation overnight. The immunoprecipitate was treated with β-mercaptoethanol (1 μL), boiled for 5 minutes, centrifuged (10,500g, 5 min), and 15 μL of sample was resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Protein was immunoblotted as described previously. Western blot and immunoprecipitation experiments were performed in duplicate or triplicate. The subcellular distribution of PKCε immunoreactivity (IR) was analyzed both by confocal microscopy and after separation of membrane and cytosol by ultracentrifugation. IR was quantified from confocal images using ImageJ software (available at: http://rsb.info.nih.gov/ij/).8Nguyen T.V. Poole D.P. Harvey J.R. et al.Investigation of PKC isoform-specific translocation and targeting of the current of the late afterhyperpolarising potential of myenteric AH neurons.Eur J Neurosci. 2005; 21: 905-913Google Scholar Membrane fluorescence was calculated by subtracting cytoplasmic pixel counts from total cell pixel counts. Three or more animals with at least 30 cells each were analyzed per treatment. To separate cytosolic and particulate fractions the samples were minced and sonicated, cellular debris was removed (10,000 rpm, 5 min), and the supernatant was centrifuged at 100,000g (1 h, 4°C). The supernatant was retained as the cytosolic fraction and the pellet was resuspended in cold lysis buffer plus Triton X-100 (1%). After centrifugation (10,000g, 10 min, 4°C) the Triton X-100 soluble supernatant was retained as the particulate fraction. Recordings were taken from submucosal neurons under current-clamp conditions,8Nguyen T.V. Poole D.P. Harvey J.R. et al.Investigation of PKC isoform-specific translocation and targeting of the current of the late afterhyperpolarising potential of myenteric AH neurons.Eur J Neurosci. 2005; 21: 905-913Google Scholar maintained at 33°C–34°C. Microelectrodes were filled with 1% biocytin (Sigma-Aldrich) in 1.0 M KCl, and had resistances in the range of 100–120 MΩ. To evaluate excitability of neurons, depolarizing pulses of 500 ms, less than 0.2 nA, applied via the recording electrode, were used to induce 1–3 action potentials before and in the presence of agonists. Small hyperpolarizing current pulses (100 ms, 0.02–0.05 nA) were injected to determine input resistance. During recording, neurons were filled with the marker substance, biocytin, and preparations subsequently were processed to reveal biocytin as a permanent deposit.13Clerc N. Furness J.B. Bornstein J.C. et al.Correlation of electrophysiological and morphological characteristics of myenteric neurons of the duodenum in the guinea-pig.Neuroscience. 1998; 82: 899-914Google Scholar The types of neurons were identified later by morphologic analysis.14Furness J.B. Alex G. Clark M.J. et al.Morphologies and projections of defined classes of neurons in the submucosa of the guinea-pig small intestine.Anat Rec. 2003; 272A: 475-483Google Scholar Inflammation of the ileum was induced by injecting trinitrobenzene sulphonate (TNBS) (30 mg/kg body mass in 30% ethanol) into the distal ileum.15Sayani F.A. Keenan C.M. Van Sickle M.D. et al.The expression and role of Fas ligand in intestinal inflammation.Neurogastroenterol Motil. 2004; 16: 61-74Google Scholar Animals were weighed daily before sacrificing them 6–7 days after injection. Segments of ileum were taken for macroscopic and histopathologic damage assessment (1.5 cm proximal to injection site) and for the immunohistochemical assay of PKCε translocation (4–7 cm proximal to injection site). The PKCε-specific activator octapeptide (HDAPIGYD) based on the PKCε anchoring protein, ε receptor for activated C kinase (RACK), pseudo-εRACK (ψεRACK) was coupled to transactivator of transduction (Tat; YARAAARQARAG).16van Baal J. de Widt J. Divecha N. et al.Translocation of diacylglycerol kinase θ cytosol to plasma membrane in response to activation of G protein-coupled receptors and protein kinase C.J Biol Chem. 2005; 280: 9870-9878Google Scholar Tat-Tat was used as a negative control. Peptide synthesis was by conventional solid-phase and Fmoc chemistry using an automated synthesizer (Symphony/Multiplex; Protein Technologies, Tucson, AZ).17Zeng W. Ghosh S. Lau Y.F. et al.Highly immunogenic and totally synthetic lipopeptides as self-adjuvanting immunocontraceptive vaccines.J Immunol. 2002; 169: 4905-4912Google Scholar 5(6)-Carboxyfluorescein (Fluka, Steinheim, Germany) labeling of peptides attached to the solid support was performed at 4-fold excess in presence of equimolar amounts of O-benzotriazole-N,N,N′,N′-tetramethyluronium-hexafluorophosphate (Novabiochem, Darmstadt, Germany), 1-hydroxybenzotriazole (Auspep, Melbourne, Australia), and 1.5 equivalents of diisopropylethylamine (Sigma-Aldrich) in N,N′-dimethylformamide in reduced O2 for 18 hours during agitation. Peptides were cleaved from supports with 88% trifluoroacetic acid, 5% phenol (BDH Chemicals, Melbourne, Australia), 5% water, and 2% triisopropylsilane and precipitated and washed in ice-cold diethyl ether followed by lyophilization. Peptides were purified by high-performance liquid chromatography. The following compounds were used: phorbol 12,13-dibutyrate (PDBu) (Sigma-Aldrich), bisindolylmaleimide 1 (Calbiochem, La Jolla, CA), forskolin (Sigma-Aldrich), ingenol 3,20-dibenzoate (LC Laboratories, Woburn, MA), calphostin C (Wako, Osaka, Japan), PKCε translocation inhibitor (PKCεI) (N-myristoyl-EAVSLKPT; BioMol Research Laboratories, Plymouth Meeting, PA), SB23537518Hay D.W.P. Giardina G.A.M. Griswold D.E. et al.Nonpeptide tachykinin receptor antagonists III. SB 235375, a low central nervous system-penetrant, potent and selective neurokinin-3 receptor antagonist, inhibits citric acid-induced cough and airways hyper-reactivity in guinea pigs.J Pharmacol Exp Ther. 2002; 300: 314-323Google Scholar (a gift from Dr Gareth Sanger, GlaxoSmithKline Laboratories, Harlow, UK), senktide (ausPep), SLIGRL-NH2 and LRGILS-NH2 (gifts from Professor Nigel Bunnett, University of California—San Francisco, San Francisco, CA), and tetrodotoxin (Alomone Laboratories, Jerusalem, Israel). Single PKCε-IR bands of 90–95 kilodaltons (predicted PKCε mass) were obtained from submucosal lysates with 2 different antibodies against PKCε (Figure 1A). Bands at other molecular weights did not occur and bands were not detected when primary antibodies were omitted. Western blots of protein immunoprecipitated with the monoclonal anti-PKCε antibody yielded 2 bands when probed using the rabbit polyclonal anti-PKCε antibody. These were at approximately 50 kilodaltons and 90–95 kilodaltons. The approximately 50-kilodalton band was protein A. It was present in immunoprecipitates of lysis buffer containing antibody plus protein A or protein A alone. A 90–95 kilodalton band also was detected when rabbit polyclonal anti-PKCε immunoprecipitates were probed with the monoclonal anti-PKCε antibody. Thus, both antibodies detect PKCε at 90–95 kilodaltons. PKCε-IR was localized to the cytoplasm but not in the nucleus of all submucosal neurons, and other cell types (Figure 1B). Cytoplasmic immunoreactivity of most neurons was distributed evenly with no apparent difference between the intensities of staining in the cytoplasm and at the plasma membrane. PDBu (100 nmol/L, 10 min) increased the intensity of PKCε-IR close to the plasma membranes of neurons and reduced the relative intensity of cytoplasmic IR (Figure 1B and C). PDBu also increased the immunoreactivity of PKC-phosphorylated proteins, particularly near the plasma membranes of neurons (Figure 1D and E). No change in PKC-dependent phosphoproteins was observed in untreated control preparations. Calphostin C (1 μmol/L) reduced the increase of phosphoprotein immunoreactivity caused by PDBu. Translocation of PKCε in response to PDBu (100 nmol/L, 10 min) was reduced significantly after pretreatment of preparations with the myristoylated PKCε translocation inhibitor peptide (PKCεI, 1 μmol/L, 20 min; Figure 2C). However, immunolabeling showed that PDBu still stimulated PKC-mediated phosphorylation in submucosal neurons in the presence of PKCεI (1 μmol/L). The activator of novel PKCs, ingenol,19Asada A. Zhao Y. Kondo S. et al.Induction of thymocyte apoptosis by Ca2+-independent protein kinase C (nPKC) activation and its regulation by calcineurin activation.J Biol Chem. 1998; 273: 28392-28398Google Scholar evoked dose- and time-dependent translocation from the cytoplasm to the vicinity of the plasma membrane (Figure 3). The maximum response was observed at 1 μmol/L, with no significant changes occurring at 1 nmol/L or 10 nmol/L (Figure 3A). Initial movement was detected after 10 minutes and was increased markedly in all neurons after 1 hour of exposure (1 μmol/L; Figure 3B). After exposure to ingenol (1 μmol/L) for 1 hour, almost all PKCε-IR was close to the surface membrane and phosphorylated PKC substrates had a similar distribution (Figure 3C and D). Dimethyl sulfoxide, the vehicle in which PDBu and ingenol were dissolved (0.002%, up to 1 h), did not have any effect on PKCε localization or PKC-mediated phosphorylation. The PKCε-specific activator peptide (Tat-ψεRACK; 5 μmo/L, 5 min) caused translocation of PKCε from the cytoplasm toward the plasma membrane (Figure 4A). Equivalent treatment with Tat-Tat had no effect. Effective entry into submucosal neurons was shown using carboxyfluorescein-coupled Tat-ψεRACK (Figure 4B and C). Translocation to organelles, such as the Golgi apparatus, or to the nuclear membrane, was not observed with any activator, PDBu, ingenol, or Tat-ψεRACK. Translocation investigated by separating cytoplasmic and particulate fractions matched observations made using the immunohistochemical method of analysis (Figure 5). A significant increase in PKCε in the particulate fraction occurred after PDBu (100 nmol/L, 1 h). A corresponding reduction in intensity of the cytosolic PKCε band was detected (Figure 5A). The changes in the subcellular distribution of PKCε were dose-dependent (10 nmol/L to 1 μmol/L), the greatest shift in PKCε distribution being observed with 1 μmol/L PDBu (Figure 5B and C). We also investigated the effect of forskolin to test whether activation of protein kinase A triggers the translocation of PKC. Preparations were exposed to forskolin at a concentration (1 μmol/L, 30 min) that causes a protein kinase A–mediated excitation of neurons of guinea pig submucosal ganglia.20Shen K.Z. Surprenant A. Common ionic mechanisms of excitation by substance P and other transmitters in guinea-pig submucosal neurones.J Physiol (Lond). 1993; 462: 483-501Google Scholar No translocation of PKCε was observed. The 4 classes of submucosal neurons in the guinea-pig small intestine are distinguished by their electrophysiologic characteristics (S or AH) and shapes (Dogiel type I, type II, type IV, and stellate), into vasoactive intestinal peptide (VIP)-containing secretomotor/vasodilator neurons (S/Dogiel type I), cholinergic secretomotor neurons (S/type IV), cholinergic secretomotor/vasodilator neurons (S/stellate), and intrinsic primary afferent neurons (AH/Dogiel type II).14Furness J.B. Alex G. Clark M.J. et al.Morphologies and projections of defined classes of neurons in the submucosa of the guinea-pig small intestine.Anat Rec. 2003; 272A: 475-483Google Scholar AH neurons were identified by the hump on the falling phase of the action potential and a prolonged afterhyperpolarizing potential (AHP), after the action potential. Neurons of all 4 types were excited by ingenol (1 μmol/L), as characterized by a significant increase in the number of action potentials evoked by a 500-ms depolarizing current pulse (Figure 6,Table 2). The IAHP of AH neurons was suppressed by ingenol (Figure 6). No change in Ih was detected in response to ingenol. Neither the number of action potentials nor the IAHP were restored to pre-ingenol levels by washout.Table 2Effects of Ingenol (1 μmol/L) on Electrophysiologic Properties of Submucosal NeuronsAH/Dogiel type II (n = 3)S/Uniaxonal (n = 9)Before ingenolPlus ingenolBefore ingenolPlus ingenolInput resistance (Rin, MOhm)172 ± 25366 ± 23aSignificantly different from before ingenol (P < .05), paired t test.337 ± 37446 ± 57aSignificantly different from before ingenol (P < .05), paired t test.Membrane potential (MP, mV)−60 ± 4−51 ± 4aSignificantly different from before ingenol (P < .05), paired t test.−65 ± 1−64 ± 1Excitability (no. of action potentials)2.7 ± 0.318 ± 1aSignificantly different from before ingenol (P < .05), paired t test.1 1 ± 0.32.9 ± 0.5aSignificantly different from before ingenol (P < .05), paired t test.Action potential first interval (ms)35 ± 520 ± 2aSignificantly different from before ingenol (P < .05), paired t test.42 ± 333 ± 2aSignificantly different from before ingenol (P < .05), paired t test.NOTE. All data are mean ± SEM.a Significantly different from before ingenol (P < .05), paired t test. Open table in a new tab NOTE. All data are mean ± SEM. All TNBS-injected animals showed reductions in weight in the 6–7 days postsurgery, relative to sham-operated animals, and showed histologic evidence of inflammation, including reduced villus height, scarring, thickening of the external muscle, and lymphocytic infiltration of enteric ganglia. A significant increase in PKCε-IR occurred at the periphery of submucosal neurons 6 and 7 days after TNBS treatment (Figure 7). Translocation was not restricted to any particular functional subclass of neuron, with evidence for translocation in all neurons examined. Preparations with the greatest degree of PKCε translocation had the greatest histologic damage. Immunohistochemical analysis of PKC-mediated phosphorylation (using anti-PKC[S]) showed that there was an increase in labeling at the plasma membrane of submucosal neurons, indicating that PKC-targeted substrates were likely to be located at this subcellular region. Trypsin (5 nmol/L to 5 mmol/L) or PAR2-activating peptide (SLIGRL-NH2, 50 μmol/L) triggered translocation of PKCε from the cytoplasm to the vicinity of the plasma membrane of about 50% of neurons (Figure 8A and B). Translocation was reduced significantly after pre-incubation with PKCεI (1 μmol/L), but was unaffected by tetrodotoxin (1 μmol/L; Figure 8C). The reverse peptide, LRGILS-NH2 (50 μmol/L), did not alter the subcellular distribution of PKCε (Figure 8C). Double-labeling studies indicated that all neurons that showed PKCε translocation in response to PAR2 activators were VIP-IR noncholinergic secretomotor neurons (trypsin: 97% of affected neurons were VIP-IR, and SLIGRL: 99% were VIP-IR; Figure 8D and E). PKCε translocation was reduced significantly after heat inactivation of trypsin or exposure to trypsin inhibitors before treatment. Senktide (10 nmol/L to 1 μmol/L), an activator of neurokinin (NK)3 receptors,21Laufer R. Gilon C. Chorev M. et al.[pGlu6,Pro9]SP6-11, a selective agonist for the substance P-receptor subtype.J Med Chem. 1986; 29: 1284-1288Google Scholar caused dose- and time-dependent translocation of PKCε to the plasma membranes of submucosal neurons (Figure 9). This response occurred in a subset of neurons, primarily located at the peripheries of submucosal ganglia (Figure 9, Figure 10). Double-labeling indicated that this response was restricted to the neuropeptide Y (NPY) and calretinin-immunoreactive subclasses of cholinergic secretomotor neurons (Figure 9B, C, F, and G). The majority of NPY-positive neurons (93% ± 3%, n = 4 animals) and calretinin-positive neurons (91% ± 6%, n = 4 animals) showed PKCε translocation. PKCε translocation in VIP-IR neurons almost never was observed (3 of 57 neurons examined), and no PKCε translocation was observed in NeuN-IR, large Dogiel type II neurons. The translocation of PKCε was rapid and significant changes were observed after 10–30 seconds of treatment with senktide (Figure 9A). There was generally a complete return of PKCε to the cytoplasm within 2 minutes of the initial treatment (Figure 10A). Tetrodotoxin (1 μmol/L) did not detectably alter PKCε translocation in response to senktide (Figure 10C), indicating that the action was direct, and not an indirect response to release of other neurotransmitters. NK3-receptor–specific inhibition by SB235375 (1 μmol/L) abolished the effects of senktide (Figures 9D, H, and 10C). Similarly, PKCεI (1 μmol/L) significantly reduced PKCε translocation after senktide treatment (Figure 10C). PKC inhibition (bisindolylmaleimide 1, 200 nmol/L) had no significant effect on senktide-evoked PKCε translocation, indicating that translocation was independent of kinase activity (Figure 10C).Figure 10Quantitative analysis of NK3-receptor–mediated PKCε translocation in NPY-IR submucosal neurons. (A) PKCε translocation in response to senktide (1 μmol/L) was rapid and transient, with maximal membrane association detected at 10 seconds and a return to the cytoplasm within 2 minutes. (B) PKCε translocation (30 sec) was dose-dependent. Significant increases in translocation to the membrane were detected at senktide concentrations of 100 nmol/L and 1 μmol/L. (C) Effects of inhibitors on senktide-evoked PKCε translocation (1 μmol/L, 15 sec). PKCε translocation was reduced significantly by pre-incubation with PKCεI (1 μmol/L) or SB235375 (1 μmol/L), but not by bisindolylmaleimide 1 (200 nmol/L) or tetrodotoxin (1 μmol/L). **P < .001.View Larg