Volume 133, Issue 4, Pages 1229-1239 (October 2007) Inflammation and Inflammatory Agents Activate Protein Kinase C ε Translocation and Excite Guinea-Pig Submucosal Neurons  Daniel P. Poole, Hayato Matsuyama, Trung V. Nguyen, Emily M.Y. Eriksson, Christopher J. Fowler, John B. Furness  Gastroenterology  Volume 133, Issue 4, Pages 1229-1239 (October 2007) DOI: 10.1053/j.gastro.2007.07.002 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 PKCε translocation and change in location of substrates for PKC-mediated phosphorylation in response to PDBu (100 nmol/L, 10 min). (A) Western blot analysis of lysates from guinea-pig submucosa (smp), external muscle-myenteric plexus (mp), and heart probed using antibodies specific for PKCε. The third column is a Western blot of protein immunoprecipitated using a mouse anti-PKCε antibody from submucosal lysates that gave a 95-kilodalton band when probed with a rabbit anti-PKCε antibody (smp–ip). These data indicate that the antibodies are specific for PKCε. (B) PKCε immunoreactivity was present in the cytoplasm of submucosal neurons in control ganglia. (C) After treatment with PDBu, much of this immunoreactivity was translocated to the vicinity of the plasma membrane (arrows). (D) Localization of proteins that are phosphorylated at the serine residue of a PKC consensus site using an antiphosphoserine-consensus peptide antibody. The control tissue shows phosphorylated protein evenly through the cytoplasm. (E) After exposure of the tissue to PDBu (100 nmol/L, 10 min), an increase in the intensity of phosphorylated protein immunoreactivity occurred, particularly near the surface membrane (arrows). Scale bar = 25 μm. Gastroenterology 2007 133, 1229-1239DOI: (10.1053/j.gastro.2007.07.002) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Concentration- and time-dependence of PKCε translocation in response to PDBu. (A) Effects of different concentrations (applied for 5 min) on the localization of PKCε. Maximum responses to PDBu were at 1 μmol/L. No significant translocation occurred at 1 nmol/L, and the effect at 10 μmol/L was less than at 1 μmol/L. (B) Translocation of PKCε immunoreactivity after PDBu (100 nmol/L) was time-dependent, with initial responses after 1 minute and maximum responses after 1 hour. (C) PDBu-evoked (100 nmol/L, 10 min) translocation was reduced significantly using a PKCε-specific inhibitor peptide, PKCεI (N-myristoyl-EAVSLKPT, 1 μmol/L). **P < .001 compared with (A and B) untreated and with PKC control or (C) PDBu plus inhibitor peptide. Gastroenterology 2007 133, 1229-1239DOI: (10.1053/j.gastro.2007.07.002) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Concentration- and time-dependent PKCε translocation in response to ingenol. (A) Translocation (1-h exposure) was detected at 100 nmol/L and 1 μmol/L, with no significant changes in the distribution of PKCε at 1 nmol/L and 10 nmol/L. (B) Ingenol (1 μmol/L) stimulated PKCε translocation after an exposure period of 10 minutes, with a greater effect being observed after treatment for 1 hour, compared with lower concentrations or shorter times. **P < .001, compared with untreated. (C) Confocal image of a submucosal ganglion showing PKCε-labeling close to the plasma membrane (arrows) after ingenol (1 μmol/L, 1 h). (D) Immunohistochemical localization of phosphorylated PKC substrates in the same ganglion as C. Scale bar = 25 μm. Gastroenterology 2007 133, 1229-1239DOI: (10.1053/j.gastro.2007.07.002) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 PKCε translocation in response to the Tat-coupled PKCε activator peptide, Tat-ψεRACK. (A) Effective uptake of carboxyfluorescein-labeled Tat- ψεRACK (5 μmol/L, 5 min) occurred in all submucosal neurons. (B and C) Significant neuronal PKCε translocation was detected in Tat-ψεRACK–treated preparations, relative to untreated controls and to Tat-Tat– (5 μmol/L, 10 min) treated preparations. Scale bar = 25 μm. **P < .001. Gastroenterology 2007 133, 1229-1239DOI: (10.1053/j.gastro.2007.07.002) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Western blot analysis of PKCε translocation in subcellular fractions from the submucosa. (A) PKCε immunoreactivity shifted from the cytosolic (cyt) to the particulate (partic) fraction after exposure to PDBu (100 nmol/L, 1 hour). Coomassie staining of gels showed similar loading of protein. (B) Western analysis (upper) shows that the shift in PKCε immunoreactivity to the particulate fraction was dose-dependent. The gel images below the Western blots show the protein loading detected by Coomassie staining. (C) Quantitation of the Western blot data for the particulate fraction after exposure to PDBu (n = 5; *P < .05, **P < .001 compared with 10 nmol/L). Data have been corrected for protein loading differences. Gastroenterology 2007 133, 1229-1239DOI: (10.1053/j.gastro.2007.07.002) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 The effect of the stimulant of novel PKCs, ingenol, on the input resistance and excitability of submucosal neurons. Excitability was measured in terms of the number of action potentials elicited by a less than 0.2-nA depolarizing current. Small hyperpolarizing pulses were used to measure input resistance. (A) Records from an AH/Dogiel type II neuron. The neuron is identified as an AH neuron by the sag (AHP) after the action potentials (arrow) and the inflection in the decay of the action potential (AP), which can be seen in the differential of the trace (arrowhead, inset). (B) Record from a VIP secretomotor neuron. (C) Record from a NPY secretomotor neuron. These neurons have typical identifying shapes. At the right are camera lucida drawings of neurons that were excited by ingenol. The neurons were filled with marker dye during recording. The neurons from which records B and C were taken are labeled accordingly. *Cell with the typical morphology of a calretinin neuron that was excited by ingenol. **Another example of a VIP neuron that was excited. Gastroenterology 2007 133, 1229-1239DOI: (10.1053/j.gastro.2007.07.002) Copyright © 2007 AGA Institute Terms and Conditions

Figure 7 PKCε translocation in submucosal neurons after TNBS-induced ileitis (6–7 days treatment). (A and B) Examples of PKCε localization in submucosal neurons in preparations of control and inflamed ileum. (C) Analysis of PKCε translocation in response to TNBS-induced inflammation. PKCε association with the plasma membrane was increased significantly relative to untreated controls and sham-operated animals (n = 5 guinea pigs for each group). Scale bar = 25 μm. **P < .001. Gastroenterology 2007 133, 1229-1239DOI: (10.1053/j.gastro.2007.07.002) Copyright © 2007 AGA Institute Terms and Conditions

Figure 8 Effects of PAR2 activators on PKCε localization in submucosal neurons. (A) Trypsin (1 h) caused dose-dependent PKCε translocation, with 5 nmol/L giving the greatest response. (B) Effects of trypsin (5 nmol/L) were time-dependent, with significant PKCε translocation detected after 10 minutes of exposure and greatest responses after 1 hour. (C) The receptor agonist, SLIGRL-NH2 (50 μmol/L, 20 min), caused a significant increase in the association of PKCε with the plasma membrane (b) in comparison with the inactive reverse peptide LRGILS-NH2 (a). The response to SLIGRL-NH2 was reduced significantly by the PKCε-specific inhibitor peptide, N-myristoyl-EAVSLKPT (1 μmol/L) (c). Tetrodotoxin (1 μmol/L) had no effect on SLIGRL-NH2–evoked translocation to the surface membrane (d). (A and B) **P < .001 compared with no trypsin control. (C) **P < .001 for b compared with a, and c compared with b. (D and E) Double labeling immunofluorescence images of a trypsin-treated (5 nmol/L, 1 h) submucosal whole mount. (D) Localization of PKCε, seen at the plasma membranes of some neurons (arrows), but not others (arrow with asterisk). (E) VIP-positive neurons (labeled with asterisk). Ninety-seven percent of neurons (n = 128) showing PKCε translocation were immunoreactive for VIP (examples at arrows). Scale bar = 25 μm. Gastroenterology 2007 133, 1229-1239DOI: (10.1053/j.gastro.2007.07.002) Copyright © 2007 AGA Institute Terms and Conditions

Figure 9 PKCε translocation in submucosal neurons in response to NK3-receptor activation. (A and E) PKCε-IR was distributed evenly within the cytoplasm of all neurons in untreated control preparations. Treatment with senktide (1 μmol/L, 15 sec) caused prominent translocation of PKCε in (B and F) calretinin- and (C and G) NPY-immunoreactive cholinergic secretomotor neurons. (D and H) Pretreatment with the NK3-receptor antagonist SB235375 (1 μmol/L) prevented senktide-evoked PKCε translocation. Scale bar = 25 μm. Gastroenterology 2007 133, 1229-1239DOI: (10.1053/j.gastro.2007.07.002) Copyright © 2007 AGA Institute Terms and Conditions

Figure 10 Quantitative 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. Gastroenterology 2007 133, 1229-1239DOI: (10.1053/j.gastro.2007.07.002) Copyright © 2007 AGA Institute Terms and Conditions