Inflammation and Inflammatory Agents Activate Protein Kinase C ε Translocation and Excite Guinea-Pig Submucosal Neurons
Department of Anatomy and Cell Biology, Centre for Neuroscience, University of Melbourne, Parkville, Victoria, Australia. Gastroenterology
(Impact Factor: 16.72).
11/2007; 133(4):1229-39. DOI: 10.1053/j.gastro.2007.07.002
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 PKCepsilon in submucosal neurons by studying translocation in response to inflammatory mediators, effects on neuron excitability, and the changes in PKCepsilon distribution in a trinitrobenzene sulphonate model of ileitis.
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.
All submucosal neurons were immunoreactive for the novel PKC, PKCepsilon, and direct PKC activators, phorbol 12,13-dibutyrate, ingenol 3,20-dibenzoate, and the PKCepsilon-specific activator, transactivator of transduction-Psiepsilon receptor for activated C kinase, all caused PKCepsilon translocation from cytoplasm to surfaces of the neurons. Electrophysiologic studies showed that the stimulant of novel PKCs, ingenol (1 micromol/L), increased excitability of all neurons. Stimulation of protease-activated receptors caused PKCepsilon 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 PKCepsilon-specific translocation inhibitor peptide. Increased PKCepsilon at the plasma membrane occurred in all neurons 6-7 days after an inflammatory stimulus.
Major targets for PKCepsilon include ion channels near the plasma membrane. PKCepsilon is likely to have a significant role in controlling the excitability of submucosal neurons and is probably an intermediate in causing hyperexcitability after inflammation.
Available from: Martin Steinhoff
- "The subcellular distribution of NK 1 R was analysed from captured images using Image J (http://rsbweb.nih.gov/ij/) as described previously (Poole et al. 2007). Single optical sections of the neuronal soma, including the nucleus, were analysed. "
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ABSTRACT: Neuropeptide signalling at the plasma membrane is terminated by neuropeptide degradation by cell-surface peptidases, and by β-arrestin-dependent receptor desensitization and endocytosis. However, receptors continue to signal from endosomes by β-arrestin-dependent processes, and endosomal sorting mediates recycling and resensitization of plasma membrane signalling. The mechanisms that control signalling and trafficking of receptors in endosomes are poorly defined. We report a major role for endothelin-converting enzyme-1 (ECE-1) in controlling substance P (SP) and the neurokinin 1 receptor (NK 1R) in endosomes of myenteric neurones. ECE-1 mRNA and protein were expressed by myenteric neurones of rat and mouse intestine. SP (10 nm, 10 min) induced interaction of NK 1R and β-arrestin at the plasma membrane, and the SP-NK 1R-β-arrestin signalosome complex trafficked by a dynamin-mediated mechanism to ECE-1-containing early endosomes, where ECE-1 can degrade SP. After 120 min, NK 1R recycled from endosomes to the plasma membrane. ECE-1 inhibitors (SM-19712, PD-069185) and the vacuolar H +ATPase inhibitor bafilomycin A 1, which prevent endosomal SP degradation, suppressed NK 1R recycling by >50%. Preincubation of neurones with SP (10 nm, 5 min) desensitized Ca 2+ transients to a second SP challenge after 10 min, and SP signals resensitized after 60 min. SM-19712 inhibited NK 1R resensitization by >90%. ECE-1 inhibitors also caused sustained SP-induced activation of extracellular signal-regulated kinases, consistent with stabilization of the SP-NK 1R-β-arrestin signalosome. By degrading SP and destabilizing endosomal signalosomes, ECE-1 has a dual role in controlling endocytic signalling and trafficking of the NK 1R: promoting resensitization of G protein-mediated plasma membrane signalling, and terminating β-arrestin-mediated endosomal signalling. © 2011 The Authors. Journal compilation
Available from: Rita Selvatici
- "compared to control (acetic acid), ○ P<0.01 compared to saline (one-way ANOVA, followed by Dunnett's multiple comparison test) Inflammation is a series of well-coordinated events that depend on sequential arrival of inflammatory leukocytes to the site of inflammation; acute inflammation is tightly linked to neutrophils that, in addition to their protective role against invading microorganisms, are also considered to be tissue-damaging cells (Selvatici et al. 2006). Activation of PKC is implicated in the pathogenesis of inflammation (Igwe 2006; Poole et al. 2007; Shin et al. 2007; Schubl et al. 2009). Several studies demonstrated that the PKC inhibitors: GF109203X, Gö 6850, Ro 31-8425, Ro 31-8830, chelerytrin, were effective anti-inflammatory agents as evaluated by several models such as ear edema, neutrophil influx and vascular permeability, experimental arthritis (Mulqueen et al. 1992; Kuchera et al. 1993; Jacobson et al. 1995; Merritt et al. 1997; Shin et al. 2007). "
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ABSTRACT: We recently characterized the PKI55 protein as an endogenous protein kinase C (PKC) inhibitor and investigated, in vitro, the potential anti-inflammatory actions of its N-terminal peptides 1-16 (peptide 5), 1-8 (peptide 8) and 1-5 (peptide 9). We showed their ability to inhibit chemotaxis in human polymorphonuclear leukocytes activated by the N-formyl tripeptide for-Met-Leu-Phe-OMe. In this work, we evaluated the anti-inflammatory and the analgesic effects of the selected peptides by in vivo experiments carried out in the mouse. The peptides 5, 8 and 9 (0.1 and 10 nmol i.c.v.) were effective in both the parameters chosen to test the anti-inflammatory activity, i.e., the xylene-induced ear edema and the acetic acid-induced infiltration of neutrophils in the peritoneal cavity. In addition, they displayed analgesic effect, evaluated by the acetic acid-induced writhing test. All the peptides' effects were shared by the reference compounds, dexamethasone and indomethacin (10 mg kg(-1) i.p.), but not by the 9-scramble peptide (10 nmol i.c.v.). The peptide 9, which represents the shortest active sequence of the PKI55 protein, was tested in the ear edema model even following intraperitoneal (i.p.) administration and proved to be effective in the range doses 3-30 mg kg(-1). Moreover, an increase in plasma corticosterone levels was detected in mice treated with the peptide 9, but not with the 9-scramble peptide (both at 10 nmol i.c.v.). The anti-inflammatory and analgesic effects of the PKI55-derived synthetic peptides, possibly related both to PKC inhibition and hypothalamic-pituitary-adrenal axis activation, deserve further investigation in view of potential therapeutic exploitation.
Available from: Graeme Cottrell
- "PKD is a known target of PKC, including PKCε (Brandlin et al., 2002; Haworth et al., 2007; Poole et al., 2008; Rey et al., 2004; Romero et al., 2006; Waldron and Rozengurt, 2003), PKCalpha , -delta and -eta (Berna et al., 2007; Song et al., 2007; Tan et al., 2003; Tinsley et al., 2004; Waldron et al., 1999). In previous studies, we and others have shown that PAR 2 stimulation activates PKCε in transfected cells, in primary spinal sensory neurons (Amadesi et al., 2006) and in enteric submucosal neurons (Poole et al., 2007). Here we showed that stimulation of PAR 2 induced activation of PKD1, PKD2 and PKD3 and this effect was reduced in cells treated with a PKCε translocation inhibitor peptide. "
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ABSTRACT: Serine proteases generated during injury and inflammation cleave protease-activated receptor 2 (PAR(2)) on primary sensory neurons to induce neurogenic inflammation and hyperalgesia. Hyperalgesia requires sensitization of transient receptor potential vanilloid (TRPV) ion channels by mechanisms involving phospholipase C and protein kinase C (PKC). The protein kinase D (PKD) serine/threonine kinases are activated by diacylglycerol and PKCs and can phosphorylate TRPV1. Thus, PKDs may participate in novel signal transduction pathways triggered by serine proteases during inflammation and pain. However, it is not known whether PAR(2) activates PKD, and the expression of PKD isoforms by nociceptive neurons is poorly characterized. By using HEK293 cells transfected with PKDs, we found that PAR(2) stimulation promoted plasma membrane translocation and phosphorylation of PKD1, PKD2, and PKD3, indicating activation. This effect was partially dependent on PKCepsilon. By immunofluorescence and confocal microscopy, with antibodies against PKD1/PKD2 and PKD3 and neuronal markers, we found that PKDs were expressed in rat and mouse dorsal root ganglia (DRG) neurons, including nociceptive neurons that expressed TRPV1, PAR(2), and neuropeptides. PAR(2) agonist induced phosphorylation of PKD in cultured DRG neurons, indicating PKD activation. Intraplantar injection of PAR(2) agonist also caused phosphorylation of PKD in neurons of lumbar DRG, confirming activation in vivo. Thus, PKD1, PKD2, and PKD3 are expressed in primary sensory neurons that mediate neurogenic inflammation and pain transmission, and PAR(2) agonists activate PKDs in HEK293 cells and DRG neurons in culture and in intact animals. PKD may be a novel component of a signal transduction pathway for protease-induced activation of nociceptive neurons and an important new target for antiinflammatory and analgesic therapies.
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