ingenol-dibenzoate and senktide

Tachykinins, acting through NK(3) receptors (NK(3)R), contribute to excitatory transmission to intrinsic primary afferent neurons (IPANs) of the small intestine. Although this transmission is dependent on protein kinase C (PKC), its maintenance could depend on protein kinase D (PKD), a downstream target of PKC. Here we show that PKD1/2-immunoreactivity occurred exclusively in IPANs of the guinea pig ileum, demonstrated by double staining with the IPAN marker NeuN. PKCepsilon was also colocalized with PKD1/2 in IPANs. PKCepsilon and PKD1/2 trafficking was studied in enteric neurons within whole mounts of the ileal wall. In untreated preparations, PKCepsilon and PKD1/2 were cytosolic and no signal for activated (phosphorylated) PKD was detected. The NK(3)R agonist senktide evoked a transient translocation of PKCepsilon and PKD1/2 from the cytosol to the plasma membrane and induced PKD1/2 phosphorylation at the plasma membrane. PKCepsilon translocation was maximal at 10 s and returned to the cytosol within 2 min. Phosphorylated-PKD1/2 was detected at the plasma membrane within 15 s and translocated to the cytosol by 2 min, where it remained active up to 30 min after NK(3)R stimulation. PKD1/2 activation was reduced by a PKCepsilon inhibitor and prevented by NK(3)R inhibition. NK(3)R-mediated PKCepsilon and PKD activation was confirmed in HEK293 cells transiently expressing NK(3)R and green fluorescent protein-tagged PKCepsilon, PKD1, PKD2, or PKD3. Senktide caused membrane translocation and activation of kinases within 30 s. After 15 min, phosphorylated PKD had returned to the cytosol. PKD activation was confirmed through Western blotting. Thus stimulation of NK(3)R activates PKCepsilon and PKD in sequence, and sequential activation of these kinases may account for rapid and prolonged modulation of IPAN function.

Topics: Acetates; Animals; Cell Line; Diterpenes; Female; Guinea Pigs; Humans; Ileum; Kinetics; Male; Myenteric Plexus; Neurons, Afferent; Peptide Fragments; Phorbol 12,13-Dibutyrate; Phosphorylation; Protein Kinase C; Protein Kinase C-epsilon; Protein Kinase D2; Protein Kinases; Protein Transport; Quinolines; Receptors, G-Protein-Coupled; Receptors, Neurokinin-3; Substance P; Transfection

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.

Topics: Action Potentials; Animals; Blotting, Western; Calbindin 2; Cell Membrane; Cytoplasm; Disease Models, Animal; Diterpenes; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Activators; Guinea Pigs; Ileitis; Ileum; In Vitro Techniques; Inflammation Mediators; Kinetics; Neuropeptide Y; Oligopeptides; Peptide Fragments; Phorbol 12,13-Dibutyrate; Protein Kinase C-epsilon; Protein Transport; Receptor, PAR-2; Receptors, Neurokinin-3; S100 Calcium Binding Protein G; Signal Transduction; Submucous Plexus; Substance P; Trinitrobenzenesulfonic Acid; Trypsin; Vasoactive Intestinal Peptide