vasoactive-intestinal-peptide and senktide

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

The contractile effect of capsaicin in the guinea-pig small intestine involves an activation of enteric cholinergic neurons. Our present data show that the P(2) purinoceptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 30 microM) significantly reduces the contractile response to capsaicin (2 microM) in the presence, but not in the absence, of the tachykinin receptor antagonists [O-Pro(9), (Spiro-gamma-lactam)Leu(10), Trp(11)]physalaemin (1-11) (GR 82334; 3 microM) and (S)-(N)-(1-(3-(1-benzoyl-3-(3, 4-dichlorophenyl)piperidin-3-yl)propyl)-4-phenylpiperidine-4-yl)-N -methylacetamide (SR 142804: 100 nM) (for blocking tachykinin NK1 and NK3 receptors, respectively). PPADS (30 microM) fails to influence submaximal cholinergic contractions evoked by cholecystokinin octapeptide (CCK-8; 2-3 nM) or senktide (1 nM), or the direct smooth muscle-contracting effect of histamine (100-200 nM). A higher concentration (300 microM) of PPADS is also without effect against the stimulatory action of cholecystokinin octapeptide. This means that PPADS can probably be safely used as a purinoceptor antagonist in intestinal preparations. The putative pituitary adenylate cyclase activating peptide (PACAP) receptor antagonist PACAP-(6-38) (3 microM) significantly reduces the contractile effect of PACAP-(1-38) (10 nM) and abolishes that of vasoactive intestinal polypeptide (VIP; 10 nM). PACAP-(6-38) (3 microM) fails to influence the effect of capsaicin (2 microM) both in the absence and in the presence of tachykinin receptor antagonists. The nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine (L-NOARG; 100 microM) also fails to inhibit the capsaicin-induced motor response. We conclude that an endogenous ligand of PPADS-sensitive P(2) purinoceptors (possibly ATP), but not a VIP/PACAP-like peptide or NO, is involved in the nontachykininergic activation of cholinergic neurons in the course of the capsaicin-induced contraction.

Topics: Acetylcholine; Adenosine Triphosphate; Animals; Capsaicin; Enzyme Inhibitors; Guinea Pigs; Ileum; In Vitro Techniques; Intestine, Small; Muscle Contraction; Muscle, Smooth; Neurokinin-1 Receptor Antagonists; Neuropeptides; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Peptide Fragments; Physalaemin; Piperidines; Pituitary Adenylate Cyclase-Activating Polypeptide; Pyridoxal Phosphate; Receptors, Neurokinin-3; Sincalide; Substance P; Vasoactive Intestinal Peptide

The mechanism of action of endogenous tachykinins [substance P (SP) and neurokinin A and B (NKA and NKB)] and of receptor-specific tachykinin analogues (SP methyl ester (SPME), [beta-Ala8]NKA-(4-10), and senktide) was examined in circular muscle of guinea pig stomach. Cross-desensitization studies confirmed that SPME and SP interacted with NK-1 receptors, [beta-Ala8]NKA-(4-10) and NKA with NK-2 receptors, and senktide and NKB with NK-3 receptors. NK-1 and NK-3-receptor agonists induced relaxation and stimulated vasoactive intestinal peptide (VIP) release and nitric oxide (NO) production: tetrodotoxin abolished VIP release, NO production, and relaxation, converting the response to NK-1-receptor agonists to contraction; the NO synthase inhibitor NG-nitro-L-arginine (L-NNA) abolished NO production, partly inhibited VIP release (56-64%, P < 0.01), and abolished relaxation; the VIP antagonist VIP-(10-28) partly inhibited NO production (73-74%, P < 0.001) and relaxation (56-58%, P < 0.01); and atropine augmented relaxation by 28-35% (P < 0.01). The pattern of inhibition implied that: 1) relaxation was mediated by VIP and NO; 2) VIP release was partly dependent on NO production, since it was strongly inhibited by L-NNA; and 3) NO was largely produced by the action of VIP on muscle cells, since it was strongly inhibited by VIP-(10-28). NK-2-receptor agonists elicited only contraction that was not affected by tetrodotoxin; these agonists also inhibited VIP release, NO production, and relaxation induced by NK-1- and NK-3-receptor agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Arginine; Dose-Response Relationship, Drug; Guinea Pigs; In Vitro Techniques; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Neurokinin A; Nitric Oxide; Nitroarginine; Peptide Fragments; Stomach; Substance P; Tachykinins; Tetrodotoxin; Vasoactive Intestinal Peptide

The tachykinin NK3 receptor agonist, senktide, produces concentration-dependent contraction of the circular muscle of the guinea-pig ileum (EC50 2.59 nM). In the presence of the blocker of neuronal type of voltage-sensitive calcium channels, omega-conotoxin (0.1 microM), the contractile response to a low concentration of senktide was converted to an inhibitory effect on spontaneous activity of the ileum. This inhibitory effect was further enhanced in the presence of atropine (1 microM) and was abolished by tetrodotoxin (1 microM), indicating its neural origin. In the presence of atropine and omega-conotoxin, the inhibitory response to senktide (1 nM) was greatly inhibited or even abolished by L-nitroarginine (30 microM), its effect being prevented by L-arginine but not by D-arginine (300 microM in each case). Apamin (0.1 microM) failed to significantly affect the inhibitory response to senktide. Apamin enhanced spontaneous activity of the preparation while L-nitroarginine had no effect. Neither apamin nor L-nitroarginine affected the inhibitory response to isoprenaline. These findings indicate that inhibition of circular muscle activity produced through NK3 receptor stimulation in the guinea-pig ileum is mediated through a neuronal pathway involving nitric oxide or a nitric oxide-like substance(s) generation.

Topics: Animals; Arginine; Guinea Pigs; Ileum; In Vitro Techniques; Male; Muscle Relaxation; Muscle, Smooth; Nitric Oxide; Nitroarginine; omega-Conotoxins; Peptide Fragments; Peptides; Receptors, Neurokinin-3; Substance P; Vasoactive Intestinal Peptide