sr-142801 and pyridoxal-phosphate-6-azophenyl-2--4--disulfonic-acid

Slow excitatory postsynaptic potentials (EPSPs) in enteric neurons arise from diverse sources, but which neurotransmitters mediate specific types of slow EPSPs is unclear. We investigated transmitters and receptors mediating slow EPSPs in myenteric neurons evoked by electrical stimulation of the mucosa in guinea pig small intestine. Segments of ileum or jejunum were dissected to allow access to the myenteric plexus adjacent to intact mucosa, in vitro. AH and S neurons were impaled with conventional intracellular electrodes. Trains of stimuli delivered to the mucosa evoked slow EPSPs in AH neurons that were blocked or depressed by the neurokinin-1 (NK1) tachykinin antagonist SR140333 (100 nM) in 10 of 11 neurons; the NK3 tachykinin receptor antagonist SR142801 (100 nM) had no effect on slow EPSPs in seven of nine AH neurons. Single pulses to the mucosa evoked fast EPSPs and slow depolarizations in S neurons. The depolarizations were divided into intermediate (durations 300-900 ms) or slow (durations 1.3-9 s) EPSPs. The slow EPSPs were blocked by pyridoxal phosphate-6-axophenyl-2-4-disulfonic acid (30 microM, N = 3) or the specific P2Y(1) antagonist MRS 2179 (10 microM, N = 6) and were predominantly in anally projecting S neurons that were immunoreactive for nitric oxide synthase (NOS). In contrast, intermediate EPSPs were predominantly evoked in NOS-negative neurons; these were abolished by MRS 2179 (N = 8). Thus activation of pathways running from the mucosa excites three different types of slow EPSP in myenteric neurons, which are mediated by either a tachykinin (NK1, AH neurons) or a purine nucleotide (P2Y(1), S neurons).

Topics: Adenosine Diphosphate; Animals; Electric Stimulation; Evoked Potentials; Excitatory Postsynaptic Potentials; Female; Guinea Pigs; Ileum; Immunohistochemistry; In Vitro Techniques; Intestinal Mucosa; Jejunum; Kinetics; Male; Myenteric Plexus; Neurokinin-1 Receptor Antagonists; Nitrergic Neurons; Nitric Oxide Synthase Type I; Piperidines; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Quinuclidines; Receptors, Neurokinin-1; Receptors, Purinergic P2; Receptors, Purinergic P2Y1

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

In functional experiments, we have investigated the effect exerted by neurotransmitters released from capsaicin-sensitive primary afferent nerve terminals in the isolated guinea-pig common bile duct. In resting preparations, capsaicin (0.1 microM) produced a quick contraction (45.1+/-4% of KCl 80mM) which was abolished by either atropine (1 microM) or tetrodotoxin (0.5 microM). The tachykinin receptor-selective antagonists GR 82334 (NK1 receptor-selective; 3 microM), MEN 11420 (NK2 receptor-selective; 1 microM) and SR 142801 (NK3 receptor-selective; 0.1 microM) administered separately failed to reduce the capsaicin-evoked contraction, whereas any combination of the three antagonists was effective: GR 82334 plus MEN 11420, 36+/-7% reduction; GR 82334 plus SR 142801, 48+/-4% reduction; MEN 11420 plus SR 142801, 55+/-3% reduction; GR 82334 plus MEN 11420 plus SR 142801, 57+/-5% reduction. Neither the CGRP1 receptor antagonist h-CGRP (8-37) (1.5 microM) nor the P2X purinoceptor antagonist PPADS (50 microM) affected the contractile response to capsaicin. The effect of capsaicin (0.1 microM) was abolished by pretreatment with capsaicin itself (10 microM for 15 min). Human calcitonin gene-related peptide (h-CGRP; 0.1 microM) mimicked the effect of capsaicin on resting preparations (contractile response =28% of KCl 80 mM). In preparations precontracted with a submaximal concentration of KCl (24 mM), and in the presence of atropine (1 microM), GR 82334 (3 microM) and MEN 11420 (3 microM), capsaicin (1 microM) produced a tetrodotoxin-insensitive long-lasting relaxation (45+/-3% reduction of tone, at 4min from administration), which was unaffected by the nitric oxide (NO) synthase inhibitor, L-NOARG (100 microM). h-CGRP (10-50 nM) produced a similar sustained relaxation of precontracted preparations (59+/-4% reduction of tone). h-CGRP (8-37) (1.5 microM) almost completely reversed the relaxations produced by both capsaicin and h-CGRP. Application of electrical field stimulation (EFS: trains of stimuli of 10Hz; 0.25ms pulse width; supramaximal voltage; for 60s) to precontracted preparations produced a sustained, tetrodotoxin (1 microM)-sensitive relaxation (32+/-4% reduction of tone). L-NOARG (100 microM) greatly reduced (69+/-5% inhibition) the EFS-elicited relaxation. A complete reversal of the relaxant response to EFS into a contraction was obtained by administering L-NOARG to preparations in which a functional blockade of capsaicin-sensitive primary afferent neur

Topics: Animals; Atropine; Calcitonin Gene-Related Peptide; Capsaicin; Common Bile Duct; Electric Stimulation; Guinea Pigs; Humans; Immunohistochemistry; In Vitro Techniques; Male; Muscle Contraction; Neurokinin-1 Receptor Antagonists; Peptide Fragments; Peptides, Cyclic; Physalaemin; Piperidines; Potassium Chloride; Pyridoxal Phosphate; Receptors, Neurokinin-2; Receptors, Neurokinin-3; Receptors, Tachykinin; Tachykinins; Tetrodotoxin