Tachykinins: receptor to effector.

Tachykinins belong to an evolutionarily conserved family of peptide neurotransmitters. The mammalian tachykinins include substance P, neurokinin A and neurokinin B, which exert their effects by binding to specific receptors. These tachykinin receptors are divided into three types, designated NK1, NK2 and NK3, respectively. Tachykinin receptors have been cloned and contain seven segments spanning the cell membrane, indicating their inclusion in the G-protein-linked receptor family. The continued development of selective agonists and antagonists for each receptor has helped elucidate roles for these mediators, ranging from effects in the central nervous system to the perpetuation of the inflammatory response in the periphery. Various selective ligands have shown both inter- and intraspecies differences in binding potencies, indicating distinct binding sites in the tachykinin receptor. The interaction of tachykinin with its receptor activates Gq, which in turn activates phospholipase C to break down phosphatidyl inositol bisphosphate into inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 acts on specific receptors in the sarcoplasmic reticulum to release intracellular stores of Ca2+, while DAG acts via protein kinase C to open L-type calcium channels in the plasma membrane. The rise in intracellular [Ca2+] induces the tissue response. With an array of actions as diverse as that seen with tachykinins, there is scope for numerous therapeutic possibilities. With the development of potent, selective non-peptide antagonists, there could be potential benefits in the treatment of a variety of clinical conditions, including chronic pain, Parkinson's disease, Alzheimer's disease, depression, rheumatoid arthritis, irritable bowel syndrome and asthma.

Effects of the cysteinyl leukotriene receptor antagonists pranlukast and zafirlukast on tracheal mucus secretion in ovalbumin-sensitized guinea-pigs in vitro.

1. We investigated the inhibitory effects of the cysteinyl leukotriene (CysLT1) receptor antagonists, pranlukast and zafirlukast, on 35SO4 labelled mucus output, in vitro, in guinea-pig trachea, induced by leukotriene D4 (LTD4) or by antigen challenge of sensitized animals. Agonists and antagonists were administered mucosally, except in selected comparative experiments where drugs were administered both mucosally and serosally to assess the influence of the epithelium on evoked-secretion. 2. LTD4 increased 35SO4 output in a concentration-related manner with a maximal increase of 23 fold above controls at 100 microM and an approximate EC50 of 2 microM. Combined mucosal and serosal addition of LTD4 did not significantly affect the secretory response compared with mucosal addition alone. Neither LTC4 nor LTE4 (10 microM each) affected 35SO4 output. Pranlukast or zafirlukast significantly inhibited 10 microM LTD4-evoked 35SO4 output in a concentration-dependent fashion, with maximal inhibitions of 83% at 10 microM pranlukast and 78% at 10 microM zafirlukast, and IC50 values of 0.3 microM for pranlukast and 0.6 microM for zafirlukast. Combined mucosal and serosal administration of the antagonists (5 microM each) gave degrees of inhibition of mucosal-serosal 10 microM LTD4-evoked 35SO4 output similar to those of the drugs given mucosally. Pranlukast (0.5 microM) caused a parallel rightward shift of the LTD4 concentration-response curve with a pKB of 7. Pranlukast did not inhibit ATP-induced 35SO4 output. 3. Ovalbumin (10-500 microg ml(-1) challenge of tracheae from guinea-pigs actively sensitized with ovalbumin caused a concentration-related increase in 35SO4 output with a maximal increase of 20 fold above vehicle controls at 200 microg ml(-1). The combination of the antihistamines pyrilamine and cimetidine (0.1 mM each) did not inhibit ovalbumin-induced 35SO4 output in sensitized guinea-pigs. Neither mucosal (10 microM or 100 microM) nor mucosal-serosal (100 microM) histamine had any significant effect on 35SO4 output. 4. Pranlukast or zafirlukast (5 microM each) significantly suppressed ovalbumin-induced secretion in tracheae from sensitized guinea-pigs by 70% and 65%, respectively. 5 We conclude that LTD4 or ovalbumin challenge of sensitized animals provokes mucus secretion from guinea-pig trachea in vitro and this effect is inhibited by the CysLT1 receptor antagonists pranlukast and zafirlukast. These antagonists may be beneficial in the treatment of allergic airway diseases in which mucus hypersecretion is a clinical symptom, for example asthma and allergic rhinitis.

Effect of vasoactive intestinal peptide (VIP)-related peptides on cholinergic neurogenic and direct mucus secretion in ferret trachea in vitro.

1 We investigated whether vasoactive intestinal peptide (VIP) and its related peptides, pituitary adenylate cyclase activating peptide (PACAP) and secretin, regulate cholinergic neural mucus secretion in ferret trachea in vitro, using 35SO4 as a mucus marker. We also studied the interaction between VIP and secretin on cholinergic mucus output. 2 VIP (1 and 10 microM) increased secretion, whereas neither PACAP1 - 27, PACAP1 - 38 nor secretin (up to 10 microM) increased mucus output. In contrast, VIP, PACAP1 - 27 and PACAP1 - 38 concentration-dependently inhibited cholinergic neural secretion, with an order of potency of VIP>PACAP 1 - 38>PACAP1 - 27. Neither PACAP1 - 27 nor PACAP1 - 38 altered the secretion induced by acetylcholine (ACh). 3 Secretin increased cholinergic neural secretion with a maximal increase of 190% at 1 microM. This potentiation was blocked by VIP or atropine. Similarly, secretin (1 microM) potentiated VIP (1 microM)-induced mucus output by 160%. Secretin did not alter exogenous ACh-induced secretion. VIP vs secretin competition curves suggested these two peptides were competing reversibly for the same receptor. 4 We conclude that, in ferret trachea in vitro, VIP and PACAPs inhibit cholinergic neural secretion via pre-junctional modulation of cholinergic neurotransmission. VIP and secretin compete for the same receptor, possibly a VIP1 receptor, at which secretin may be a receptor antagonist.

Nitric oxide inhibition of basal and neurogenic mucus secretion in ferret trachea in vitro.

1. In order to examine the role of nitric oxide (NO) on airway mucus secretion we studied the effects of the nitric oxide synthase (NOS) inhibitor L-N(G)-monomethyl-L-arginine (L-NMMA), a novel nitric oxide donor, (+/-)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide (FK409), and the NO precursor L-arginine on basal mucus secretion in the ferret trachea in vitro in Ussing chambers. We also determined the effects of these agents upon secretion induced by electrical stimulation of nerves or by acetylcholine (ACh). We used 35SO4 as a mucus marker. 2. L-NMMA (0.01-1 mM) increased basal output of 35SO4-labelled macromolecules with a maximal increase above baseline of 248% at 0.1 mM L-NMMA. L-Arginine (1 mM) alone had no significant effect on basal secretion but reversed the potentiating effect of L-NMMA on basal secretion. L-NMMA-induced increases in basal mucus secretion were sustained for at least 30 min in the continuing presence of the NOS inhibitor. In contrast to the potentiating effects of L-NMMA, FK409 (100 nM) reduced basal secretion by 60% (at 1 nM and at 10 nM it was without effect). 3. Electrical stimulation (50 V, 10 Hz, 0.5 ms for 5 min) increased 35SO4 output by 174%. L-NMMA (1 and 10 mM) present during stimulation of tracheal segments resulted in significant potentiations of 214% and 116%, respectively, of the neurogenic response. The potentiated response to 10 mM L-NMMA was reversed by L-arginine (1 mM). At this dose L-arginine had no effect itself on basal secretion. In contrast to the potentiating effects of L-NMMA on neurogenic secretion, FK409 at 10 nM and 100 nM inhibited the neurogenic response by 98% and 99%. 4. At all concentrations tested, neither L-NMMA (0.01 mM-1 mM) nor FK409 (1-100 mM) had any significant effect on ACh-induced mucus secretion. 5. These observations lead us to conclude that nitric oxide, derived from constitutive NO synthase, acts as an endogenous inhibitor of both basal and neurogenic mucus secretion in ferret trachea in vitro.

Neuroregulation by vasoactive intestinal peptide (VIP) of mucus secretion in ferret trachea: activation of BK(Ca) channels and inhibition of neurotransmitter release.

1. The aims of this study were to determine: (1) whether vasoactive intestinal peptide (VIP) regulates cholinergic and 'sensory-efferent' (tachykininergic) 35SO4 labelled mucus output in ferret trachea in vitro, using a VIP antibody, (2) the class of potassium (K+) channel involved in VIP-regulation of cholinergic neural secretion using glibenclamide (an ATP-sensitive K+ (K(ATP)) channel inhibitor), iberiotoxin (a large conductance calcium activated K+ (BK(ca)) channel blocker), and apamin (a small conductance K(ca) (SK(ca)) channel blocker), and (3) the effect of VIP on cholinergic neurotransmission using [3H]-choline overflow as a marker for acetylcholine (ACh) release. 2. Exogenous VIP (1 and 10 microM) alone increased 35SO4 output by up to 53% above baseline, but suppressed (by up to 80% at 1 microM) cholinergic and tachykininergic neural secretion without altering secretion induced by ACh or substance P (1 microM each). Endogenous VIP accounted for the minor increase in non-adrenergic, non-cholinergic (NANC), non-tachykininergic neural secretion, which was compatible with the secretory response of exogenous VIP. 3. Iberiotoxin (3 microM), but not apamin (1 microM) or glibenclamide (0.1 microM), reversed the inhibition by VIP (10 nM) of cholinergic neural secretion. 4. Both endogenous VIP (by use of the VIP antibody; 1:500 dilution) and exogenous VIP (0.1 microM), the latter by 34%, inhibited ACh release from cholinergic nerve terminals and this suppression was completely reversed by iberiotoxin (0.1 microM). 5. We conclude that, in ferret trachea in vitro, endogenous VIP has dual activity whereby its small direct stimulatory action on mucus secretion is secondary to its marked regulation of cholinergic and tachykininergic neurogenic mucus secretion. Regulation is via inhibition of neurotransmitter release, consequent upon opening of BK(Ca) channels. In the context of neurogenic mucus secretion, we propose that VIP joins NO as a neurotransmitter of i-NANC nerves in ferret trachea.

Effect of the long-acting tachykinin NK(1) receptor antagonist MEN 11467 on tracheal mucus secretion in allergic ferrets.

1. We investigated the effect of MEN 11467 ((1R,2S)-2-N[1(H)indol-3-yl-carbonyl]-1-N-[N(alpha)(p-tolylacetyl)-N(alpha)(methyl)-D-3-(2-naphthyl)alanyl]diaminocyclohexane) on tachykinin-induced mucus secretion in ferret trachea in vitro and determined its effect on secretion by tracheae from allergic ferrets in response to allergen challenge. 2. Repeated administration of [Sar(9),Met(O(2))(11)]-substance P ([Sar(9)]SP, 1 microM) maintained mucus output above control values for at least 1.75 h. MEN 11467 inhibited secretion in a concentration-dependent manner with maximal inhibition at 10 microM and an approximate IC(50) of 0.3 microM. Inhibition by MEN 11467 (0.1--10 microM) was maintained, to varying degree, for at least 1.75 h after washout in the continued presence of [Sar(9)]SP. 3. In electrically stimulated tracheae, tachykininergic neural secretion was virtually abolished by 1 microM MEN 11467. 4. In tracheae from ovalbumin-sensitised animals, repeated administration of ovalbumin maintained mucus output above controls for 1.5 h. MEN 11467 inhibited ovalbumin-induced secretion in a concentration-dependent manner, with complete inhibition at 1 microM. Inhibition by MEN 11467 (1 and 10 microM) was maintained, to varying degree, after drug washout for the 1.5 h of ovalbumin stimulation. 5. MEN 11467 1 microM did not affect secretion induced by either acetylcholine or histamine, whereas 10 microM MEN 11467 did inhibit agonist-induced secretion. 6. We conclude that, in ferret trachea in vitro, MEN 11467 at concentrations of 0.1--1 microM is a long acting and selective inhibitor of tachykininergic-induced mucus secretion, and may have therapeutic potential for bronchial hypersecretion associated with allergic conditions, for example in asthma.

Cutaneous permeability responses to bradykinin and histamine in the guinea-pig: possible differences in their mechanism of action.

1. Plasma protein extravasation (PPE) responses in guinea-pig skin have been measured using accumulation of intravenously injected 125I-labelled human serum albumin (125I-HSA). 2. The nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME; 0.1 mumol/site) significantly reduced responses to bradykinin (BK; 0.5 nmol/site) or histamine (4.5 nmol/site) when co-injected with the inflammatory mediators. D-NAME (0.1 mumol/site) had no significant effect. 3. L-NAME (0.01-0.1 mumol/site) appeared to produce greater shifts of the dose-response curve to BK (0.1-3 nmol/site) than of that to histamine (2.3-27 nmol/site). Both 0.01 and 0.1 mumol L-NAME/site significantly reduced the response to BK (0.5 nmol/site) whereas only the higher dose of L-NAME produced a significant reduction in the response to histamine (4.5 nmol/site). 4. The inhibitory effect of L-NAME (0.1 mumol/site) on the response to BK but not on that to histamine was significantly reversed by L-arginine (L-Arg; 10 mumol/site). D-arginine (D-Arg; 10 mumol/site) had no significant effect in either case. 5. L-Arg (10 mumol/site) significantly enhanced the response to BK but inhibited that to histamine. D-Arg (10 mumol/site) had no significant effect on BK but significantly inhibited histamine. L-Lysine (L-Lys: 10 mumol/site) had no significant effect on the response to either BK or histamine. 6. L-Arg (100 mM) had a significant inhibitory effect on isometric contractions to histamine, but not BK in guinea-pig ileum in vitro. D-Arg (100 mM) also significantly inhibited histamine responses whereas L-Lys (100 mM) had no effect. 7. The alpha-adrenoceptor agonist, phenylephrine (0.3 or 6 nmol/site) inhibited matched responses to BK (0.5 nmol/site) or histamine (5.4 nmol/site) to comparable degrees, but gave significant inhibition only at the higher dose. 8. The Beta-adrenoceptor agonist, isoprenaline (0.5 or 10 nmol/site) had a significant inhibitory effect on the response to histamine (5.4 nmol/site) whereas a comparable response to BK (0.5 nmol/site) was significantly reduced by the higher dose only.9. Our results with L-NAME suggest that local production of NO is involved in the modulation of mediator-induced vascular permeability. It is possible that NO may play a greater role in the extravasation response to BK than to that induced by histamine.10. The differential effects of L-NAME and isoprenaline on BK- and histamine-induced PPE raise the possibility that BK and histamine may induce vascular permeability via different mechanisms in guinea-pig skin.

Effect of non-peptide tachykinin NK(1) receptor antagonists on non-adrenergic, non-cholinergic neurogenic mucus secretion in ferret trachea.

We investigated, in ferret trachea in vitro, the binding characteristics and the inhibition of non-adrenergic, non-cholinergic (NANC) neural mucus secretion of four tachykinin receptor antagonists: the non-peptide tachykinin NK(1) receptor antagonists CGP 49823 ((2R,4S)-2-benzyl-1-(3, 5-dimethylbenzoyl)-4-(quinolin-micro-ylmethyl amino) piperidine), CGP 55000 ((2R,4S)-2-benzyl-1-(3, 5-bistrifluoromethyl-benzoyl)-4-(quinolinyl-methylamino)piperidine ) and CP 99,994 ((+)-(2S,3S)-3-methoxybenzyl amino)-2-phenylpiperidine), and the peptide tachykinin NK(2) receptor antagonist MEN 10,627 (cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2beta-5beta)). CGP 49823, CGP 55000 and CP 99,994 concentration-dependently displaced [125I]Bolton-Hunter substance P binding in tracheal membranes with Hill coefficients not different from unity and IC(50) values of 1.4, 1.7 and 1.3 nM, respectively. In contrast, MEN 10,627 displaced binding according to a two-site model, with IC(50)s of 0.2 nM and 1. 3 microM. Electrical stimulation of tracheal segments with adrenoceptor and cholinoceptor blockade increased output of the mucus marker 35SO(4) by 59% above baseline (representing the NANC neural secretory response). CGP 49823, CGP 55000 or CP 99,994 concentration-dependently inhibited NANC neural secretion with IC(50) values of 30, 8 and 120 nM, respectively. In contrast, MEN 10, 627 (3 microM) did not inhibit secretion. The NK(1) antagonists, but not the NK(2) antagonist, inhibited [Sar(9)]substance P-induced secretion, while none of the antagonists affected acetylcholine-induced secretion. We conclude that NANC neural secretion in ferret trachea in vitro is a useful test system for tachykinin NK(1) receptor antagonists with therapeutic potential in conditions of the airways in which tachykininergic mechanisms and mucus hypersecretion are implicated in pathophysiology, for example asthma and chronic bronchitis.

Persistence of effects of nitric oxide synthase inhibitors: comparisons on blood flow and plasma exudation in guinea pig skin.

Plasma protein extravasation has been measured in guinea pig skin using 125I-albumin and blood flow using 133Xenon (133Xe) clearance. The nitric oxide (NO) synthase inhibitors N(G)-nitro-L-arginine methyl ester (L-NAME), N(G)-monomethyl-L-arginine (l-NMMA) and N(G)-nitro-L-arginine (L-NOArg) and the alpha-adrenoceptor agonist, phenylephrine, inhibited bradykinin induced plasma protein extravasation when co-injected with the peptide. The inhibitory effects of L-NAME and L-NOArg lasted for up to 8 and 4 h, respectively, whereas phenylephrine and L-NMMA had no persistent inhibitory effects. When co-injected with 133Xe, L-NAME, L-NMMA, L-NOArg and phenylephrine, but not D-NAME, produced significant reductions in skin blood flow. When injected prior to 133Xe, L-NAME and L-NOArg, but not phenylephrine or L-NMMA, significantly reduced flow. The effect of L-NAME on flow was not significant at 8 h. Thus, although the inhibitory effects of the NO synthase inhibitors on mediator induced plasma protein extravasation show correlations with their effects on blood flow, the persistent effect of L-NAME on exudation appears to extend beyond its effect on flow.

Effect of fenspiride, a non-steroidal antiinflammatory agent, on neurogenic mucus secretion in ferret trachea in vitro.

Neural mechanisms contribute to control of mucus secretion in the airways. Fenspiride is a non-steroidal antiinflammatory agent which has a variety of actions, including inhibition of neurogenic bronchoconstriction. The effect of fenspiride on neurally-mediated mucus secretion was investigated in vitro in electrically-stimulated ferret trachea, using(35)SO(4)as a mucus marker. Cholinergic secretory responses were isolated using adrenoceptor and tachykinin receptor antagonists. Tachykinin responses were isolated using cholinoceptor and adrenoceptor antagonists. Electrical stimulation increased cholinergic secretion by;90% and tachykininergic secretion by;40%. Fenspiride (1 microM-1 mM) tended to inhibit cholinergic secretion in a concentration-dependent manner, although only at 1 mM was inhibition (by 87%) significant. Inhibition by fenspiride of tachykininergic secretion was not concentration-dependent, and again significant inhibition (by 85%) was only at 1 mM. Inhibition was not due to loss of tissue viability, as assessed by restitution of secretory response after washout. Fenspiride also inhibited secretion induced by acetylcholine, but did not inhibit substance P-induced secretion. Histamine receptor antagonists increased basal secretion by 164%, whereas fenspiride did not affect basal secretion. We conclude that, in ferret trachea in vitro, fenspiride inhibits neurally-mediated mucus secretion, with antimuscarinic action the most plausible mechanism of action, but not necessarily the only mechanism.

On muscarinic control of neurogenic mucus secretion in ferret trachea.

1. Muscarinic receptor subtypes mediating neurogenic mucus secretion in ferret trachea were characterized in vitro and in vivo using 35SO4 as a label for secreted mucus, and the muscarinic receptor antagonists telenzepine for the M1 receptor subtype, methoctramine for the M2 subtype and 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) for the M3 receptor. We also performed receptor binding and mapping studies. 2. Each muscarinic antagonist displaced [N-methyl-3H]scopolamine binding with high-affinity binding constant (KH) values of 1.9, 2.7 and 5.0 nM for telenzepine, methoctramine and 4-DAMP, respectively. Muscarinic M1 and M3 receptors localized to submucosal glands, whereas M2 receptors did not. 3. In vitro, electrical stimulation (50 V, 10 Hz, 0.5 ms for 5 min) increased 35SO4 output by 160%. Telenzepine did not inhibit the neurogenic secretory response at concentrations two-or twentyfold its KH value, nor did it inhibit secretion induced by acetylcholine (ACh). 4-DAMP inhibited neurogenic secretion by 80 and 95%, respectively, at concentrations two-and twentyfold its KH value, and also inhibited ACh-induced secretion. Methoctramine potentiated neurogenic secretion induced at 2.5 Hz (50 V, 0.5 ms for 5 min) in a dose-related (5.4-100 nM) manner with increases of 33-451% above electrically stimulated values. Methoctramine did not potentiate secretion induced at 10 Hz and did not have any effect on ACh-induced secretion. 4. In vivo, vagal stimulation (10 V, 10 Hz, 2 ms for 8 min) increased output of 35SO4 by approximately 120%. Telenzepine had no significant effect on neurogenic secretion. Methoctramine approximately doubled the stimulated response, whereas 4-DAMP abolished the stimulated secretory response. 5. We conclude that in ferret trachea, cholinergic nerve stimulation increases mucus secretion via muscarinic M3 receptors on the submucosal glands. The magnitude of the secretory response is regulated by neuronal M2 muscarinic receptors. The muscarinic M1 receptors localized to the submucosal glands do not appear to be involved with mucus secretion.