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.     

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 of mucus secretion by opioid receptors and KATP and BKCa channels in ferret trachea in vitro

1. Opioid agonists inhibit neurogenic mucus secretion in the airways. The mechanism of the inhibition is unknown but may be via opening of potassium (K⁺) channels. We studied the effect on neurogenic secretion in ferret trachea in vitro of the OP₁ receptor (formerly known as δ opioid receptor) agonist [D-Pen^2,5]enkephalin (DPDPE), the OP₂ receptor (formely κ) agonist U-50,488H, the OP₃ receptor (formerly μ) agonist [D-Ala², N-Me-Phe, Gly-ol⁵]enkephalin (DAMGO), the ATP-sensitive K⁺ (K_ATP) channel inhibitor glibenclamide, the large conductance calcium activated K⁺ (BK_Ca) channel blocker iberiotoxin, the small conductance K_Ca (SK_Ca) channel blocker apamin, the K_ATP channel opener levcromakalim, a putative K_ATP channel opener RS 91309, and the BK_Ca channel opener NS 1619. Secretion was quantified by use of ³⁵SO₄ as a mucus marker.
2. Electrical stimulation increased tracheal secretion by up to 40 fold above sham-stimulated levels. DAMGO or DPDPE (10 μM each) significantly inhibited neurogenic secretion by 85% and 77%, respectively, effects which were reversed by naloxone. U-50,488H had no significant inhibitory effect on neurogenic secretion, and none of the opioids had any effect on ACh-induced or [Sar⁹]substance P-induced secretion.
3. Inhibition of neurogenic secretion by DAMGO or DPDPE was reversed by iberiotoxin (3 μM) but not by either glibenclamide or apamin (0.1 μM each). Iberiotoxin alone did not affect the neurogenic secretory response.
4. Levcromakalim, RS 91309 or NS 1619 (3 nM–3 μM) inhibited neurogenic secretion with maximal inhibitions at 3 μM of 68%, 72% and 96%, respectively. Neither levcromakalim nor RS 91309 at any concentration tested significantly inhibited acetylcholine (ACh)-induced secretion, whereas inhibition (60%) was achieved at the highest concentration of NS 1619, a response which was blocked by iberiotoxin.
5. Inhibition of neurogenic secretion by levcromakalim (3 μM) or RS 91309 (30 nM) was inhibited by glibenclamide but not by iberiotoxin. In contrast, inhibition by NS 1619 (30 nM and 3 μM) was blocked by iberiotoxin but not by glibenclamide.
6. We conclude that, in ferret trachea in vitro, OP₁ or OP₃ opioid receptors inhibit neurogenic mucus secretion at a prejunctional site and that the mechanism of the inhibition is via opening of BK_Ca channels. Direct opening of BK_Ca channels or K_ATP channels also inhibits neurogenic mucus secretion. In addition, opening of BK_Ca channels inhibits ACh-evoked secretion of mucus. Drugs which open BK_Ca channels may have therapeutic anti-secretory activity in bronchial diseases in which neurogenic mechanisms and mucus hypersecretion are implicated in pathophysiology, for example asthma and chronic bronchitis.

     

Action of a novel drug (Zy 16039) on mucus secretion in the ferret isolated trachea in vitro

1. The effect of 4-H-2-carboxamido-4-phenyl-thieno-[3,2c]-[1]-benzopyran (Zy 16039) was examined on the smooth muscle contraction, mucus secretion and albumin transudation in the ferret whole trachea in vitro. 2. Zy 16039 (0.1-20 microM) produced a concentration-dependent relaxation of the ferret trachea contracted by methacholine (1 microM) and phenylephrine (10 microM). The relaxations were about 20% of the full contractions. 3. Zy 16039 has no effect on the resting (zero) output of mucus in the ferret trachea. Methacholine-induced mucus secretion was significantly inhibited by Zy 16039, whereas phenylephrine-induced secretion was significantly increased. 4. Methacholine-induced secretion of lysozyme, a marker of serous cell secretion, was inhibited by Zy 16039 both with regard to output and concentration of lysozyme. In contrast, Zy 16039 significantly increased the output of lysozyme due to phenylephrine, with no effect on concentration. 5. Zy 16039 had no significant effect on the rate of output of fluorescent albumin through the tracheal wall. However the concentration of albumin in the mucus samples was changed because of the effect of Zy 16039 on mucus secretion induced by methacholine and phenylephrine. 6. We conclude that Zy 16039 relaxes airway smooth muscle, and either promotes or inhibits mucus secretion depending on its source. It has qualitatively similar actions to vasoactive intestinal peptide.     

The effect of vasoactive intestinal peptide on smooth muscle tone and mucus secretion from the ferret trachea.

The effect of vasoactive intestinal peptide (VIP) was examined on the smooth muscle contraction and mucus secretion produced by methacholine and phenylephrine in the ferret whole trachea in vitro. VIP (0.5 to 800 nM) produced a concentration-dependent relaxation of the ferret trachea contracted by methacholine (1 microM) and phenylephrine (10 microM). The concentration-response curves for methacholine- and phenylephrine-induced contractions were both shifted to the right by VIP (0.1 microM). Methacholine-induced secretion was inhibited in a concentration-dependent manner by VIP, whereas that due to phenylephrine was enhanced. The concentration-response curve for methacholine-induced secretion was shifted to the right by VIP, whereas the curve for phenylephrine was shifted to the left. Methacholine produced a concentration-dependent increase in the rate of output of lysozyme from the ferret trachea with no corresponding increase in the concentration of lysozyme in the mucus. Phenylephrine produced a concentration-dependent increase in the rate of output and in the concentration of lysozyme. VIP (0.1 microM) significantly increased the concentration of lysozyme in the mucus produced by methacholine with no increase in the rate of lysozyme output. However, the rate of lysozyme output due to phenylephrine was significantly increased by VIP (0.1 microM) with no increase in concentration. We suggest that VIP inhibits secretion from mucous cells stimulated by methacholine, and enhances the secretion produced by phenylephrine from serous cells.     

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.     

The effect of hydrogen peroxide on smooth muscle tone,mucus secretion and epithelial albumin transport of the ferret trachea in vitro

The effect of hydrogen peroxide (H₂O₂) was examined on baseline and on methacholine- and phenylephrine-stimulated smooth muscle tone, mucus volume and lysozyme outputs, and epithelial albumin transport of the ferret whole trachea in vitro. H₂O₂ (10 μM–10 mM) had no significant effect on tracheal smooth muscle tone but produced concentration-dependent increases in mucus volume, lysozyme and albumin outputs. The potential difference (P.D.) across the trachea was not changed by H₂O₂. Exposure of the trachea to H₂O₂ (1 mM) for 2 h reduced the smooth muscle contractions and lysozyme outputs due to methacholine (1 μM) and phenylephrine (10 μM). Methacholine-induced albumin output was significantly increased by H₂O₂ but that due to phenylephrine was not significantly affected. Exposure to H₂O₂ had no significant effect on the mucus volume output produced by methacholine or phenylephrine. Thus H₂O₂ directly stimulates submucosal gland secretion, including secretion from serous cells, and epithelial albumin transport across the ferret trachea but has no effect on tracheal smooth muscle tone. H₂O₂ reduces methacholine- and phenylephrine-induced smooth muscle contractions and serous cell secretion. H₂O₂ causes hyperresponsiveness of albumin output to methacholine but not to phenylephrine.     

The effects of peptides and mediators on mucus secretion rate and smooth muscle tone in the ferret trachea

The effects of a number of peptides and mediators were measured on the secretion rate of tracheal mucus and tracheal smooth muscle tone in the ferret in vitro whole trachea. The comparison of secretion rate and smooth muscle tone, measured simultaneously in the same preparation, shows that there are wide differences in sensitivity between the two systems; there appears to be no relationship between mucus volume output and smooth muscle contraction. The comparison of mucus secretion rate and glycoprotein output in other models and species to these drugs in the same concentrations indicates that there may be mucus glycoprotein output without an increase in volume output.     

Comparison of mucus flow rate, radiolabelled glycoprotein output and smooth muscle contraction in the ferret trachea in vitro.

1. The concentration-response curves for rate of mucus output, labelled-glycoprotein output and smooth muscle contraction in response to methacholine, phenylephrine and salbutamol were determined in the ferret trachea in vitro. 2. The potencies of methacholine and phenylephrine are both in order: smooth muscle contraction, glycoprotein output, rate of mucus output. 3. At lower concentrations methacholine is more potent than is phenylephrine on smooth muscle contraction, glycoprotein output and rate of mucus output. 4. Concentration-response curves for salbutamol show very little change in rate of mucus output but a large increase in glycoprotein output. 5. It is concluded that the glycoprotein output induced by salbutamol may come from a source different from those induced by methacholine and phenylephrine.     

The actions of methacholine, phenylephrine, salbutamol and histamine on mucus secretion from the ferret in-vitro trachea

Methacholine, phenylephrine and histamine produced highly significant and salbutamol significant increases in the rate of mucus secretion from the ferret trachea. Methacholine, phenylephrine and histamine all produced highly significant increases in the rate of output of lysozyme, but the concentration of lysozyme in the mucus was significantly increased only by phenylephrine. Salbutamol produced no significant change in the output of lysozyme, and the concentration of lysozyme in the mucus was significantly decreased. It is concluded that methacholine, phenylephrine and histamine are potent stimulators of serous cell secretion whereas salbutamol has only a weak secretory action on these cells. Methacholine, histamine and salbutamol probably stimulate secretion from mucous cells as well as from serous cells. The increase in the concentration of lysozyme produced by phenylephrine may be due to stimulation of a fluid reabsorption mechanism.     

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 μM and an approximate EC₅₀ of 2 μM. Combined mucosal and serosal addition of LTD₄ did not significantly affect the secretory response compared with mucosal addition alone. Neither LTC₄ nor LTE₄ (10 μM each) affected ³⁵SO₄ output. Pranlukast or zafirlukast significantly inhibited 10 μM LTD₄-evoked ³⁵SO₄ output in a concentration-dependent fashion, with maximal inhibitions of 83% at 10 μM pranlukast and 78% at 10 μM zafirlukast, and IC₅₀ values of 0.3 μM for pranlukast and 0.6 μM for zafirlukast. Combined mucosal and serosal administration of the antagonists (5 μM each) gave degrees of inhibition of mucosal-serosal 10 μM LTD₄-evoked ³⁵SO₄ output similar to those of the drugs given mucosally. Pranlukast (0.5 μM) caused a parallel rightward shift of the LTD₄ concentration-response curve with a pK_B of 7. Pranlukast did not inhibit ATP-induced ³⁵SO₄ output.
3. Ovalbumin (10–500 μg 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 μg 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 μM or 100 μM) nor mucosal-serosal (100 μM) histamine had any significant effect on ³⁵SO₄ output.
4. Pranlukast or zafirlukast (5 μM 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.

     

Competitive antagonists discriminate between NK2 tachykinin receptor subtypes.

1. We have compared the ability of various tachykinins and selective tachykinin receptor agonists to induce contraction of the endothelium-denuded rabbit pulmonary artery (RPA) and hamster trachea (HT) and have estimated the affinity of some newly developed NK2 selective antagonists in the same tissues. 2. In confirmation of previous findings, experiments with the agonists indicated that NK2 receptors are the main if not the sole mediators of the response to tachykinins in both RPA and HT. No evidence for significant degradation of neurokinin A (NKA) was found in either tissue when experiments were repeated in the presence of a mixture of peptidase inhibitors (thiorphan, captopril and bestatin, 1 microM each). 3. The peptide antagonists tested were: Peptide I = [Tyr5, D-Trp6,8,9, Arg10]-NKA(4-10); Peptide II = [Tyr5, D-Trp6,8,9, Arg10]-NKA(3-10); Peptide III = Ac-Leu-Asp-Gln-Trp-Phe-Gly-NH2. The three peptides produced a concentration-dependent rightward shift of the concentration-response curve to NKA in both RPA and HT with no significant depression of the maximal response attainable. The slopes of the Schild plots were not significantly different from unity, indicating a competitive antagonism. Peptides I and II were about 100 times more potent in the RPA than in the HT, while Peptide III was about 100 times more potent in the HT than RPA. 4. The pA2 values obtained in these two tissues with the three antagonists were not significantly different when tested in the absence or presence of peptidase inhibitors, or when a selective NK2 receptor agonist, [beta Ala8]-NKA(4-10) was used instead of NKA.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of calcitonin gene-related peptide on submucosal gland secretion and epithelial albumin transport in the ferret trachea in vitro.

1. We have examined the effect of calcitonin gene-related peptide (CGRP) on basal mucus volume, lysozyme and albumin outputs from the ferret whole trachea in vitro, and on the outputs produced by methacholine and substance P (SP). We have also examined the effect of inhibiting neutral enkephalinase with thiorphan on the responses to CGRP. 2. CGRP (1-100 nM) produced small concentration-dependent increases in basal mucus volume, lysozyme and albumin outputs. These effect of CGRP were enhanced by thiorphan. The increases in basal outputs with CGRP and the potentiation by thiorphan were considerably less than previously observed with SP and neurokinin A (NKA). CGRP had no significant effect on potential difference (PD) across the trachea. 3. CGRP produced a concentration-dependent inhibition of methacholine- and SP-induced lysozyme output but a concentration-dependent increase in methacholine- and SP-induced albumin output. The effects of CGRP on methacholine-induced lysozyme and albumin outputs were enhanced by thiorphan. CGRP weakly inhibited methacholine-induced mucus volume output and weakly enhanced SP-induced mucus volume output. 4. Thus, CGRP weakly stimulates basal serous cell secretion and epithelial albumin transport, but does not alter epithelial integrity. CGRP inhibits the serous cell secretion due to methacholine or SP, but potentiates the epithelial albumin transport produced by these agents. The interaction between CGRP and other sensory neuropeptides or muscarinic agonists on airway submucosal glands and epithelium may be important in the normal airway and in inflammatory airway diseases where release of sensory neuropeptides is enhanced.     

Airway inflammation and tachykinins: prospects for the development of tachykinin receptor antagonists.

The tachykinins substance P and neurokinin A are contained within sensory airway nerves. Immune cells form an additional source of tachykinins in inflamed airways. Elevated levels of tachykinins have been recovered from the airways of patients with asthma and chronic obstructive pulmonary disease. Airway inflammation leads to an upregulation of tachykinin NK(1) and NK(2) receptors. Preclinical studies have indicated a role for the tachykinin NK(1), NK(2) and NK(3) receptors in bronchoconstriction, airway hyperresponsiveness and airway inflammation caused by allergic and nonallergic stimuli. Compounds that are able to block two or three tachykinin receptors hold promise for the treatment of airways diseases such as asthma and/or chronic obstructive pulmonary disease.     

Small molecule antagonists of the tachykinin NK 2 receptor

The NK(2) receptor (member of the tachykinin receptor family) is mainly located in the smooth muscle of the urinary, respiratory and gastrointestinal tracts, with limited presence in the CNS. This has raised interest in tachykinin NK(2) receptor antagonists for the treatment of urological disorders, asthma. This review outlines progress done after 1998 in the field of NK(2) small molecule antagonists, both acting on the NK(1)/NK(2), NK(2)/NK(3), NK(1)/NK(2)/NK(3) receptors and selective for the NK(2) one.     

Receptors mediating the effects of substance P and neurokinin A on mucus secretion and smooth muscle tone of the ferret trachea: potentiation by an enkephalinase inhibitor.

1. The effects of substance P (SP) and neurokinin A (NKA) were examined on tracheal smooth muscle tone, mucus volume output, lysozyme output and albumin transport across the ferret in vitro whole trachea in the presence and absence of the enkephalinase inhibitor, thiorphan. 2. SP (0.001-3 microM) and NKA (0.01-10 microM) contracted the tracheal smooth muscle and increased mucus volume, lysozyme and albumin outputs into the tracheal lumen. The EC50 values for SP and NKA for all of the variables measured were significantly reduced, and all of the maximum responses were significantly enhanced by thiorphan (10 microM). 3. In the presence of thiorphan, SP (1 microM) and NKA (10 microM) produced albumin concentrations in the secreted mucus (8.9 and 7.2 micrograms microliters-1) which were greater than those in the submucosal buffer (4.2 micrograms microliters-1). 4. In the presence of thiorphan, NKA was approximately 5 times more potent than SP at contracting the tracheal smooth muscle. Conversely SP was 23, 15 and 22 times more potent than NKA at stimulating mucus volume, lysozyme and albumin outputs respectively. 5. Thus, there is neutral endopeptidase in the ferret trachea in vitro which cleaves exogenously applied SP and NKA, thereby reducing the magnitude and potency of their actions. SP and NKA contract the ferret tracheal muscle probably by an action at NK2 (or NK3)-receptors but stimulate mucus volume output, lysozyme output and albumin transport across the tracheal wall probably by an action on NK1 receptors.     

The effects of peptide histidine isoleucine and neuropeptide Y on mucus volume output from the ferret trachea.

1. The effects of peptide histidine isoleucine (PHI) and neuropeptide Y (NPY) were examined on the mucus volume output produced by methacholine and phenylephrine in the ferret whole trachea in vitro. 2. Sustained application of methacholine (5 microM) or phenylephrine (20 microM) produced a maintained volume output of mucus from the trachea. Both these agonists also increased the output of lysozyme (a marker for serous cell secretion). 3. PHI inhibited the maintained mucus volume output produced by methacholine but had no effect on that due to phenylephrine. The output of lysozyme produced by methacholine or phenylephrine was not significantly changed by PHI. 4. NPY enhanced the volume output of mucus produced by methacholine or phenylephrine; however, the rate of output of lysozyme in mucus produced by both agonists was reduced by NPY. 5. We suggest that PHI has no effect on serous cell secretion but inhibits secretion from another source, possibly mucous cells. NPY inhibits serous cell secretion but has a stronger stimulant action on secretion from another source, again possibly mucous cells. 6. PHI and NPY may be important physiological modulators of mucus volume output in the 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.     

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.     

Nonpeptidic urotensin-II receptor antagonists I: in vitro pharmacological characterization of SB-706375

1. SB-706375 potently inhibited [(125)I]hU-II binding to both mammalian recombinant and 'native' UT receptors (K(i) 4.7+/-1.5 to 20.7+/-3.6 nM at rodent, feline and primate recombinant UT receptors and K(i) 5.4+/-0.4 nM at the endogenous UT receptor in SJRH30 cells). 2. Prior exposure to SB-706375 (1 microM, 30 min) did not alter [(125)I]hU-II binding affinity or density in recombinant cells (K(D) 3.1+/-0.4 vs 5.8+/-0.9 nM and B(max) 3.1+/-1.0 vs 2.8+/-0.8 pmol mg(-1)) consistent with a reversible mode of action. 3. The novel, nonpeptidic radioligand [(3)H]SB-657510, a close analogue of SB-706375, bound to the monkey UT receptor (K(D) 2.6+/-0.4 nM, B(max) 0.86+/-0.12 pmol mg(-1)) in a manner that was inhibited by both U-II isopeptides and SB-706375 (K(i) 4.6+/-1.4 to 17.6+/-5.4 nM) consistent with the sulphonamides and native U-II ligands sharing a common UT receptor binding domain. 4. SB-706375 was a potent, competitive hU-II antagonist across species with pK(b) 7.29-8.00 in HEK293-UT receptor cells (inhibition of [Ca(2+)](i)-mobilization) and pK(b) 7.47 in rat isolated aorta (inhibition of contraction). SB-706375 also reversed tone established in the rat aorta by prior exposure to hU-II (K(app) approximately 20 nM). 5. SB-706375 was a selective U-II antagonist with >/=100-fold selectivity for the human UT receptor compared to 86 distinct receptors, ion channels, enzymes, transporters and nuclear hormones (K(i)/IC(50)>1 microM). Accordingly, the contractile responses induced in isolated aortae by KCl, phenylephrine, angiotensin II and endothelin-1 were unaltered by SB-706375 (1 microM). 6. In summary, SB-706375 is a high-affinity, surmountable, reversible and selective nonpeptide UT receptor antagonist with cross-species activity that will assist in delineating the pathophysiological actions of U-II in mammals.