No abstract

1 Muscarinic receptor subtypes mediating neurogenic mucus secretion in ferret trachea were characterized in vitro and in vivo using 35sQ, 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 twoor 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 twoand 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 35S04 by -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.Cholinergic nerves are the dominant neural pathway involved in control of a number of airway functions, including mucus secretion (Rogers, 1996). Physiological responses to cholinergic nerve stimulation are mediated via cholinergic muscarinic receptors (Barnes, 1993). Four subtypes of muscarinic receptor (M1-M3, and possibly M4) can be demonstrated pharmacologically, although the muscarinic antagonist drugs currently available are not highly selective for the different receptor subtypes (Eglen, Reddy & Watson, 1994). In the airways, Ml receptors are localized to parasympathetic ganglia, submucosal glands and, in humans, to the alveolar walls (Barnes, 1993

1 Plasma protein extravasation (PPE) responses in guinea-pig skin have been measured using accumulation of intravenously injected '25I-labelled human serum albumin ('25I-HSA).2 The nitric oxide (NO) synthase inhibitor, N0-nitro-L-arginine methyl ester (L-NAME; 0.1 mol/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 pmol/site) had no significant effect.3 L-NAME (0.01-0.1 gmol/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 fmol 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 tmol/site) on the response to BK but not on that to histamine was significantly reversed by L-arginine (L-Arg; 10 pmol/site). D-arginine (D-Arg; 10 gpmol/site) had no significant effect in either case. D-Arg (10 l mol/site) had no significant effect on BK but significantly inhibited histamine. L-Lysine (L-Lys: limol/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 a-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 P-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.

1 We investigated the inhibitory e ects of the cysteinyl leukotriene (CysLT 1 ) receptor antagonists, pranlukast and za®rlukast, on 35 SO 4 labelled mucus output, in vitro, in guinea-pig trachea, induced by leukotriene D 4 (LTD 4 ) 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 in¯uence of the epithelium on evoked-secretion. 2 LTD 4 increased 35 SO 4 output in a concentration-related manner with a maximal increase of 23 fold above controls at 100 mM and an approximate EC 50 of 2 mM. Combined mucosal and serosal addition of LTD 4 did not signi®cantly a ect the secretory response compared with mucosal addition alone. Neither LTC 4 nor LTE 4 (10 mM each) a ected 35 SO 4 output. Pranlukast or za®rlukast signi®cantly inhibited 10 mM LTD 4 -evoked 35 SO 4 output in a concentration-dependent fashion, with maximal inhibitions of 83% at 10 mM pranlukast and 78% at 10 mM za®rlukast, and IC 50 values of 0.3 mM for pranlukast and 0.6 mM for za®rlukast. Combined mucosal and serosal administration of the antagonists (5 mM each) gave degrees of inhibition of mucosal-serosal 10 mM LTD 4 -evoked 35 SO 4 output similar to those of the drugs given mucosally. Pranlukast (0.5 mM) caused a parallel rightward shift of the LTD 4 concentrationresponse curve with a pK B of 7. Pranlukast did not inhibit ATP-induced 35 SO 4 output. 3 Ovalbumin (10 ± 500 mg ml 71 ) challenge of tracheae from guinea-pigs actively sensitized with ovalbumin caused a concentration-related increase in 35 SO 4 output with a maximal increase of 20 fold above vehicle controls at 200 mg ml 71 . The combination of the antihistamines pyrilamine and cimetidine (0.1 mM each) did not inhibit ovalbumin-induced 35 SO 4 output in sensitized guinea-pigs. Neither mucosal (10 mM or 100 mM) nor mucosal-serosal (100 mM) histamine had any signi®cant e ect on 35 SO 4 output. 4 Pranlukast or za®rlukast (5 mM each) signi®cantly suppressed ovalbumin-induced secretion in tracheae from sensitized guinea-pigs by 70% and 65%, respectively. 5 We conclude that LTD 4 or ovalbumin challenge of sensitized animals provokes mucus secretion from guinea-pig trachea in vitro and this e ect is inhibited by the CysLT 1 receptor antagonists pranlukast and za®rlukast. These antagonists may be bene®cial in the treatment of allergic airway diseases in which mucus hypersecretion is a clinical symptom, for example asthma and allergic rhinitis.

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-N0-monomethyl-L-arginine (L-NMMA), a novel nitric oxide donor, (± )-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide (FK409), and the NO precursor Larginine 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-NMMAinduced 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.