Carolina Elzinga

Muscarinic M3 receptors mediate contraction of human central and peripheral airway smooth muscle

The muscarinic receptor subtype involved in human airway smooth muscle contraction was characterised for the first time, using subtype-selective muscarinic antagonists. It was demonstrated that methacholine-induced contraction of central (trachea) and peripheral (small bronchi) airway smooth muscle preparations was antagonised by pirenzepine, AF-DX 116, 4-DAMP methobromide, hexahydrosiladifenidol, and methoctramine with pA2-values characteristic of M3 (smooth muscle/glandular) muscarinic receptors. Since these pA2-values demonstrate significant correlations with those found in bovine and guinea-pig tracheal smooth muscle contraction, it is concluded that these animal tissues provide a good model for the study of M3 subtype-selective muscarinic antagonists to be used as bronchodilators.

Muscarinic M2 receptors in bovine tracheal smooth muscle: discrepancies between binding and function

Previous work showing that AF-DX 116, a cardioselective muscarinic antagonist in functional experiments, does not discriminate between muscarinic receptors in bovine cardiac and tracheal membranes has been extended. In addition to AF-DX 116 we used the muscarinic antagonists, atropine, pirenzepine, 4-DAMP methobromide, gallamine, hexahydrosiladifenidol and methoctramine, in radioligand binding experiments on bovine cardiac left ventricular and tracheal smooth muscle membranes. The functional antagonism of the methacholine-induced contraction of bovine tracheal smooth muscle strips was also evaluated. An excellent correlation was found for all compounds between the binding affinities for muscarinic receptors in cardiac and tracheal smooth muscle membranes; moreover, the affinities found in cardiac membranes correspond with the pA2 values reported for atrial preparations of rat and guinea pig. However, significant and occasionally marked discrepancies were found between binding and functional affinities of these muscarinic antagonists on bovine tracheal smooth muscle.

Characterization of the muscarinic receptor subtype involved in phosphoinositide metabolism in bovine tracheal smooth muscle

1 The muscarinic receptor subtype involved in the methacholine‐induced enhancement of phosphoinositide metabolism in bovine tracheal smooth muscle was identified by using the M2‐selective antagonist AF‐DX 116 and the M3‐selective antagonist 4‐diphenylacetoxy‐N‐methylpiperidine (4‐DAMP) methobromide, in addition to the M1‐selective antagonist pirenzepine, in a classical Schild analysis. 2 All the antagonists shifted the methacholine dose‐response curve to the right in a parallel and concentration‐dependent fashion, yielding Schild plots with slopes not significantly different from unity. The pA2 values (6.94, 6.32 and 8.54 for pirenzepine, AF‐DX 116 and 4‐DAMP methobromide respectively) indicate that it is the M3 (smooth muscle/glandular), but not the M2 (cardiac) muscarinic receptor subtype, present in this tissue, that mediates phosphoinositide turnover, in accordance with our previous contractile studies. 3 The results provide additional evidence for the involvement of phosphoinositide turnover in the pharmacomechanical coupling between muscarinic receptor stimulation and contraction in (bovine tracheal) smooth muscle.

EVIDENCE FOR A DIRECT RELATIONSHIP BETWEEN PHOSPHOINOSITIDE METABOLISM AND AIRWAY SMOOTH-MUSCLE CONTRACTION INDUCED BY MUSCARINIC AGONISTS

The relationship between bovine tracheal muscle contraction and phosphoinositide metabolism was studied with the muscarinic agonists, methacholine, oxotremorine, and McN-A-343. Analysis of the dose-response curves for contraction and inositol phosphates accumulation with these agonists demonstrated a direct relationship between the two parameters, with a considerable reserve of inositol phosphate production for the full contractile agonists, methacholine and oxotremorine, and no reserve for the partial agonist, McN-A-343.

Epac as a novel effector of airway smooth muscle relaxation

Dysfunctional regulation of airway smooth muscle tone is a feature of obstructive airway diseases such as asthma and chronic obstructive pulmonary disease. Airway smooth muscle contraction is directly associated with changes in the phosphorylation of myosin light chain (MLC), which is increased by Rho and decreased by Rac. Although cyclic adenosine monophosphate (cAMP)‐elevating agents are believed to relieve bronchoconstriction mainly via activation of protein kinase A (PKA), here we addressed the role of the novel cAMP‐mediated exchange protein Epac in the regulation of airway smooth muscle tone. Isometric tension measurements showed that specific activation of Epac led to relaxation of guinea pig tracheal preparations pre‐contracted with methacholine, independently of PKA. In airway smooth muscle cells, Epac activation reduced methacholine‐induced MLC phosphorylation. Moreover, when Epac was stimulated, we observed a decreased methacholine‐induced RhoA activation, measured by both stress fibre formation and pull‐down assay whereas the same Epac activation prevented methacholine‐induced Rac1 inhibition measured by pull‐down assay. Epac‐driven inhibition of both methacholine‐induced muscle contraction by Toxin B‐1470, and MLC phosphorylation by the Rac1‐inhibitor NSC23766, were significantly attenuated, confirming the importance of Rac1 in Epac‐mediated relaxation. Importantly, human airway smooth muscle tissue also expresses Epac, and Epac activation both relaxed pre‐contracted human tracheal preparations and decreased MLC phosphorylation. Collectively, we show that activation of Epac relaxes airway smooth muscle by decreasing MLC phosphorylation by skewing the balance of RhoA/Rac1 activation towards Rac1. Therefore, activation of Epac may have therapeutical potential in the treatment of obstructive airway diseases.

Modulation of agonist-induced phosphoinositide metabolism, Ca2+ signalling and contraction of airway smooth muscle by cyclic AMP-dependent mechanisms.

1 The effects of increased cellular cyclic AMP levels induced by isoprenaline, forskolin and 8‐bromoadenosine 3′:5′‐cyclic monophosphate (8‐Br‐cyclic AMP) on phosphoinositide metabolism and changes in intracellular Ca2+ elicited by methacholine and histamine were examined in bovine isolated tracheal smooth muscle (BTSM) cells. 2 Isoprenaline (pD2 (‐log10 EC50) = 6.32±0.24) and forskolin (pD2 = 5.6±0.05) enhanced cyclic AMP levels in a concentration‐dependent fashion in these cells, while methacholine (pD2 = 5.64±0.12) and histamine (pD2 = 4.90±0.04) caused a concentration‐related increase in [3H]‐inositol phosphates (IP) accumulation in the presence of 10 mM LiCl. 3 Preincubation of the cells (5 min, 37°C) with isoprenaline (1 μm), forskolin (10 μm) and 8‐Br‐cyclic AMP (1 mM) did not affect the IP accumulation induced by methacholine, but significantly reduced the maximal IP production by histamine (1 mM). However, the effect of isoprenaline was small (15.0±0.6% inhibition) and insignificant at histamine concentrations between 0.1 and 100 μm. 4 Both methacholine and histamine induced a fast (max. in 0.5‐2 s) and transient increase of intracellular Ca2+ concentration ([Ca2+]i) followed by a sustained phase lasting several minutes. EGTA (5 mM) attenuated the sustained phase, indicating that this phase depends on extracellular Ca2+. 5 Preincubation of the cells (5 min, 37°C) with isoprenaline (1 μm), forskolin (10 μm) and 8‐Br‐cyclic AMP (1 μm) significantly attenuated both the Ca2+‐transient and the sustained phase generated at equipotent IP producing concentrations of 1 μm methacholine and 100 μm histamine (approx. 40% of maximal methacholine‐induced IP response), but did not affect changes in [Ca2+]i induced by 100 μm methacholine (95.2±3.5% of maximal methacholine‐induced IP response). 6 Significant correlations were found between the isoprenaline‐induced inhibition of BTSM contraction and inhibition of Ca2+ mobilization or influx induced by methacholine and histamine, that were similar for each contractile agonist. 7 These data indicate that (a) cyclic AMP‐dependent inhibition of Ca2+ mobilization in BTSM cells is not primarily caused by attenuation of IP production, suggesting that cyclic AMP induced protein kinase A (PKA) activation is effective at a different level in the [Ca2+]i homeostasis, (b) that attenuation of intracellular Ca2+ concentration plays a major role in β‐adrenoceptor‐mediated relaxation of methacholine‐ and histamine‐induced airway smooth muscle contraction, and (c) that the relative resistance of the muscarinic agonist‐induced contraction to β‐adrenoceptor agonists, especially at (supra) maximal contractile concentrations is largely determined by its higher potency in inducing intracellular Ca2+ changes.

Allosteric interactions of three muscarine antagonists at bovine tracheal smooth muscle and cardiac M2 receptors

The kinetics of [3H]dexetimide dissociation from muscarine receptors in bovine cardiac left ventricular and tracheal smooth muscle membranes were studied in the absence and presence of three muscarine antagonists. It was found that [3H]dexetimide dissociation from cardiac muscarine receptors was monophasic and very fast (half life less than 1 min) and was slowed by the cardioselective muscarine antagonists, gallamine, methoctramine and AF-DX 116, concentration dependently. [3H]Dexetimide dissociation from tracheal muscarine receptors was biphasic, with a fast phase (half-life less than 1 min) followed after 4-5 min by a slow phase (half-life = 38.5 min). The fast component, but not the slow component, was slowed by the muscarine antagonists with concentration dependencies very similar to those found in the heart. We conclude from these data that the major population of tracheal smooth muscle muscarine receptors resembles the cardiac M2 type not only with respect to equilibrium binding affinities but also with respect to the secondary, allosteric binding site on the muscarine receptor. The results also imply that the cardiac receptor subtype is much more sensitive to allosteric modulation than the glandular/smooth muscle receptor subtype.

No evidence for a role of muscarinic M2 receptors in functional antagonism in bovine trachea.

1 The functional antagonism between methacholine‐ or histamine‐induced contraction and β‐adrenoceptor‐mediated relaxation was evaluated in bovine tracheal smooth muscle in vitro. In addition, the putative contribution of muscarinic M2 receptors mediating inhibition of β‐adrenoceptor‐induced biochemical responses to this functional antagonism was investigated with the selective muscarinic antagonists, pirenzepine (M1 over M2), AF‐DX 116 and gallamine (M2 over M3), and hexahydrosiladiphenidol (M3 over M2). 2 By use of isotonic tension measurement, contractions were induced with various concentrations of methacholine or histamine, and isoprenaline concentration‐relaxation curves were obtained in the absence or presence of the muscarinic antagonists. Antagonist concentrations were chosen so as to produce selective blockade of M2 receptors (AF‐DX 116 0.1 μm, gallamine 30 μm), or half‐maximal blockade of M3 receptors (pirenzepine 0.1 μm, AF‐DX 116 0.5 μm, hexahydrosiladiphenidol 0.03 μm). Since these latter antagonist concentrations mimicked KB values towards bovine tracheal smooth muscle M3 receptors, antagonist‐induced decreases in contractile tone were compensated for by doubling the agonist concentration. 3 It was found that isoprenaline‐induced relaxation of bovine tracheal smooth muscle preparations was dependent on the nature and the concentration of the contractile agonist used. Thus, isoprenaline pD2 (—log EC50) values were decreased 3.7 log units as a result of increasing cholinergic tone from 22 to 106%, and 2.4 log units by increasing histamine tone over a similar range. Furthermore, maximal relaxability of cholinergic tone decreased gradually from 100% at low to only 1.3% at supramaximal contraction levels, whereas with histamine almost complete relaxation was maintained at all concentrations applied. As a result, isoprenaline relaxation was clearly hampered with methacholine compared to histamine at equal levels of contractile tone. 4 In the presence of gallamine, isoprenaline relaxation was facilitated for most concentrations of methacholine, and for all concentrations of histamine. These changes could be explained by the decreased contraction levels for both contractile agonists in the presence of gallamine. 5 Isoprenaline‐induced relaxation of cholinergic contraction was also facilitated by AF‐DX 116 as well as by pirenzepine and hexahydrosiladiphenidol, and these (small) changes were again related to the (small) decreases in cholinergic contraction levels that were present in these experiments despite the additional administration of the agonist to readjust contractile tone. Similarly, changes in isoprenaline relaxation of histam0ine‐induced tone could be explained by different contraction levels. 6These results can be explained by the sole involvement of muscarinic M3 receptors, and provide no evidence for a role of muscarinic M2 receptors in functional antagonism in bovine trachea. Furthermore, they stress the importance of taking into account noncholinergic controls as well as contraction levels in these experiments.

Muscarinic M2 receptors do not participate in the functional antagonism between methacholine and isoprenaline in guinea pig tracheal smooth muscle

We investigated whether muscarinic M2 receptors, known to inhibit adenylyl cyclase activity in airway smooth muscle, also inhibit isoprenaline-induced relaxation of guinea pig tracheal smooth muscle, as has recently been described for the dog (Fernandes et al., 1992, J. Pharmacol. Exp. Ther. 262, 119). Smooth muscle strips were contracted with various concentrations of methacholine or histamine (which served as a control) in the absence or presence of the M2-selective muscarinic receptor antagonist, gallamine (30 microM), and cumulative isoprenaline-relaxation curves were obtained. It was found that muscarinic M2 receptor blockade had no significant effect on isoprenaline pD2 and Emax values, neither with histamine nor with methacholine. The results show that, in guinea pig trachea, muscarinic M2 receptors do not significantly influence the functional antagonism of cholinergic smooth muscle contraction by isoprenaline.

Cholinergic contraction of the guinea pig lung strip is mediated by muscarinic M2-like receptors

The muscarinic receptor subtype mediating contraction of the guinea pig lung strip preparation was investigated and compared with that in guinea pig tracheal and human peripheral airway (small bronchi) smooth muscle preparations, using a number of subtype selective muscarinic receptor antagonists. It was found that guinea pig lung strip contraction was not mediated by a homogeneous class of muscarinic M3 receptors, in contrast to guinea pig tracheal and human peripheral airway smooth muscle. The affinities of the M1- and M3/M2-selective muscarinic receptor antagonists on the guinea pig lung strip were between 0.35 and 1.94 log units lower than in the M3 receptor tissues (respective pA2 values on guinea pig lung strip and trachea: pirenzepine 6.36/6.71, AF-DX 474 6.39/7.11, AQ-RA 721 6.93/7.96, DAU 5884 6.78/8.72, UH-AH 371 7.04/8.20), whereas the affinities of the M2/M3-selective antagonists were between 0.63 and 1.97 log units higher (AF-DX 116 6.63/6.00, AQ-RA 741 7.48/6.63, gallamine 5.44/3.47, methoctramine 7.30/5.38). As a result, a good correlation was obtained when pA2 values from guinea pig lung strip were compared to pKi values towards bovine cardiac muscarinic M2 receptors, though it was noticed that pirenzepine and the M3/M2-selective antagonists showed a closer relationship than the M2-selective compounds. These results suggest that cholinergic contraction of the guinea pig lung strip is mediated by muscarinic M2-like receptors, possibly representing a novel subtype or a mixture of M2 (cardiac) and M3 (or M4) subtypes. It remains to be established, however, on what structure in the lung these contractile M2-like receptors are located and also by which transduction mechanism they produce contraction.