Smooth Muscle Muscarinic Receptors Research Articles

Lung Rejection and Bronchial Hyperresponsiveness to Methacholine and Ultrasonically Nebulized Distilled Water in Heart-Lung Transplantation Patients

Acute denervation of the lungs occurs after heart-lung transplantation (HLT), affecting both afferent and efferent nerves below the tracheal anastomosis. After surgery, the carina and main bronchi are perfused by mediastinal collaterals derived from the coronary arteries, and the intrapulmonary airways by retrograde blood flow from pulmonary artery collaterals. During acute rejection, the lungs are subjected to inflammation, particularly perivascular lymphocytic infiltrates. Rejection can be diagnosed by transbronchial biopsy (TBB). We report the bronchial responses to inhaled methacholine and ultrasonically nebulized distilled water (USNDW) in 16 HLT patients 2 wk to 43 months after surgery, relating them to the lung histopathology from concurrent TBB. Methacholine bronchial hyperresponsiveness was common, but it was not associated with airway epithelial or submucosal inflammation or perivascular lymphocytic infiltration. Six patients had a modest response to USNDW (fall in FEV1 greater than 10%). The responsiveness to USNDW was not associated with enhanced methacholine responsiveness or epithelial and mucosal inflammation. However, it was more commonly seen in patients with lung rejection and perivascular infiltrates. Methacholine hyperresponsiveness in HLT patients could therefore reflect denervation hypersensitivity of airway smooth muscle muscarinic receptors. The modest response to USNDW in some patients cannot be a result of a vagal reflex but could reflect a pathologic vascular response associated with lung rejection. These observations offer insight into the possible mechanisms of bronchial hyperresponsiveness in disease.

Methods for analyzing and interpreting cooperativity in dose-response curves—II. Partial agonists acting on muscarinic receptors in smooth muscle

1. 1. Dose-response curves for eight muscarinic agonists contracting guinea pig ileum were transformed into Hanes-Woolf plots and Hill plots respectively. 2. 2. Hill plots for three full agonists were linear with slopes of 1.6, illustrating positive homotropic cooperativity involving two or more agonist binding sites. 3. 3. Five partial agonists gave curved Hill plots with Hill coefficients of 1.2–1.7 at low agonist concentration and Hill coefficients of 0.3–0.5 at high agonist concentrations. 4. 4. Hanes-Woolf transformations were used to delineate the transition from positive to negative cooperativity and to assist in the interpolation of Hill plots. 5. 5. The results show that all of the agonists studied induce positive cooperativity, and that the difference between full agonists and partial agonists results from the ability of the latter to induce pronounced negative cooperativity at higher concentrations.

The interaction of methoctramine and himbacine at atrial, smooth muscle and endothelial muscarinic receptors in vitro.

1. The action of methoctramine and himbacine at muscarinic receptors has been studied using guinea-pig isolated trachea, oesophageal muscularis mucosae, paced left atria, and rat aortic preparations. 2. Methoctramine (1 x 10(-6)-3.2 x 10(-4) M), but not himbacine, elicited positive inotropic responses. These responses were enhanced by pretreating the animals with reserpine. The responses in reserpine-treated animals were not antagonized by phentolamine (1 x 10(-6) M) but were antagonized by propranolol (1 x 10(-6) M). 3. Methoctramine, but not himbacine, exhibited allosteric inhibitory effects at cardiac muscarinic receptors, resulting in a curvilinear Schild plot. Deviations from competitive antagonism were also observed in combination dose-ratio experiments using atropine and methoctramine. At 1 x 10(-6) M, the pKB value for methoctramine was 7.88 +/- 0.15 (mean +/- s.e.mean, n = 5). The pA2 value for himbacine at cardiac muscarinic receptors was 8.52 +/- 0.06 (n = 3). 4. At tracheal and oesophageal muscularis mucosal smooth muscle receptors, the Schild plots for both antagonists were linear. The pA2 values for methoctramine at receptors in these two preparations were similar (6.08 +/- 0.05 and 6.03 +/- 0.09 respectively, n = 4) and were approximately 60 fold less than those values observed at atrial receptors. Himbacine, also exhibited similar values at muscarinic receptors in the trachea and oesophageal muscularis mucosae (7.61 +/- 0.05 and 7.57 +/- 0.04 respectively, n = 4). 5. Muscarinic receptors mediating relaxation of the rat aortic endothelium exhibited pA2 values for methoctramine (5.87 +/- 0.12, n = 6) which were similar to those observed in the smooth muscle, but not the atria. The pA2 values for himbacine at endothelial muscarinic receptors were approximately 0.5 pA2 units lower than those observed at muscarinic receptors in smooth muscle (6.92 + 0.80, n = 6). In addition, the Schild slopes for methoctramine and himbacine at these receptors were significantly (P < 0.05) less than unity. 6. Methoctramine, and to a lesser extent himbacine, are potent and selective antagonists for cardiac muscarinic receptors. However, caution should be used in interpretation of the data with methoctramine in view of the inhibitory allosteric properties and direct inotropic actions of this compound.

An autoradiographic study of muscarinic cholinoceptors in blood vessels: no localization on vascular endothelium

In vitro labelling and autoradiographic techniques were used to examine the localization of [ 3H]quinuclidinyl benzilate ([ 3H]QNB) and [ 125I]4-iodo-QNB ([ 125I]4IQNB) to slide-mounted sections of rabbit aorta and pulmonary artery, cat aorta, pulmonary and superior mesenteric arteries. These vessels all respond to acetylcholine (ACh) with endothelium-dependent relaxation, yet there was no evidence for endothelium-related binding of either [ 3H]QNB or [ 125I]4IQNB. Muscarinic receptors were localized over the medial smooth muscle and, in the rabbit pulmonary artery, the density of binding increased towards the adventitia. Binding of either radioligand to sections of rabbit pulmonary artery was not affected by the muscarinic M 1 receptor antagonist pirenzepine (20 nM) but was markedly reduced by the muscarinic M 2 antagonist 4DAMP (4-diphenylacetoxy-N-methyl-piperidine methobromide) (1 nM). This study provides evidence for muscarinic receptors located directly on smooth muscle cells, indicating that endothelium-dependent relaxation to ACh results from an indirect mechanism involving smooth muscle muscarinic receptors.

Differential effects of pertussis toxin on muscarinic responses in isolated atria and smooth muscle.

1. The effect of pretreatment with pertussis toxin has been studied on responses to muscarinic agonists in guinea-pig atria and smooth muscle in vitro. 2. 48 h after a single intravenous injection of pertussis toxin (3.2-100 micrograms.kg-1), muscarinic receptor-mediated negative inotropic responses in the atria were inhibited in a dose-dependent manner, with complete abolition of responses occurring after administration of 100 micrograms.kg-1. 3. In contrast, there was no effect on atrial positive inotropic responses to isoprenaline. In addition, no effect was observed on contractile responses to carbachol and pilocarpine in the ileum, trachea, oesophageal muscularis mucosae and urinary bladder, either in terms of potency or maximal response, at all dose levels of pertussis toxin studied. 4. It is concluded that muscarinic receptors in the atria, but not smooth muscle, are probably coupled to the inhibitory regulatory protein Ni, which is functionally inactivated by pertussis toxin. The differences in coupling between atrial and smooth muscle muscarinic receptors provide further evidence for muscarinic receptor heterogeneity in these two tissues.

Autoradiographic visualization of muscarinic receptors in pulmonary nerves and ganglia

We investigated autoradiographically the distribution of muscarinic receptors in bovine airways using (−)-[ 3H]quinuclidinyl benzilate as radioligand. The autoradiographs demonstrated the presence of muscarinic receptors in smooth muscle as well as neuronal muscarinic receptors in pulmonary nerves and ganglia. It is reasonable to believe that the neuronal muscarinic receptors participate in the regulation of neurotransmitter release at the peripheral nerve terminals innervating the bronchial smooth muscle.

Differential neuronal survival in the avian ciliary ganglion after chronic acetylcholine receptor blockade

We have described in the preceding 2 papers the development of the pharmacological and contractile properties of all targets of the ciliary ganglion: the iris and ciliary body (Pilar et al., 1987), and the choroidal coat (Meriney and Pilar, 1987). In this paper, we examine the chronic effects of ACh receptor (AChR) blockade on ciliary ganglion neuron survival. Nicotinic or muscarinic AChR blockers were administered daily to developing chicken embryos during the normal neuronal death period in the ciliary ganglion. The effects of the blockers on ganglionic and neuromuscular transmission were assessed, and neuronal survival was assayed by counting both the total number of ganglion neurons and the selectively HRP-labeled ciliary neurons after the normal neuronal death period. Blockade of ganglionic transmission decreases survival in both populations of neurons. Blockade of neuromuscular muscular transmission increases survival in the ciliary population, which innervates the striated iris and ciliary body muscle. In contrast, blockade of synaptic activity has various influences on the survival of the choroid population, which innervates the smooth muscle of the choroid coat. Smooth muscle muscarinic receptor blockade with atropine does not influence survival. At higher doses (which block ganglionic transmission), atropine decreases choroid survival. Survival of the choroid population is increased by nicotinic blockade with 75 micrograms alpha bungarotoxin (alpha BTX), but decreased by 12.5 micrograms alpha BTX. Two main conclusions arise from these studies. Activation of postsynaptic AChRs in both the ganglion and the periphery are important in the regulation of neuronal survival. These effects usually occur in opposite directions: Blockade of ganglionic transmission decreases neuronal survival, while paralysis of neuromuscular transmission increases neuronal survival. This embodies the "balance" hypothesis (Cunningham, 1982) for neuronal survival, which states that motoneurons must balance afferent and target interactions during a critical period after synapses are formed in both regions. The present observations support this hypothesis. However, although both ciliary and choroid neurons have been shown to depend on the presence of the periphery for survival, target muscle paralysis via AChR blockade rescues the ciliary neurons but does not influence survival in the choroid population. Target-dependent regulation of choroid neuron survival during the normal neuronal death period is clearly different from the regulation of ciliary neuron survival.

Pharmacological differences between atrial and smooth muscle muscarinic receptors

Pharmacological differences between atrial and smooth muscle muscarinic receptors

Contractile effects of cysteamine on the guinea-pig ileum.

Cysteamine (beta-mercaptoethylamine HCl) (1.0-40.0 mM) induced a concentration-dependent increase in tonic and phasic contractions of segments of guinea-pig ileum in vitro. Myenteric plexus-longitudinal muscle (MPLM) preparations also responded with an increase in tonic contractions but phasic contractions were either greatly reduced or absent, indicating that these were a response of the circular muscle. Atropine (5 microM) inhibited the cysteamine-induced contractions, whereas hexamethonium and guanethidine had no effect, suggesting that cysteamine was acting at least partly via a cholinergic mechanism involving muscarinic receptors. Tetrodotoxin increased the phasic contractions of ileal segments, but had no effect on the tonic component. Treatment of MPLM preparations with morphine (1 microM) resulted in a small reduction in responsiveness to cysteamine, and blocked electrically-induced contractions by at least 90%. Since morphine acts by inhibiting acetylcholine release via hyperpolarization of intrinsic neurones, a small but significant part of the cysteamine-induced contractions probably resulted from stimulation of acetylcholine release from intrinsic neurones. Following a response to high cysteamine concentrations (greater than 15 mM) tissues were refractory to subsequent cysteamine administration. Cross-desensitization between cysteamine and acetylcholine also occurred, as short-term (1-3 min) incubation of MPLM preparations with high concentrations of either compound (1-10 microM acetylcholine or 20 mM cysteamine) resulted in a reduced responsiveness to both. A reduced sensitivity to acetylcholine or cysteamine was obtained following long-term (45 min) incubation with acetylcholine (1 microM). Removal of Na+ from the incubation medium negated this effect. In contrast, the refractoriness to acetylcholine or cysteamine following long-term (45 min) incubation with cysteamine (20 mM) was accentuated in low Na+ medium. It is suggested that cysteamine induces a contraction of both the circular and longitudinal muscle of the guinea-pig ileum by stimulating the release of acetylcholine from intrinsic neurones, by an action at the level of the smooth muscle muscarinic receptor, and possibly by a non-cholinergic mechanism. However, the mechanisms by which acetylcholine and cysteamine induce tissue refractoriness probably differ.

Cholinergic control mechanisms for immunoreactive motilin release and motility in the canine duodenum.

The relationship of immunoreactive (IR) motilin release from the duodenum to duodenal motility changes was investigated in anaesthetized dogs. Stimulation of one or both vagi at 5 or 15 Hz or field stimulation of intrinsic duodenal nerves produced significant increases in duodenal vein IR motilin concentrations and accompanying increases in duodenal motility. However, only stimulation of both vagi at 15 Hz produced significant changes in peripheral venous concentrations of IR motilin. These occurred after a delay at a time when both the duodenum and the antrum were quiescent. Either hexamethonium or atropine blocked IR motilin release induced by stimulation of intrinsic or extrinsic nerves while only atropine inhibited the release induced by intraarterial carbachol. The response stimulated by carbachol and blocked by atropine was tetrodotoxin insensitive and the muscarinic receptor involved was presumably located on a nonneural structure. The site sensitive to hexamethonium was presumably the neural pathway which terminated at the muscarinic receptor. Concomitant studies of duodenal motility responses to vagal and field stimulation suggested a conventional neural pathway with preganglionic cholinergic nerves in the vagus, postganglionic cholinergic nerves in the duodenum (activated by field stimulation) and a smooth muscle muscarinic receptor. Activation of antral motility by stimulation of the abdominal vagus or intraarterial carbachol injections to the antrum increased duodenal IR motilin release in the absence of duodenal motility. Thus activation of the intrinsic nerves which cross the pylorus initiated IR motilin release as well as inhibited duodenal motility.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscarinic cholinergic receptor structure. Receptor size, membrane orientation, and absence of major phylogenetic structural diversity.

The structure of the muscarinic acetylcholine receptor was investigated by comparing polypeptides identified by sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis with the size of the intact receptor in cell membranes as determined by target size analysis. Muscarinic receptors from human, dog, and rat brain, rat and dog cardiac muscle, and guinea pig ileum longitudinal smooth muscle labeled with [3H] propylbenzilylcholine mustard, a covalent affinity reagent, appeared as single polypeptides with molecular weights of 80,000 on NaDodSO4-polyacrylamide gels. NaDodSO4-polyacrylamide gels of ileum smooth muscle muscarinic receptor also consistently displayed smaller peptides of 64, 52, 42, 36, 23, and 18 kDa. In order to determine whether the 80-kDa protein represented all or only a portion of the muscarinic receptor, target size analysis was undertaken. Radiation-induced receptor inactivation was measured by loss of [3H]quinuclidinyl benzilate specific binding and by loss of [3H]propylbenzilylcholine mustard-labeled receptor protein on NaDodSO4 gels. Target size analysis of rat and human brain, canine heart, and guinea pig ileum smooth muscle muscarinic receptors all indicated that the intact membrane-bound receptor has an average molecular mass of 80,000 daltons. These data demonstrate that the protein isolated on NaDodSO4 gels represents the intact receptor molecule. The question of whether structurally distinct receptors exist in different tissues and species was answered, in part, by limited proteolysis studies of the 80-kDa protein isolated from the above tissues. Trypsin and papain produce peptides of 64, 52, 42, 36, 23, and 18 kDa from all receptors studied, indicating a lack of major structural diversity and the absence of multiple structural forms of the muscarinic receptor. Limited proteolysis of the membrane-bound receptor produces a major peptide of 42,000 daltons and minor peptides of 36, 23, and 18 kDa, all of which contain the ligand binding site and protrude from the membrane into the extracellular space.

Developmental loss of a smooth muscle muscarinic receptor population

The radioligand [ 3H]quinuclidinyl benzilate has been used to examine the muscarinic receptor population of the chick expansor secundariorum muscle during its post-hatch development. At hatch the muscle bound 0.165 fmol of the ligand, but during the following two weeks binding decreased rapidly. Further reduction in ligand binding to the adult stage was observed, but at a much slower rate. The developmental loss of binding sites within this tissue corresponds to previously demonstrated reduction in acetylcholine sensitivity.

The effect of harmaline and related β-carbolines on the acetylcholine-stimulated contractions of guinea-pig ileum

Five members of the Harmala family of alkaloids, four commercially available and one synthetic homologue, were tested for their actions on the acetylcholine-elicited contractions of the guinea-pig ileum. Under normal Tyrode conditions over comparable concentration ranges harmalol was without effect, harmaline and harmaline inhibited competitive-noncompetitive inhibition whilst the related 2-methylated derivatives effected purely competitive antagonism. Binding studies using [ 3H]QNB have further confirmed the competitive aspect of this antagonism. The competitive-noncompetitive inhibitory property of harmaline on smooth muscle muscarinic receptors has not been previously reported. Harmaline is known to compete with Na + binding sites and significantly complete substitution of Na + by K + in the Tyrode medium was found to abolish the noncompetitive component of harmaline inhibition. Explanations are offered for both the competitive and noncompetitive components of the inhibition produced by the Harmala alkaloids.

The labelling and isolation of neuroreceptors

Publisher Summary The true identity of receptors is to some extent a semantic problem that has come about because the term receptor was first introduced to account for a phenomenon rather than to name a specific physiological entity. With the enormous advances in biochemistry and physiology, much was learnt about the molecular basis of physiological processes and this led to speculation as to the biochemical nature of receptors. In practice, the receptor is generally considered to be the drug binding macromolecule and it remains to be seen whether this consists of a single polypeptide chain or an oligomer. The sequence of events in the action of a drug at its receptor is thought to be (1) the access of the drug to the receptor, (2) the interaction of the drug with the receptor, (3) the interaction of an agonist with its receptor causes a conformational change in the receptor that leads directly or indirectly to a variety of responses depending on the receptor type, (4) these responses lead by one or more steps to the ultimate response of the cell that may be muscle contraction or relaxation, impulse generation, secretion, or metabolic activity. This chapter discusses receptor labeling and catecholamine receptors. It also discusses nicotinic receptors in striated muscle and in brain and describes muscarinic receptors in smooth muscle.