Kristen E. Belmonte

Neuronal eotaxin and the effects of ccr3 antagonist on airway hyperreactivity and M2 receptor dysfunction

Eosinophils cluster around airway nerves in patients with fatal asthma and in antigen-challenged animals. Activated eosinophils release major basic protein, which blocks inhibitory M2 muscarinic receptors (M2Rs) on nerves, increasing acetylcholine release and potentiating vagally mediated bronchoconstriction. We tested whether GW701897B, an antagonist of CCR3 (the receptor for eotaxin as well as a group of eosinophil active chemokines), affected vagal reactivity and M2R function in ovalbumin-challenged guinea pigs. Sensitized animals were treated with the CCR3 antagonist before inhaling ovalbumin. Antigen-challenged animals were hyperresponsive to vagal stimulation, but those that received the CCR3 antagonist were not. M2R function was lost in antigen-challenged animals, but not in those that received the CCR3 antagonist. Although the CCR3 antagonist did not decrease the number of eosinophils in lung tissues as assessed histologically, CCR3 antagonist prevented antigen-induced clustering of eosinophils along the nerves. Immunostaining revealed eotaxin in airway nerves and in cultured airway parasympathetic neurons from both guinea pigs and humans. Both IL-4 and IL-13 increased expression of eotaxin in cultured airway parasympathetic neurons as well as in human neuroblastoma cells. Thus, signaling via CCR3 mediates eosinophil recruitment to airway nerves and may be a prerequisite to blockade of inhibitory M2Rs by eosinophil major basic protein.

Regulation of TNF-α and IFN-γ induced CXCL10 expression: participation of the airway smooth muscle in the pulmonary inflammatory response in chronic obstructive pulmonary disease

The chemokine CXCL10 is produced by many inflammatory cells found in the diseased lung and has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). The present study demonstrates elevated CXCL10 protein in the lungs of COPD patients, which appears histologically in airway smooth muscle (hASM). In primary cultured hASM cells taken from normal donors, CXCL10 protein expression was induced by IFN‐γ and TNF‐α, cytokines reported as elevated in COPD, and a synergistic response was obtained when they were combined. TNF‐α stimulation of hASM enhanced accumulation of CXCL10 mRNA, indicating regulation at the transcriptional level, while IFN‐γ stimulation resulted in a smaller accumulation of CXCL10 mRNA. When these cytokines were applied simultaneously, an additive effect was obtained. TNF‐α ‐induced CXCL10 expression in hASM was dependent on NFκB activation, and a salicylanilide NFκB inhibitor blocked the CXCL10 expression. In contrast, IFN‐γ stimulation resulted in transient NFκB activation, and the inhibitor had little effect on CXCL10 expression. When these cytokines were added simultaneously, NFκB was activated earlier and lasted longer, and the effect was blocked by the inhibitor. These data demonstrate a potential active role for hASM in pulmonary inflammatory diseases such as COPD by producing CXCL10.

Antigen-induced hyperreactivity to histamine: role of the vagus nerves and eosinophils.

M2 muscarinic receptors limit acetylcholine release from the pulmonary parasympathetic nerves. M2 receptors are dysfunctional in antigen-challenged guinea pigs, causing increased vagally mediated bronchoconstriction. Dysfunction of these M2 receptors is due to eosinophil major basic protein, which is an antagonist for M2 receptors. Histamine-induced bronchoconstriction is composed of a vagal reflex in addition to its direct effect on airway smooth muscle. Because hyperreactivity to histamine is seen in antigen-challenged animals, we hypothesized that hyperreactivity to histamine may be due to increased vagally mediated bronchoconstriction caused by dysfunction of M2 receptors. In anesthetized, antigen-challenged guinea pigs, histamine-induced bronchoconstriction was greater than that in control guinea pigs. After vagotomy or atropine treatment, the response to histamine in antigen-challenged animals was the same as that in control animals. In antigen-challenged animals, blockade of eosinophil influx into the airways or neutralization of eosinophil major basic protein prevented the development of hyperreactivity to histamine. Thus hyperreactivity to histamine in antigen-challenged guinea pigs is vagally mediated and dependent on eosinophil major basic protein.M2muscarinic receptors limit acetylcholine release from the pulmonary parasympathetic nerves. M2receptors are dysfunctional in antigen-challenged guinea pigs, causing increased vagally mediated bronchoconstriction. Dysfunction of these M2 receptors is due to eosinophil major basic protein, which is an antagonist for M2 receptors. Histamine-induced bronchoconstriction is composed of a vagal reflex in addition to its direct effect on airway smooth muscle. Because hyperreactivity to histamine is seen in antigen-challenged animals, we hypothesized that hyperreactivity to histamine may be due to increased vagally mediated bronchoconstriction caused by dysfunction of M2 receptors. In anesthetized, antigen-challenged guinea pigs, histamine-induced bronchoconstriction was greater than that in control guinea pigs. After vagotomy or atropine treatment, the response to histamine in antigen-challenged animals was the same as that in control animals. In antigen-challenged animals, blockade of eosinophil influx into the airways or neutralization of eosinophil major basic protein prevented the development of hyperreactivity to histamine. Thus hyperreactivity to histamine in antigen-challenged guinea pigs is vagally mediated and dependent on eosinophil major basic protein.

Increased function of inhibitory neuronal M2 muscarinic receptors in diabetic rat lungs

The function of inhibitory neuronal M2 muscarinic receptors in diabetic rat lungs was investigated. Neuronal M2 muscarinic receptors inhibit acetylcholine release from parasympathetic nerves. Thus, stimulation of neuronal M2 muscarinic receptors with muscarinic agonists, such as pilocarpine, inhibits acetylcholine release and vagally induced bronchoconstriction. In contrast, blockade of neuronal M2 muscarinic receptors with selective M2 muscarinic antagonists, such as AF‐DX 116, potentiates acetylcholine release and vagally induced bronchoconstriction. Rats were made diabetic by streptozotocin (65 mg kg−1, i.v.). After 7–14 days the rats were anaesthetized with urethane (1.5 g kg−1, i.p.), tracheostomized, vagotomized, ventilated and paralysed with suxamethonium (30 mg kg−1, i.v.). Some 7 day diabetic rats were treated with low doses of long acting (NPH) insulin (2 units day−1, s.c.) for 7 days before experimentation. This dose of insulin was not sufficient to restore normoglycaemia in diabetic rats. Thus, insulin‐treated diabetic rats remained hyperglycaemic. Distal electrical stimulation (5–70 Hz, 6 s, 40 V, 0.4 ms) of the vagi caused bronchoconstriction, measured as an increase in inflation pressure and bradycardia. In diabetic rats, vagally induced bronchoconstriction was significantly depressed vs controls. In contrast, bronchoconstriction caused by i.v. acetylcholine was similar in diabetic and control animals. The function of neuronal M2 muscarinic receptors was tested with the muscarinic agonist pilocarpine (0.001–100.0 μg kg−1, i.v.) and the antagonist AF‐DX 116 (0.01–3.0 mg kg−1, i.v.). Pilocarpine inhibited vagally‐induced bronchoconstriction (30 Hz, 20–40 V, 0.4 ms at 6 s) and AF‐DX 116 potentiated vagally‐induced bronchoconstriction (20 Hz, 20–40 V, 0.4 ms at 6 s) to a significantly greater degree in diabetic rats compared to controls. Both frequency‐dependent vagally‐induced bronchoconstriction and M2 muscarinic receptor function could be restored to nearly control values in diabetic rats treated with low doses of insulin. Displacement of [3H]QNB (1 nM) with the agonist carbachol (10.0 nM–10.0 mM) from diabetic cardiac M2 muscarinic receptors revealed a half log increase in agonist binding affinity at both the high and low affinity binding sites vs controls. In contrast, M2 receptors from insulin‐treated diabetic rat hearts showed no significant difference in binding affinity vs controls. These data show that neuronal M2 muscarinic receptors in the lungs have increased function in diabetic rats, suggesting that insulin modulates M2 muscarinic receptor function.

Effects of tachykinin NK1 receptor antagonists on vagal hyperreactivity and neuronal M2 muscarinic receptor function in antigen challenged guinea-pigs.

The role of tachykinin NK1 receptors in the recruitment of eosinophils to airway nerves, loss of inhibitory neuronal M2 muscarinic receptor function and the development of vagal hyperreactivity was tested in antigen‐challenged guinea‐pigs. In anaesthetized guinea‐pigs, the muscarinic agonist, pilocarpine (1–100 μg kg−1, i.v), inhibited vagally induced bronchoconstriction, in control, but not in antigen‐challenged guinea‐pigs 24 h after antigen challenge. This indicates normal function of neuronal M2 muscarinic receptors in controls and loss of neuronal M2 receptor function in challenged guinea‐pigs. Pretreatment of sensitized guinea‐pigs with the NK1 receptor antagonists CP99994 (4 mg kg−1, i.p.), SR140333 (1 mg kg−1, s.c.) or CP96345 (15 mg kg−1, i.p.) before antigen challenge, prevented M2 receptor dysfunction. Neither administration of the NK1 antagonists after antigen challenge, nor pretreatment with an NK2 receptor antagonist, MEN10376 (5 μmol kg−1, i.p.), before antigen challenge, prevented M2 receptor dysfunction. Electrical stimulation of the vagus nerves caused a frequency‐dependent (2–15 Hz, 10 V, 0.2 ms for 5 s) bronchoconstriction that was significantly increased following antigen challenge. Pretreatment with the NK1 receptor antagonists CP99994 or SR140333 before challenge prevented this increase. Histamine (1–20 nmol kg−1, i.v.) caused a dose‐dependent bronchoconstriction, which was vagally mediated, and was significantly increased in antigen challenged guinea‐pigs compared to controls. Pretreatment of sensitized animals with CP99994 before challenge prevented the increase in histamine‐induced reactivity. Bronchoalveolar lavage and histological studies showed that after antigen challenge significant numbers of eosinophils accumulated in the airways and around airway nerves. This eosinophilia was not altered by pretreatment with the NK1 receptor antagonist CP99994. These data indicate that pretreatment of antigen‐sensitized guinea‐pigs with NK1, but not with NK2 receptor antagonists before antigen challenge prevented the development of hyperreactivity by protecting neuronal M2 receptor function. NK1 receptor antagonists do not inhibit eosinophil accumulation around airway nerves.

Discovery of novel 1-azoniabicyclo[2.2.2]octane muscarinic acetylcholine receptor antagonists.

A novel 4-hydroxyl(diphenyl)methyl substituted quinuclidine series was discovered as a very promising class of muscarinic antagonists. The structure-activity relationships of the connectivity of the diphenyl moiety to the quinuclidine core and around the ring nitrogen side chain are described. Computational docking studies using an homology model of the M(3) receptor readily explained the observed structure-activity relationship of the various compounds. Compound 14o was identified as a very potent, slowly reversible M(3) antagonist with a very long in vivo duration of bronchoprotection.

Fluorescent styryl dyes FM1-43 and FM2-10 are muscarinic receptor antagonists: intravital visualization of receptor occupancy

The fluorescent styryl dyes FM1‐43 and FM2‐10 have been used to visualize the endocytic and exocytic processes involved in neurotransmission in a variety of central and peripheral nerve preparations. Their utility is limited to some extent by a poorly understood vesicular‐independent labelling of cells and tissues. We show here that one likely cause of this troublesome background labelling is that FM1‐43 and FM2‐10 are selective and competitive antagonists at both cloned and endogenously expressed muscarinic acetylcholine receptors. In radioligand binding studies, FM1‐43 and FM2‐10 bound with moderate affinity (23–220 nm) to membranes of Chinese hamster ovary (CHO) cells expressing cloned human muscarinic receptors (M1–M5). In functional studies in vitro, FM1‐43 and FM2‐10 inhibited electrical field stimulation (EFS) and acetylcholine‐induced cholinergic contractions of guinea‐pig tracheal strips (IC50: FM1‐43, 0.4 ± 0.1; FM2‐10, 1.6 ± 0.1 μm; concentration of antagonist producing a 2‐fold leftward shift in the acetylcholine concentration–response curve (Kb): FM1‐43, 0.3 ± 0.1; FM2‐10, 15.8 ± 10.1 μm). Neither compound inhibited EFS‐evoked, non‐adrenergic non‐cholinergic nerve‐mediated relaxations or contractions of the airways, or contractions mediated by histamine H1 receptor or tachykinin NK2 receptor activation. Incubating freshly excised tracheal whole‐mount preparations with 5 μm FM1‐43 resulted in intense fluorescence labelling of the smooth muscle that was reduced by up to 90% in the presence of selective M2 and M3 receptor antagonists. The potency of the FM dyes as muscarinic receptor antagonists is within the concentration range used to study vesicular cycling at nerve terminals. Given that muscarinic receptors play a key role in the regulation of neurotransmitter release from a variety of neurones, the anticholinergic properties of FM dyes may have important implications when studying vesicular events in the nervous system. In addition, these dyes may provide a novel tool for visualizing muscarinic receptor occupancy in living tissue or cell preparations.

Discovery of biphenyl piperazines as novel and long acting muscarinic acetylcholine receptor antagonists.

A series of novel biphenyl piperazines was discovered as highly potent muscarinic acetylcholine receptor antagonists via high throughput screening and subsequent optimization. Compound 5c with respective 500- and 20-fold subtype selectivity for M3 over M2 and M1 exhibited excellent inhibitory activity and long duration of action in a bronchoconstriction in vivo model in mice via intranasal administration. The novel inhaled mAChR antagonists are potentially useful therapeutic agents for the treatment of chronic obstructive pulmonary disease.

Design, Synthesis, and Structure-Activity Relationship of Tropane Muscarinic Acetylcholine Receptor Antagonists

Novel tropane derivatives were characterized as muscarinic acetylcholine receptor antagonists (mAChRs). Through optimization of the structure-activity relationship around the tropane scaffold, the quaternary ammonium salt 34 was identified as a very potent M(3) mAChR antagonist. The compound was functionally active and displayed greater than 24 h duration of action in a mouse model of bronchoconstriction.

Discovery of novel and long acting muscarinic acetylcholine receptor antagonists.

High throughput screening and subsequent optimization led to the discovery of novel quaternary ammonium salts as highly potent muscarinic acetylcholine receptor antagonists with excellent selectivity. Compounds 8a, 13a, and 13b showed excellent inhibitory activity and long duration of action in bronchoconstriction in vivo models in two species via intranasal or intratracheal administration. The novel inhaled muscarinic receptor antagonists are potentially useful therapeutic agents for the treatment of chronic obstructive pulmonary disease and other bronchoconstriction disorders.