Airway hyperresponsiveness and inflammation are fundamental hallmarks of allergic asthma that are accompanied by increases in certain polycations, such as eosinophil cationic protein. Levels of these cations in body fluids correlate with asthma severity. We show that polycations and elevated extracellular calcium activate the human recombinant and native calcium-sensing receptor (CaSR), leading to intracellular calcium mobilization, cyclic adenosine monophosphate breakdown, and p38 mitogen-activated protein kinase phosphorylation in airway smooth muscle (ASM) cells. These effects can be prevented by CaSR antagonists, termed calcilytics. Moreover, asthmatic patients and allergen-sensitized mice expressed more CaSR in ASMs than did their healthy counterparts. Indeed, polycations induced hyper-reactivity in mouse bronchi, and this effect was prevented by calcilytics and absent in mice with CaSR ablation from ASM. Calcilytics also reduced airway hyperresponsiveness and inflammation in allergen-sensitized mice in vivo. These data show that a functional CaSR is up-regulated in asthmatic ASM and targeted by locally produced polycations to induce hyperresponsiveness and inflammation. Thus, calcilytics may represent effective asthma therapeutics.

1 The cannabinoid arachidonyl ethanolamide (anandamide) caused concentration-dependent relaxation of 5-HT-precontracted, myograph-mounted, segments of rat left anterior descending coronary artery. 2 This relaxation was endothelium-independent, una ected by the fatty acid amide hydrolase inhibitor, arachidonyl tri¯uoromethyl ketone (10 mM), and mimicked by the non-hydrolysable anandamide derivative, methanandamide. 3 Relaxations to anandamide were attenuated by the cannabinoid receptor antagonist, SR 141716A (3 mM), but una ected by AM 251 (1 mM) and AM 630 (1 mM), more selective antagonists of cannabinoid CB 1 and CB 2 receptors respectively. Palmitoylethanolamide, a selective CB 2 receptor agonist, did not relax precontracted coronary arteries. 4 Anandamide relaxations were not a ected by inhibition of sensory nerve transmission with capsaicin (10 mM) or blockade of vanilloid VR1 receptors with capsazepine (5 mM). Nevertheless capsaicin relaxed coronary arteries in a concentration-dependent and capsazepine-sensitive manner, con®rming functional sensory nerves were present. In contrast, capsazepine and capsaicin did inhibit anandamide relaxations in methoxamine-precontracted rat small mesenteric arteries. 5 Relaxations to anandamide were inhibited by TEA (1 mM) or iberiotoxin (50 nM), blockers of large conductance, Ca 2+ -activated K + channels (BK Ca ). Gap junction inhibition with 18a-glycyrrhetinic acid (100 mM) did not a ect anandamide relaxations. 6 This study shows anandamide relaxes the rat coronary artery by a novel mechanism. Anandamide-induced relaxations do not involve the endothelium, degradation into active metabolites, or activation of cannabinoid CB 1 or CB 2 receptors, but may involve activation of BK Ca . Vanilloid receptor activation also has no role in the e ects of anandamide in coronary arteries, even though functional sensory nerves are present. British Journal of Pharmacology (2001) 134, 921 ± 929

1 Cannabinoids are known to cause coronary vasodilatation and reduce left ventricular developed pressure (LVDP) in isolated hearts although the identity of the receptor(s) mediating these responses is unknown. Our objective was to pharmacologically characterize cannabinoid receptors mediating cardiac responses to the endocannabinoid, anandamide. 2 Dose-response curves for coronary perfusion pressure (CPP) and LVDP were constructed to anandamide, R-(+)-methanandamide, palmitoylethanolamide (PEA) and JWH015 in isolated Langendor -perfused rat hearts. Anandamide dose-response curves were also constructed in the presence of antagonists selective for CB 1 , CB 2 or VR 1 receptors. 3 Anandamide and methanadamide signi®cantly reduced CPP and LVDP but the selective CB 2 receptor agonists, PEA and JWH015 had no signi®cant e ect, compared with equivalent vehicle doses. 4 Single bolus additions of the selective CB 1 -receptor agonist, ACEA (5 nmol), decreased LVDP and CPP. When combined with JWH015 (5 nmol) these responses were not augmented. 5 Anandamide-mediated reductions in CPP were signi®cantly blocked by the selective CB 1 receptor antagonists SR 141716A (1 mM) and AM251 (1 mM) and the selective CB 2 receptor antagonist SR 144528 (1 mM) but not by another selective CB 2 receptor antagonist AM630 (10 mM) nor the vanilloid VR 1 receptor antagonist capsazepine (10 mM). 6 SR 141716A, AM281 and SR 144528 signi®cantly blocked negative inotropic responses to anandamide that were not signi®cantly a ected by AM251, AM630 and capsazepine. 7 One or more novel sites mediate negative inotropic and coronary vasodilatatory responses to anandamide. These sites can be distinguished from classical CB 1 and CB 2 receptors, as responses are sensitive to both SR 141716A and SR 144528.

1 Although the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide share a similar pharmacology, 2-AG reportedly limits myocardial ischaemia-reperfusion injury whereas anandamide does not. We therefore investigated whether or not anandamide reduces infarct size and which, if any, of the known cannabinoid-signalling pathways are involved. 2 Rat isolated perfused hearts were subjected to global, no-flow ischaemia (30 min) and reperfusion (1 h). Agonists were present from 5 min before ischaemia until the end of reperfusion. Antagonists, where used, were present throughout the protocol. Recovery of left ventricular developed pressure and coronary flow was incomplete in control hearts and not significantly affected by any drug treatment. 3 In vehicle-treated hearts, 2673% (n ¼ 13) of the left ventricle was infarcted at the end of reperfusion. Infarction of the left ventricle was significantly reduced after 1 mM anandamide (1071%, n ¼ 7) or 1 mM methanandamide (1274%, n ¼ 6) but not 1 mM HU210. Neither ACPA (1 mM; CB 1 receptor agonist) nor JWH133 (1 mM; CB 2 receptor agonist), individually or combined significantly affected infarct size. 4 Anandamide (1 mM) did not reduce infarct size in the presence of the CB 1 receptor antagonist rimonabant (SR141716A, 1 mM) or the CB 2 receptor antagonist, SR144528 (1 mM). 5 Despite sensitivity to CB 1 and CB 2 receptor antagonists, the infarct-limiting action of anandamide was not mimicked by agonists selective for CB 1 or CB 2 receptors suggesting the involvement of a novel cannabinoid site of action.

This is the first demonstration that short-term treatment with a selective AT1 versus AT2 antagonist exerts different effects on recovery of mechanical function after ischemia-reperfusion: the AT2 antagonist was cardioprotective, whereas the AT1 antagonist was not. These data suggest that AT2 antagonists and AT1 agonists may offer novel approaches for the treatment of mechanical dysfunction after ischemia-reperfusion.