Differential Inhibitory Effects of CysLT1 Receptor Antagonists on P2Y6 Receptor-Mediated Signaling and Ion Transport in Human Bronchial Epithelia

Abstract

BackgroundCysteinyl leukotriene (CysLT) is one of the proinflammatory mediators released by the bronchi during inflammation. CysLTs exert their biological effects via specific G-protein-coupled receptors. CysLT1 receptor antagonists are available for clinical use for the treatment of asthma. Recently, crosstalk between CysLT1 and P2Y6 receptors has been delineated. P2Y receptors are expressed in apical and/or basolateral membranes of virtually all polarized epithelia to control the transport of fluid and electrolytes. Previous research suggests that CysLT1 receptor antagonists inhibit the effects of nucleotides acting at P2Y receptors. However, the detailed molecular mechanism underlying the inhibition remains unresolved.Methodology/Principal FindingsIn this study, western blot analysis confirmed that both CysLT1 and P2Y6 receptors were expressed in the human bronchial epithelial cell line 16HBE14o-. All three CysLT1 antagonists inhibited the uridine diphosphate (UDP)-evoked ISC, but only montelukast inhibited the UDP-evoked [Ca2+]i increase. In the presence of forskolin or 8-bromoadenosine 3′5′ cyclic monophosphate (8-Br-cAMP), the UDP-induced ISC was potentiated but was reduced by pranlukast and zafirlukast but not montelukast. Pranlukast inhibited the UDP-evoked ISC potentiated by an Epac activator, 8-(4-Chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (8-CPT-2′-O-Me-cAMP), while montelukast and zafirlukast had no such effect. Pranlukast inhibited the real-time increase in cAMP changes activated by 8-CPT-2′-O-Me-cAMP as monitored by fluorescence resonance energy transfer imaging. Zafirlukast inhibited the UDP-induced ISC potentiated by N6- Phenyladenosine- 3′, 5′- cyclic monophosphorothioate, Sp- isomer (Sp-6-Phe-cAMP; a PKA activator) and UDP-activated PKA activity.Conclusions/SignificanceIn summary, our data strongly suggest for the first time that in human airway epithelia, the three specific CysLT1 receptor antagonists exert differential inhibitory effects on P2Y6 receptor-coupled Ca2+ signaling pathways and the potentiating effect on ISC mediated by cAMP and Epac, leading to the modulation of ion transport activities across the epithelia.

Nobiletin Stimulates Chloride Secretion in Human Bronchial Epithelia via a cAMP/PKA-Dependent Pathway

“…2C and D). The replacement of apical normal KH solution by a low Cl - concentration solution generated a favorable serosal to the mucosal Cl - gradient, and the increase in I SC response could be due to Cl - secretion as shown in our previous studies [21,25,27]. To confirm that nobiletin-stimulated I SC responses were due to Cl - secretion, ion substitution experiments were performed.…”

“…FRET imaging experiments were performed using the MetaFluor Imaging System with FRET module (Molecular Devices, Downingtown, PA, USA) as described previously [24,25]. In brief, 16HBE14o- cells were transfected with the Epac-based cAMP sensor with Lipofectamine 2000 according to the manufacturer's protocol.…”

HAMI 3379, a CysLT2 Receptor Antagonist, Attenuates Ischemia-Like Neuronal Injury by Inhibiting Microglial Activation

“…Inhibition of PDEs by montelukast may be beneficial to ischemic neuronal injury, because the resultant accumulation of cAMP protects neurons from ischemic brain injury (Tsukada et al, 2004;Lin et al, 2009) and inhibitors of PDE3 (cilostazol) and PDE4 (rolipram) have protective effects on neurons (Tanaka et al, 2010;Schaal et al, 2012). In addition, its inhibitory effects on P2Y receptors (Mamedova et al, 2005;Pugliese et al, 2009;Lau et al, 2011) may be protective, because downregulation of the novel P2Y-like receptor GPR17 protects from ischemic neuronal injury after focal cerebral ischemia in rats (Ciana et al, 2006;Zhao et al, 2012). Moreover, montelukast has antioxidative effects in peripheral tissues (Muthuraman and Sood, 2010;Coskun et al, 2011;Mohamadin et al, 2011); in a preliminary study, we also found its moderate CysLT 1 Rindependent antioxidative activity in primary neurons (unpublished data).…”

Cordyceps Extracts and the Major Ingredient, Cordycepin: Possible Cellular Mechanisms of Their Therapeutic Effects on Respiratory Disease

“…Therefore, both CFTR Cl -channels and cAMP-dependent K + channels expressed in 16HBE14o-cells could by stimulated by cordycepin through the activation of the cAMP/protein kinase A signaling pathway (Bleich and Warth, 2000;Li and Naren, 2010). In addition to the activation of the cAMP-dependent pathway, cordycepin evoked a concentration-dependent increase in intracellular [Ca 2+ ] as measured by Fura-2 imaging (Lau et al, 2011) (Fig. 4).…”