ABSTRACT
P2Y receptor activation causes the release of inflammatory cytokines in the bronchial epithelium, whereas G protein-coupled estrogen receptor (GPER), a novel estrogen (E2) receptor, may play an anti-inflammatory role in this process. We investigated the cellular mechanisms underlying the inhibitory effect of GPER activation on the P2Y receptor-mediated Ca(2+) signaling pathway and cytokine production in airway epithelia. Expression of GPER in primary human bronchial epithelial (HBE) or 16HBE14o- cells was confirmed on both the mRNA and protein levels. Stimulation of HBE or 16HBE14o- cells with E2 or G1, a specific agonist of GPER, attenuated the nucleotide-evoked increases in [Ca(2+)]i, whereas this effect was reversed by G15, a GPER-specific antagonist. G1 inhibited the secretion of two proinflammatory cytokines, interleukin (IL)-6 and IL-8, in cells stimulated by adenosine 5'-(γ-thio)triphosphate (ATPγS). G1 stimulated a real-time increase in cAMP levels in 16HBE14o- cells, which could be inhibited by adenylyl cyclase inhibitors. The inhibitory effects of E2 or G1 on P2Y receptor-induced increases in Ca(2+) were reversed by treating the cells with a protein kinase A (PKA) inhibitor. These results demonstrated that the inhibitory effects of G1 or E2 on P2Y receptor-mediated Ca(2+) mobilization and cytokine secretion were due to GPER-mediated activation of a cAMP-dependent PKA pathway. This study has reported, for the first time, the expression and function of GPER as an anti-inflammatory component in human bronchial epithelia, which may mediate through its opposing effects on the pro-inflammatory pathway activated by the P2Y receptors in inflamed airway epithelia.
Subject(s)
Calcium/metabolism , Epithelium/metabolism , Receptors, Estrogen/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Purinergic P2Y/metabolism , Signal Transduction/physiology , Bronchi/metabolism , Cell Line , Cyclic AMP-Dependent Protein Kinases/metabolism , Estrogens/metabolism , Humans , Interleukin-6/metabolism , Interleukin-8/metabolism , Respiratory Mucosa/metabolismABSTRACT
BACKGROUND/AIMS: Nobiletin, a citrus flavonoid isolated from tangerines, alters ion transport functions in intestinal epithelia, and has antagonistic effects on eosinophilic airway inflammation of asthmatic rats. The present study examined the effects of nobiletin on basal short-circuit current (I(SC)) in a human bronchial epithelial cell line (16HBE14o-), and characterized the signal transduction pathways that allowed nobiletin to regulate electrolyte transport. METHODS: The I(SC) measurement technique was used for transepithelial electrical measurements. Intracellular calcium ([Ca(2+)]i) and cAMP were also quantified. RESULTS: Nobiletin stimulated a concentration-dependent increase in I(SC), which was due to Cl- secretion. The increase in I(SC) was inhibited by a cystic fibrosis transmembrane conductance regulator inhibitor (CFTR(inh)-172), but not by 4,4'-diisothiocyano-stilbene-2,2'-disulphonic acid (DIDS), Chromanol 293B, clotrimazole, or TRAM-34. Nobiletin-stimulated I(SC) was also sensitive to a protein kinase A (PKA) inhibitor, H89, and an adenylate cyclase inhibitor, MDL-12330A. Nobiletin could not stimulate any increase in I(SC) in a cystic fibrosis (CF) cell line, CFBE41o-, which lacked a functional CFTR. Nobiletin stimulated a real-time increase in cAMP, but not [Ca(2+)]i. CONCLUSION: Nobiletin stimulated transepithelial Cl- secretion across human bronchial epithelia. The mechanisms involved activation of adenylate cyclase- and cAMP/PKA-dependent pathways, leading to activation of apical CFTR Cl- channels.
Subject(s)
Bronchi/drug effects , Chlorides/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP/metabolism , Epithelial Cells/drug effects , Flavones/pharmacology , Signal Transduction/drug effects , Adenylyl Cyclases/metabolism , Bronchi/metabolism , Calcium/metabolism , Cell Line , Cystic Fibrosis/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Epithelial Cells/metabolism , Humans , Imines/pharmacology , Ion Transport/drug effects , Isoquinolines/pharmacology , Respiratory Mucosa/drug effects , Respiratory Mucosa/metabolism , Sulfonamides/pharmacologyABSTRACT
BACKGROUND: Fluid secretion by sweat glands in response to heat and exercise is underpinned by increases in intracellular calcium. In horses, this is primarily via ß2-adrenoceptors, but studies in equine sweat gland cell lines have indicated a possible role for purinergic agonists. Knowledge of equine sweating stimulus-secretion mechanisms in intact glands from healthy animals would allow future comparison to determine whether these mechanisms are affected in equine anhidrosis. HYPOTHESIS/OBJECTIVES: To determine whether purinergic agonists can induce changes in intracellular calcium in intact, freshly isolated equine sweat glands. ANIMALS: Eleven healthy thoroughbred horses from the Hong Kong Jockey Club were used in this study. METHODS: Freshly isolated equine sweat glands were loaded with the calcium-sensitive fluorescent dye fura-2 AM, and changes in intracellular calcium were recorded before, during and after stimulation by purinergic agonists. RESULTS: Purinergic agonists ATP and UTP generated significant increases in intracellular calcium. CONCLUSIONS AND CLINICAL IMPORTANCE: The results show that it is possible to investigate stimulus-secretion coupling mechanisms by fluorescence imaging in equine sweat glands that have been isolated from fresh skin samples. Such isolated glands retain functional ß2-adrenoceptors and P2Y purinergic receptors that couple to a calcium-signalling pathway. Using isolated, intact sweat glands therefore offers a very useful model for the further study of secretory processes in equine sweat glands, and using this experimental approach could facilitate a better understanding of how these mechanisms are affected in equine anhidrosis.
