G protein-coupled estrogen receptor inhibits the P2Y receptor-mediated Ca(2+) signaling pathway in human airway epithelia.

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.

Evidence of purinergic neurotransmission in isolated, intact horse sweat glands.

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.