Ingrid P. Fricks

17β-Estradiol inhibits Ca2+-dependent homeostasis of airway surface liquid volume in human cystic fibrosis airway epithelia

Normal airways homeostatically regulate the volume of airway surface liquid (ASL) through both cAMP- and Ca2+-dependent regulation of ion and water transport. In cystic fibrosis (CF), a genetic defect causes a lack of cAMP-regulated CFTR activity, leading to diminished Cl- and water secretion from airway epithelial cells and subsequent mucus plugging, which serves as the focus for infections. Females with CF exhibit reduced survival compared with males with CF, although the mechanisms underlying this sex-related disadvantage are unknown. Despite the lack of CFTR, CF airways retain a limited capability to regulate ASL volume, as breathing-induced ATP release activates salvage purinergic pathways that raise intracellular Ca2+ concentration to stimulate an alternate pathway to Cl- secretion. We hypothesized that estrogen might affect this pathway by reducing the ability of airway epithelia to respond appropriately to nucleotides. We found that uridine triphosphate-mediated (UTP-mediated) Cl- secretion was reduced during the periovulatory estrogen maxima in both women with CF and normal, healthy women. Estrogen also inhibited Ca2+ signaling and ASL volume homeostasis in non-CF and CF airway epithelia by attenuating Ca2+ influx. This inhibition of Ca2+ signaling was prevented and even potentiated by estrogen antagonists such as tamoxifen, suggesting that antiestrogens may be beneficial in the treatment of CF lung disease because they increase Cl- secretion in the airways.

Quantification of Gi-Mediated Inhibition of Adenylyl Cyclase Activity Reveals That UDP Is a Potent Agonist of the Human P2Y14 Receptor

The P2Y14 receptor was initially identified as a G protein-coupled receptor activated by UDP-glucose and other nucleotide sugars. We have developed several cell lines that stably express the human P2Y14 receptor, allowing facile examination of its coupling to native Gi family G proteins and their associated downstream signaling pathways (J Pharmacol Exp Ther 330:162–168, 2009). In the current study, we examined P2Y14 receptor-dependent inhibition of cyclic AMP accumulation in human embryonic kidney (HEK) 293, C6 glioma, and Chinese hamster ovary (CHO) cells stably expressing this receptor. Not only was the human P2Y14 receptor activated by UDP-glucose, but it also was activated by UDP. The apparent efficacies of UDP and UDP-glucose were similar, and the EC50 values (74, 33, and 29 nM) for UDP-dependent activation of the P2Y14 receptor in HEK293, CHO, and C6 glioma cells, respectively, were similar to the EC50 values (323, 132, and 72 nM) observed for UDP-glucose. UDP and UDP-glucose also stimulated extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in P2Y14 receptor-expressing HEK293 cells but not in wild-type HEK293 cells. A series of analogs of UDP were potent P2Y14 receptor agonists, but the naturally occurring nucleoside diphosphates, CDP, GDP, and ADP exhibited agonist potencies over 100-fold less than that observed with UDP. Two UDP analogs were identified that selectively activate the P2Y14 receptor over the UDP-activated P2Y6 receptor, and these molecules stimulated phosphorylation of ERK1/2 in differentiated human HL-60 promyeloleukemia cells, which natively express the P2Y14 receptor but had no effect in wild-type HL-60 cells, which do not express the receptor. We conclude that UDP is an important cognate agonist of the human P2Y14 receptor.

UDP Is a Competitive Antagonist at the Human P2Y14 Receptor

G protein-coupled P2Y receptors (P2Y-R) are activated by adenine and uracil nucleotides. The P2Y14 receptor (P2Y14-R) is activated by at least four naturally occurring UDP sugars, with UDP-glucose (UDP-Glc) being the most potent agonist. With the goal of identifying a competitive antagonist for the P2Y14-R, UDP was examined for antagonist activity in COS-7 cells transiently expressing the human P2Y14-R and a chimeric Gα protein that couples Gi-coupled receptors to stimulation of phosphoinositide hydrolysis. UDP antagonized the agonist action of UDP-Glc, and Schild analysis confirmed that the antagonism was competitive (pKB = 7.28). Uridine 5′-O-thiodiphosphate also antagonized the human P2Y14-R (hP2Y14-R) with an apparent affinity similar to that of UDP. In contrast, no antagonist activity was observed with ADP, CDP, or GDP, and other uracil analogs also failed to exhibit antagonist activity. The antagonist activity of UDP was not observed at other hP2Y-R. In contrast to its antagonist action at the hP2Y14-R, UDP was a potent agonist (EC50 = 0.35 μM) at the rat P2Y14-R. These results identify the first competitive antagonist of the P2Y14-R and demonstrate pharmacological differences between receptor orthologs.

Gi-Dependent Cell Signaling Responses of the Human P2Y14 Receptor in Model Cell Systems

Eight G protein-coupled receptors comprise the P2Y receptor family of cell signaling proteins. The goal of the current study was to define native cell signaling pathways regulated by the uridine nucleotide sugar-activated P2Y14 receptor (P2Y14-R). The P2Y14-R was stably expressed in human embryonic kidney (HEK) 293 and C6 rat glioma cells by retroviral infection. Nucleotide sugar-dependent P2Y14-R activation was examined by measuring inhibition of forskolin-stimulated cAMP accumulation. The effect of P2Y14-R activation on mitogen-activated protein kinase signaling also was studied in P2Y14-HEK293 cells and in differentiated HL-60 human myeloid leukemia cells. UDP-Glc, UDP-galactose, UDP-glucuronic acid, and UDP-N-acetylglucosamine promoted inhibition of forskolin-stimulated cAMP accumulation in P2Y14-HEK293 and P2Y14-C6 cells, and this signaling effect was abolished by pretreatment of cells with pertussis toxin. Inhibition of cAMP formation by nucleotide sugars also was observed in direct assays of adenylyl cyclase activity in membranes prepared from P2Y14-C6 cells. UDP-Glc promoted concentration-dependent and pertussis toxin-sensitive extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in P2Y14-HEK293 cells. P2Y14-R mRNA was not observed in wild-type HL-60 cells but was readily detected in dimethyl sulfoxide-differentiated cells. Consistent with this observation, no effect of UDP-Glc was observed in wild-type HL-60 cells, but UDP-Glc-promoted pertussis toxin-sensitive activation of ERK1/2 occurred after differentiation. These results illustrate that the human P2Y14-R signals through Gi to inhibit adenylyl cyclase, and P2Y14-R activation also leads to ERK1/2 activation. This work also identifies two stable P2Y14-R-expressing cell lines and differentiated HL-60 cells as model systems for the study of P2Y14-R-dependent signal transduction.