Stable knockdown of CFTR establishes a role for the channel in P2Y receptor‐stimulated anion secretion

Abstract

P2Y receptor regulation of anion secretion was investigated in porcine endometrial gland (PEG) epithelial cells. P2Y2, P2Y4, and P2Y6 receptors were detected in monolayers of PEG cells and immunocytochemistry indicated that P2Y4 receptors were located in the apical membrane. Apical membrane current measurements showed that Ca2+-dependent and PKC-dependent Cl- channels were activated following treatment with uridine triphosphate (UTP) (5 microM). Current-voltage relationships comparing calcium-dependent and PKC-dependent UTP responses under biionic conditions showed significant differences in selectivity between Cl-)and I- for the PKC-dependent conductance (P(I)/P(Cl) = 0.76), but not for Ca2+-dependent conductance (PI/P(Cl) = 1.02). The I-/Cl- permeability ratio for the PKC-dependent conductance was identical to that measured for 8-cpt cAMP. Furthermore, PKC stimulation using phorbol 12-myristate 13-acetate (PMA) activated an apical membrane Cl- conductance that was blocked by the CFTR selective inhibitor, CFTRinh-172. CFTR silencing, accomplished by stable expression of small hairpin RNAs (shRNA), blocked the PKC-activated conductance associated with UTP stimulation and provided definitive evidence of a role for CFTR in anion secretion. CFTR activation increased the initial magnitude of Cl- secretion, and provided a more sustained secretory response compared to conditions where only Ca2+-activated Cl- channels were activated by UTP. Measurements of [cAMP]i following UTP and PMA stimulation were not significantly different than untreated controls. Thus, these results demonstrate that UTP and PMA activation of CFTR occurs independently of increases in intracellular cAMP and extend the findings of earlier studies of CFTR regulation by PKC in Xenopus oocytes to a mammalian anion secreting epithelium.

Activation of Chloride Secretion by Isoflavone Genistein in Endometrial Epithelial Cells

“…This finding suggests the possibility that genistein may stimulate Cl - secretion directly via CFTR as has been reported by others [17,20]. Although UTP has been shown to activate CFTR [34,38], it is likely that the UTP-induced transient increase in I sc could be a result of the stimulation of Cl - secretion, primarily through Ca 2+ -activated Cl - channels. Apart from a direct interaction with CFTR, genistein may increase CFTR localization in the plasma membrane [25,26].…”

“…Previous studies done on porcine endometrial epithelial cells have reported that UTP activates Cl - secretion via an increase in the intracellular Ca 2+ and in PKC stimulation [33,34]. We further examined the effects of genistein in the presence of UTP.…”

“…Therefore the aim of this study is to investigate the acute effects and the mechanism of action of genistein on ion transport function in an immortalized porcine endometrial epithelial cell line. These cells express estrogen receptors both alpha and beta subtype (unpublished data) and exhibit functionally characterized CFTR and other transport proteins for Cl - and K + secretion [34,35]. This provides a suitable model for studying the transport-related properties of estrogen-like substances.…”

Cystic fibrosis transmembrane conductance regulator is involved in airway epithelial wound repair

“…Normal human bronchial epithelial cells (NHBE) purchased from Lonza (Allendale, NJ) were passage extended by transfection with the catalytic subunit of human telomerase as previously described (32,33). NHBE cells were maintained in complete bronchial epithelial growth media (bronchial epithelial basal medium ϩ BEGM Bullet Kit).…”

“…CFTR was silenced in Calu-3 cells using the Sleeping Beauty transposon vectors pT2/si-PuroV2 for short hairpin RNA (shRNA) expression as previously described (32,33). Positive transfectancts were selected using puromycin (4 g/ml).…”