P2Y receptor regulation of sodium transport in human mammary epithelial cells

Primary human mammary epithelial (HME) cells were immortalized by stable, constitutive expression of the catalytic subunit of human telomerase. Purinergic receptors were identified by RT-PCR and quantitative RT-PCR from mRNA isolated from primary and immortalized cells grown to confluence on membrane filters. Several subtypes of P2Y receptor mRNA were identified including P2Y1, P2Y2, P2Y4, and P2Y6 receptors. RT-PCR experiments also revealed expression of A2b adenosine receptor mRNA in primary and immortalized cells. Confluent monolayers of HME cells exhibited a basal short-circuit current (Isc) that was abolished by amiloride and benzamil. When monolayers were cultured in the presence of hydrocortisone, mRNA expression of Na+ channel (ENaC) -, β-, and -subunits increased approximately threefold compared with that in cells grown without hydrocortisone. In addition, basal benzamil-sensitive Na+ transport was nearly twofold greater in hydrocortisone-treated monolayers. Stimulation with UTP, UDP, or adenosine 5'-O-(3-thiotriphosphate) (ATPS) produced increases in intracellular calcium concentration that were significantly reduced following pretreatment with the calcium-chelating agent BAPTA-AM. Concentration-response relationships indicated that the rank order of potency for these agonists was UTP > UDP > ATPS. Basolateral stimulation with UTP produced a rapid but transient increase in Isc that was significantly reduced if cells were pretreated with BAPTA-AM or benzamil. Moreover, basolateral treatment with either charybdotoxin or clotrimazole significantly inhibited the initial UTP-dependent increase in Isc and eliminated the sustained current response. These results indicate that human mammary epithelial cells express multiple P2 receptor subtypes and that Ca2+ mobilization evoked by P2Y receptor agonists stimulates Na+ absorption by increasing the activity of Ca2+-activated K+ channels located in the basolateral membrane.This work was partly supported by Korea Research Foundation Grant KRF-005-E00076 (to S. Y. Lee) and National Institutes of Health Grants AI-50494 and DK-74010 (to S. M. O'Grady)

High-throughput mutational analysis of TOR1A in primary dystonia

<p>Abstract</p> <p>Background</p> <p>Although the c.904_906delGAG mutation in Exon 5 of <it>TOR1A </it>typically manifests as early-onset generalized dystonia, DYT1 dystonia is genetically and clinically heterogeneous. Recently, another Exon 5 mutation (c.863G>A) has been associated with early-onset generalized dystonia and some ΔGAG mutation carriers present with late-onset focal dystonia. The aim of this study was to identify <it>TOR1A </it>Exon 5 mutations in a large cohort of subjects with mainly non-generalized primary dystonia.</p> <p>Methods</p> <p>High resolution melting (HRM) was used to examine the entire <it>TOR1A </it>Exon 5 coding sequence in 1014 subjects with primary dystonia (422 spasmodic dysphonia, 285 cervical dystonia, 67 blepharospasm, 41 writer's cramp, 16 oromandibular dystonia, 38 other primary focal dystonia, 112 segmental dystonia, 16 multifocal dystonia, and 17 generalized dystonia) and 250 controls (150 neurologically normal and 100 with other movement disorders). Diagnostic sensitivity and specificity were evaluated in an additional 8 subjects with known ΔGAG DYT1 dystonia and 88 subjects with ΔGAG-negative dystonia.</p> <p>Results</p> <p>HRM of <it>TOR1A </it>Exon 5 showed high (100%) diagnostic sensitivity and specificity. HRM was rapid and economical. HRM reliably differentiated the <it>TOR1A </it>ΔGAG and c.863G>A mutations. Melting curves were normal in 250/250 controls and 1012/1014 subjects with primary dystonia. The two subjects with shifted melting curves were found to harbor the classic ΔGAG deletion: 1) a non-Jewish Caucasian female with childhood-onset multifocal dystonia and 2) an Ashkenazi Jewish female with adolescent-onset spasmodic dysphonia.</p> <p>Conclusion</p> <p>First, HRM is an inexpensive, diagnostically sensitive and specific, high-throughput method for mutation discovery. Second, Exon 5 mutations in <it>TOR1A </it>are rarely associated with non-generalized primary dystonia.</p

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

The role of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) in airway epithelial wound repair was investigated using normal human bronchial epithelial (NHBE) cells and a human airway epithelial cell line (Calu-3) of serous gland origin. Measurements of wound repair were performed using continuous impedance sensing to determine the time course for wound closure. Control experiments showed that the increase in impedance corresponding to cell migration into the wound was blocked by treatment with the actin polymerization inhibitor, cytochalasin D. Time lapse imaging revealed that NHBE and Calu-3 cell wound closure was dependent on cell migration, and that movement occurred as a collective sheet of cells. Selective inhibition of CFTR activity with CFTRinh-172 or short hairpin RNA silencing of CFTR expression produced a significant delay in wound repair. The CF cell line UNCCF1T also exhibited significantly slower migration than comparable normal airway epithelial cells. Inhibition of CFTR-dependent anion transport by treatment with CFTRinh-172 slowed wound closure to the same extent as silencing CFTR protein expression, indicating that ion transport by CFTR plays a critical role in migration. Moreover, morphologic analysis of migrating cells revealed that CFTR inhibition or silencing significantly reduced lamellipodia protrusion. These findings support the conclusion that CFTR participates in airway epithelial wound repair by a mechanism involving anion transport that is coupled to the regulation of lamellipodia protrusion at the leading edge of the cell

Airway Exposure to Polyethyleneimine Nanoparticles Induces Type 2 Immunity by a Mechanism Involving Oxidative Stress and ATP Release

Polyethyleneimine (PEI) induced immune responses were investigated in human bronchial epithelial (hBE) cells and mice. PEI rapidly induced ATP release from hBE cells and pretreatment with glutathione (GSH) blocked the response. PEI activated two conductive pathways, VDAC-1 and pannexin 1, which completely accounted for ATP efflux across the plasma membrane. Moreover, PEI increased intracellular Ca2+ concentration ([Ca2+]i), which was reduced by the pannexin 1 inhibitor, 10Panx (50 μM), the VDAC-1 inhibitor, DIDS (100 μM), and was nearly abolished by pretreatment with GSH (5 mM). The increase in [Ca2+]i involved Ca2+ uptake through two pathways, one blocked by oxidized ATP (oATP, 300 μM) and another that was blocked by the TRPV-1 antagonist A784168 (100 nM). PEI stimulation also increased IL-33 mRNA expression and protein secretion. In vivo experiments showed that acute (4.5 h) PEI exposure stimulated secretion of Th2 cytokines (IL-5 and IL-13) into bronchoalveolar lavage (BAL) fluid. Conjugation of PEI with ovalbumin also induced eosinophil recruitment and secretion of IL-5 and IL-13 into BAL fluid, which was inhibited in IL-33 receptor (ST2) deficient mice. In conclusion, PEI-induced oxidative stress stimulated type 2 immune responses by activating ATP-dependent Ca2+ uptake leading to IL-33 secretion, similar to allergens derived from Alternaria

Protease-activated receptor regulation of Cl– secretion in Calu-3 cells requires prostaglandin release and CFTR activation

Human lung epithelial (Calu-3) cells were used to investigate the effects of protease-activated receptor (PAR) stimulation on Cl– secretion. Quantitative RT-PCR (QRT-PCR) showed that Calu-3 cells express PAR-1, -2, and -3 receptor mRNAs, with PAR-2 mRNA in greatest abundance. Addition of either thrombin or the PAR-2 agonist peptide SLIGRL to the basolateral solution of monolayers mounted in Ussing chambers produced a rapid increase in short-circuit current (Isc: thrombin, 21 ± 2 µA; SLIGRL, 83 ± 22 µA), which returned to baseline within 5 min after stimulation. Pretreatment of monolayers with the cell-permeant Ca2+-chelating agent BAPTA-AM (50 µM) abolished the increase in Isc produced by SLIGRL. When monolayers were treated with the cyclooxygenase inhibitor indomethacin (10 µM), nearly complete inhibition of both the thrombin- and SLIGRL-stimulated Isc was observed. In addition, basolateral treatment with the PGE2 receptor antagonist AH-6809 (25 µM) significantly inhibited the effects of SLIGRL on Isc. QRT-PCR revealed that Calu-3 cells express mRNAs for CFTR, the Ca2+-activated KCNN4 K+ channel, and the KCNQ1 K+ channel subunit, which, in association with KCNE3, is known to be regulated by cAMP. Stimulation with SLIGRL produced an increase in apical Cl– conductance that was blocked in cells expressing short hairpin RNAs designed to target CFTR. These results support the conclusion that PAR stimulation of Cl– secretion occurs by an indirect mechanism involving the synthesis and release of prostaglandins. In addition, PAR-stimulated Cl– secretion requires activation of CFTR and at least two distinct K+ channels located in the basolateral membrane