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BackgroundAltered epithelial physical and functional barrier properties along with TH1/TH2 immune dysregulation are features of allergic asthma. Regulation of junction proteins to improve barrier function of airway epithelial cells has the potential for alleviation of allergic airway inflammation.ObjectiveWe sought to determine the immunomodulatory effect of knob protein of the adenoviral capsid on allergic asthma and to investigate its mechanism of action on airway epithelial junction proteins and barrier function.MethodsAirway inflammation, including junction protein expression, was evaluated in allergen-challenged mice with and without treatment with knob. Human bronchial epithelial cells were exposed to knob, and its effects on expression of junction proteins and barrier integrity were determined.ResultsAdministration of knob to allergen-challenged mice suppressed airway inflammation (eosinophilia, airway hyperresponsiveness, and IL-5 levels) and prevented allergen-induced loss of airway epithelial occludin and E-cadherin expression. Additionally, knob decreased expression of TH2-promoting inflammatory mediators, specifically IL-33, by murine lung epithelial cells. At a cellular level, treatment of human bronchial epithelial cells with knob activated c-Jun N-terminal kinase, increased expression of occludin and E-cadherin, and enhanced epithelial barrier integrity.ConclusionIncreased expression of junction proteins mediated by knob leading to enhanced epithelial barrier function might mitigate the allergen-induced airway inflammatory response, including asthma.

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 [CFTR.sub.inh]-172 or short hairpin RNA silencing of CPTR 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 [CFTR.sub.inh]-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 CFFR 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. wound healing; cell migration; lamellipodia doi: 10.1152/ajpcell.00215.2010.

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 [P2Y.sub.1], [P2Y.sub.2], [P2Y.sub.4], and [P2Y.sub.6] receptors. RT-PCR experiments also revealed expression of [A.sub.2b] adenosine receptor mRNA in primary and immortalized cells. Confluent monolayers of HME cells exhibited a basal short-circuit current ([I.sub.sc]) that was abolished by amiloride and benzamil. When monolayers were cultured in the presence of hydrocortisone, mRNA expression of [Na.sup.+] channel (ENaC) [alpha]-, [beta]-, and [gamma]-subunits increased approximately threefold compared with that in cells grown without hydrocortisone. In addition, basal benzamil-sensitive [Na.sup.+] transport was nearly twofold greater in hydrocortisone-treated monolayers. Stimulation with UTP, UDP, or adenosine 5'-O-(3-thiotriphosphate) (ATP-[gamma]S) 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 > ATP[gamma]S. Basolateral stimulation with UTP produced a rapid but transient increase in [I.sub.sc] 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 [I.sub.sc] and eliminated the sustained current response. These results indicate that human mammary epithelial cells express multiple P2 receptor subtypes and that [Ca.sup.2+] mobilization evoked by P2Y receptor agonists stimulates [Na.sup.+] absorption by increasing the activity of [Ca.sup.2+]-activated [K.sup.+] channels located in the basolateral membrane. adenosine receptors; epithelial sodium channels; KCNN4; SK4; potassium channels

Human lung epithelial (Calu-3) cells were used to investigate the effects of protease-activated receptor (PAR) stimulation on [Cl.sup.-] 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 ([I.sub.sc]: thrombin, 21 [+ or -] 2 [micro]A; SLIGRL, 83 [+ or -] 22 [micro]A), which returned to baseline within 5 min after stimulation. Pretreatment of monolayers with the cell-permeant [Ca.sup.2+]-chelating agent BAPTA-AM (50 [micro]M) abolished the increase in [I.sub.sc]. produced by SLIGRL. When monolayers were treated with the cyclooxygenase inhibitor indomethacin (10 [micro]M), nearly complete inhibition of both the thrombin- and SLIGRL-stimulated [I.sub.sc] was observed. In addition, basolateral treatment with the PG[E.sub.2] receptor antagonist AH-6809 (25 [micro]M) significantly inhibited the effects of SLIGRL on [I.sub.sc]. QRT-PCR revealed that Calu-3 cells express mRNAs for CFTR, the [Ca.sup.2+]-activated KCNN4 [K.sup.+] channel, and the KCNQ1 [K.sup.+] channel subunit, which, in association with KCNE3, is known to be regulated by cAMP. Stimulation with SLIGRL produced an increase in apical [Cl.sup.-] conductance that was blocked in cells expressing short hairpin RNAs designed to target CFTR. These results support the conclusion that PAR stimulation of [Cl.sup.-] secretion occurs by an indirect mechanism involving the synthesis and release of prostaglandins. In addition, PAR-stimulated [Cl.sup.-] secretion requires activation of CFTR and at least two distinct [K.sup.+] channels located in the basolateral membrane. cystic fibrosis transmembrane conductance regulator; KCNQ1; calcium-activated potassium channels; KCNN4; cAMP

Kv4/K channel-interacting protein (KChIP) potassium channels are a major class of rapidly inactivating K channels in brain and heart. Considering the importance of alternative splicing to the quantitative features of KChIP gating modulation, a previously uncharacterized splice form of KChIP1 was functionally characterized. The KChIP1b splice variant differs from the previously characterized KChIP1a splice form by the inclusion of a novel amino-terminal region that is encoded by an alternative exon that is conserved in mouse, rat, and human genes. The expression of KChIP1b mRNA was high in brain but undetectable in heart or liver by RT-PCR. In cerebellar tissue, KChIP1b and KChIP1a transcripts were expressed at nearly equal levels. Coexpression of KChIP1b or KChIP1a with Kv4.2 channels in oocytes slowed K current decay and destabilized open-inactivated channel gating. Like other KChIP subunits, KChIP1b increased Kv4.2 current amplitude and KChIP1b also shifted Kv4.2 conductance-voltage curves by -10 mV. The development of Kv4.2 channel inactivation accessed from closed gating states was faster with KChIP1b coexpression. Deletion of the novel amino-terminal region in KChIP1b selectively altered the subunit's modulation of Kv4.2 closed inactivation gating. The role of the KChIP1b N[H.sub.2]-terminal region was further confirmed by direct comparison of the properties of the N[H.sub.2]-terminal deletion mutant and the KChIP1a subunit, which is encoded by a transcript that lacks the novel exon. The features of KChIP1b modulation of Kv4 channels are likely to be conserved in mammals and demonstrate a role for the KChIP1 N[H.sub.2]-terminal region in the regulation of closed inactivation gating. inactivation; gating; mutagenesis; K channel-interacting protein; exon

Whole cell perforated patchclamp experiments were performed with adult rat alveolar epithelial cells. The holding potential was -60 mV, and depolarizing voltage steps activated voltage-gated [K.sup.+] (Ky) channels. The voltage-activated currents exhibited a mean reversal potential of -32 reV. Complete activation was achieved at -10 mV. The currents exhibited slow inactivation, with significant variability in the time course between cells. Tail current analysis revealed cell-to-cell variability in [K.sup.+] selectivity, suggesting contributions of multiple Kv [alpha]-subunits to the whole cell current. The Kv channels also displayed steady-state inactivation when the membrane potential was held at depolarized voltages with a window current between -30 and 5 mV. Analysis of RNA isolated from these cells by RT-PCR revealed the presence of eight Kv [alpha]-subunits (Kv1.1, Kv1.3, Kv 1.4, Kv2.2, Kv4.1, Kv4.2, Kv4.3, and Kv9.3), three [beta]-subunits (Kv[beta]l.1, Kv[beta]2.1, and Kv[beta]3.1), and two [K.sup.+] channel interacting protein (KChIP) isoforms (KChIP2 and KChIP3). Western blot analysis with available Kv [alpha]-subunit antibodies (Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3) showed labeling of 50-kDa proteins from alveolar epithelial cells grown in monolayer culture. Immunocytochemical analysis of cells from monolayers showed that Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3 were localized to the apical membrane. We conclude that expression of multiple Kv [alpha]-, [beta]-, and KChIP subunits explains the variability in inactivation gating and [K.sup.+] selectivity observed between cells and that Kv channels in the apical membrane may contribute to basal [K.sup.+] secretion across the alveolar epithelium. voltage-gated potassium channels; potassium ion secretion; oxygen-sensitive potassium channels; alveolar fluid clearance

Electrolyte transport across the adult alveolar epithelium plays an important role in maintaining a thin fluid layer along the apical surface of the alveolus that facilitates gas exchange across the epithelium. Most of the work published on the transport properties of alveolar epithelial cells has focused on the mechanisms and regulation of [Na.sup.+] transport and, in particular, the role of amiloride-sensitive Na+ channels in the apical membrane and the [Na.sup.+]-[K.sup.+]-ATPase located in the basolateral membrane. Less is known about the identity and role of [Cl.sup.-] and [K.sup.+] channels in alveolar epithelial cells, but studies are revealing important functions for these channels in regulation of alveolar fluid volume and ionic composition. The purpose of this review is to examine previous work published on [Cl.sup.-] and [K.sup.+] channels in alveolar epithelial cells and to discuss the conclusions and speculations regarding their role in alveolar cell transport function. alveolar fluid clearance; [Cl.sup.-] absorption; [K.sup.+] secretion; epithelial ion transport

The objective of this study was to investigate the mechanism of uridine 5'-triphosphate (UTP)-dependent inhibition of [Na.sup.+] absorption in porcine endometrial epithelial cells. Acute stimulation with UTP (5 [micro]M) produced inhibition of sodium absorption and stimulation of chloride secretion. Experiments using basolateral membrane-permeabilized cell monolayers demonstrated a reduction in benzamil-sensitive [Na.sup.+] conductance in the apical membrane after UTP stimulation. The UTP-dependent inhibition of sodium transport could be mimicked by PMA (1 [micro]M). Several PKC inhibitors, including GF109203X and Go6983 (both nonselective PKC inhibitors) and rottlerin (a PKC[delta] selective inhibitor), were shown to prevent the UTP-dependent decrease in benzamil-sensitive current. The PKC[alpha]-selective inhibitors, Go6976 and PKC inhibitor 20-28, produced a partial inhibition of the UTP effect on benzamil-sensitive Isc. Inhibition of the benzamil-sensitive Isc by UTP was observed in the presence of BAPTA-AM (50 [micro]M), confirming that activation of PKCs, and not increases in [[[Ca.sup.2+]].sub.i], were directly responsible for the inhibition of apical [Na.sup.+] channels and transepithelial [Na.sup.+] absorption. KEY WORDS: P2Y receptor * ENaC * benzamil * chloride secretion * calcium-activated chloride channel

The objective of this study was to investigate the effects of insulin and insulin-like growth factor I on transepithelial Na(super +) transport across porcine glandular endometrial epithelial cells grown in primary culture. Insulin and insulin-like growth factor I acutely stimulated Na(super +) transport two- to threefold by increasing Na(super +)-K(super +) ATPase transport activity and basolateral membrane K(super +) conductance without increasing the apical membrane amiloride-sensitive Na(super +) conductance. Long-term exposure to insulin for 4 d resulted in enhanced Na(super +) absorption with a further increase in Na(super +)-K(super +) ATPase transport activity and an increase in apical membrane amiloride-sensitive Na(super +) conductance. The effect of insulin on the Na(super +)-K(super +) ATPase was the result of an increase in V(sub max) for extracellular K(super +) and intracellular Na(super +), and an increase in affinity of the pump for Na(super +). Immunohistochemical localization along with Western blot analysis of cultured porcine endometrial epithelial cells revealed the presence of (alpha)-1 and (alpha)-2 isoforms, but not the (alpha)-3 isoform of Na(super +)-K(super +) ATPase, which did not change in the presence of insulin. Insulin-stimulated Na(super +) transport was inhibited by hydroxy-2-naphthalenylmethylphosphonic acid tris-acetoxymethyl ester [HNMPA-(AM)(sub 3)], a specific inhibitor of insulin receptor tyrosine kinase activity, suggesting that the regulation of Na(super +) transport by insulin involves receptor autophosphorylation. Pretreatment with wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase as well as okadaic acid and calyculin A, inhibitors of protein phosphatase activity, also blocked the insulin-stimulated increase in short circuit and pump currents, suggesting that activation of phosphatidylinositol 3-kinase and subsequent stimulation of a protein phosphatase mediates the action of insulin on Na(super +)-K(super +) ATPase activation. Keywords: insulin-like growth factor I; epithelial ion transport; membrane transport; ouabain; amiloride

Electrolyte transport research shows that gastrin-releasing peptide (GRP) acts as an autocrine and paracrine regulatory peptide in the endometrium. GRP and substance P stimulate the release of potassium via calcium-dependent potassium conduction in the surface endometrial epithelial cells' apical membrane. GRP receptor antibody labelling in epithelial and stromal cells was identified during the regulation of electrolyte transport.

The biochemical role of Na-K-adenosine triphosphatase (ATPase) and Na+ pump expression were analyzed in alveolar type II (ATII) cells isolated from the lungs of adult rats after acute exposure to oxygen. Chronic hyperoxic injury in rat ATII cells did not alter normal Na-K-ATPase ion transport. However, hyperoxia-induced acute lung injury decreased maximal enzyme activity and levels of alpha1-subunit antigenic protein.

The gastrin-releasing peptide (GRP) was applied in combination with prostaglandin E2 (PGE2) to investigate whether PGE2 contributes to the regulation of colonic ion transport. Electrical and transepithelial flux measurements revealed an increase in short-circuit current. This increase was greater than that induced by either GRP or PGE2 treatment alone. There was also increased chlorine (Cl) secretion, although PGE2 does not activate the Cl pathway. It was therefore suggested that GRP acts as modulator of PG action on colonic ion transport.

Pig tissues derived from the distal colon were incubated in medium containing the gastrin-releasing peptide (GRP) to investigate the regulation of electrolyte transport in the colon. A transient increase in short-circuit current, followed by a sustained decrease, was observed. Co-treatment with bombesin enhanced the decrease of short-circuit current. Transepithelial flux measurements indicated that GRP increased colonic ion transport by stimulating sodium and potassium secretion across the epithelium.

The effects of prostaglandins on electrolyte transport in endometrial epithelial cells isolated from porcine uterine tissues were investigated. Addition of amiloride to the procine uterine tissues inhibited basal short-circuit current. Benzamil, amiloride and 5-(N-methyl-N-isobutyl)-amiloride blocked basal short-circuit current in that order of potency. In contrast, addition of prostaglandin F2alpha or 8-(chlorophenylthio)-adenosine 3',5'-cyclic monophosphate stimulated an increase in basal short-circuit current.

A study of neuroregulation of electrolyte transport in the porcine distal colon using electrical transmural stimulation (ETS) shows that cholinergic submucosal neurons regulate epithelial ion transport in the porcine distal colon and neuropeptide Y(NPY) inhibits neural activity of leukotriene-sensitive neurons in the colon. ETS of the colonic mucosa submucosa incubated on Ussing chamber enhances short-circuit current indicating that part of response is mediated by Cl secretion.

Use of Ussing chambers to examine Ca2+ and guanosine 3',5'-cyclic monophosphate (cGMP) modulation of Cl, Na and K transport in isolated porcine distal colon epithelium shows that cGMP activates a bumetanide-sensitive transport pathway to enhance its release while rises in intracellular Ca2+ stimulates multiple anion secretory pathways. HCO3 and bumetanide replacement affect ionomycin-induced Cl- ion release while amiloride has no effect on ionomycin-induced Na absorption. cGMP suppresses the amiloride-inhibited Na+ absorption pathway.

The investigation of the mechanisms of K secretion in the porcine gallbladder epithelium from the neck and the fundus of the organ shows that K is secreted under basal conditions by the porcine gallbladder. The gallbladder was placed in Ussing chambers for investigation after its serosal muscle had been removed. The elevated intracellular calcium concentration, which prohibits the basolateral K effuse, stimulates the K secretion by an additional TEA-insensitive sepical K permeability.

Regional transport characteristics of porcine gallbladder epithelium bathed in Ringer solution in Ussing chamber were studied. Norepinephrine, salbutamol, isoproterenol and dobutamine were administered and their effects on the Na and Cl transport activity observed. Results of the studies confirm regional differences in basal Cl and Na transport. The results also verified alpha2- and beta2-adrenoceptor mediation across the isolated gallbladder epithelium were also noted