Apical Cystic Fibrosis Transmembrane Regulator Research Articles

Cellular mechanism underlying leptin-induced anion secretion of rat epididymal epithelial cells.

A large number of studies have shown that leptin plays an important role in the regulation of fertility via the hypothalamus-pituitary-gonad axis. However, its peripheral function in epididymis was still elusive. The purpose of this study was to determine the pro-secretion effect of leptin on the rat epididymal epithelium. In the present study, real-time quantitative polymerase chain reaction, western blot, and immunohistochemical analysis were employed to detect the expression pattern of leptin receptors in rat epididymis. The pro-secretion effect of leptin on epididymal epithelial cells was measured by short-circuit current, and the prostaglandin E2 and cyclic adenosine monophosphate level was evaluated by enzyme-linked immunosorbent assay. We verified that the leptin receptor was located on the epididymal epithelium, with a relatively high expression level in corpus and cauda epididymis. Ussing chamber experiments showed that leptin stimulated a significant rise of the short-circuit current in rat epididymal epithelial cells, which could be abolished by the specific leptin receptor antagonist peptide Allo-aca, or by removing the ambient Cl- and HCO3 -. Furthermore, the leptin-stimulated short-circuit current response could be abrogated by blocking the apical cystic fibrosis transmembrane regulator or the basolateral Na+-K+-2Cl- cotransporter. Our pharmacological experiments manifested that interfering with the prostaglandin H synthase-2-prostaglandin E2-EP2/EP4-adenylate cyclase pathways could significantly blunt the cystic fibrosis transmembrane regulator-mediated anion secretion induced by leptin. The enzyme-linked immunosorbent assay demonstrated that leptin could induce a substantial increase in prostaglandin E2 release and cyclic adenosine monophosphate synthesis of primary cultured rat cauda epididymal epithelial cells. Our data also suggested that JAK2, ERK, and PI3K-dependent phosphorylation may be involved in the activation of prostaglandin H synthase-2 and the subsequent prostaglandin E2 production. The present study demonstrated the pro-secretion function of leptin in rat epididymal epithelium via the activation of cystic fibrosis transmembrane regulator and Na+-K+-2Cl- cotransporter, which was dependent on the paracrine/autocrine prostaglandin E2 stimulated EP2/EP4-adenylate cyclase pathways, and thus contributed to the formation of an appropriate microenvironment essential for sperm maturation.

Effects of carbon monoxide on ion transport across rat distal colon

The aim of the present study was to investigate whether carbon monoxide (CO) induces changes in ion transport across the distal colon of rats and to study the mechanisms involved. In Ussing chamber experiments, tricarbonyldichlororuthenium(II) dimer (CORM-2), a CO donor, evoked a concentration-dependent increase in short-circuit current (I(sc)). A maximal response was achieved at a concentration of 2.5·10(-4) mol/l. Repeated application of CORM-2 resulted in a pronounced desensitization of the tissue. Anion substitution experiments suggest that a secretion of Cl(-) and HCO(3)(-) underlie the CORM-2-induced current. Glibenclamide, a blocker of the apical cystic fibrosis transmembrane regulator channel, inhibited the I(sc) induced by the CO donor. Similarly, bumetanide, a blocker of the basolateral Na(+)-K(+)-2Cl(-) cotransporter, combined with 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid sodium salt, an inhibitor of the basolateral Cl(-)/HCO(3)(-) exchanger, inhibited the CORM-2-induced I(sc). Membrane permeabilization experiments indicated an activation of basolateral K(+) and apical Cl(-) channels by CORM-2. A partial inhibition by the neurotoxin, tetrodotoxin, suggests the involvement of secretomotor neurons in this response. In imaging experiments at fura-2-loaded colonic crypts, CORM-2 induced an increase of the cytosolic Ca(2+) concentration. This increase depended on the influx of extracellular Ca(2+), but not on the release of Ca(2+) from intracellular stores. Both enzymes for CO production, heme oxygenase I and II, are expressed in the colon as observed immunohistochemically and by RT-PCR. Consequently, endogenous CO might be a physiological modulator of colonic ion transport.

DETANO and Nitrated Lipids Increase Chloride Secretion across Lung Airway Cells

We investigated the cellular mechanisms by which nitric oxide (NO) increases chloride (Cl-) secretion across lung epithelial cells in vitro and in vivo. Addition of (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate (DETANONOate [DETANO];1-1,000 microM) into apical compartments of Ussing chambers containing Calu-3 cells increased short-circuit currents (I(sc)) from 5.2 +/- 0.8 to 15.0 +/- 2.1 microA/cm(2) (X +/- 1 SE; n = 7; P < 0.001). NO generated from two nitrated lipids (nitrolinoleic and nitrooleic acids; 1-10 microM) also increased I(sc) by about 100%. Similar effects were noted across basolaterally, but not apically, permeabilized Calu-3 cells. None of these NO donors increased I(sc) in Calu-3 cells pretreated with 10 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase). Scavenging of NO either prevented or reversed the increase of I(sc). These data indicate that NO stimulation of soluble guanylyl cyclase was sufficient and necessary for the increase of I(sc) via stimulation of the apical cystic fibrosis transmembrane regulator (CFTR). Both Calu-3 and alveolar type II (ATII) cells contained CFTR, as demonstrated by in vitro phosphorylation of immunoprecipitated CFTR by protein kinase (PK) A. PKGII (but not PKGI) phosphorylated CFTR immuniprecipitated from Calu-3 cells. Corresponding values in ATII cells were below the threshold of detection. Furthermore, DETANO, 8-Br-cGMP, or 8-(4-chlorophenylthio)-cGMP (up to 2 mM each) did not increase Cl- secretion across amiloride-treated ATII cells in vitro. Measurements of nasal potential differences in anesthetized mice showed that perfusion of the nares with DETANO activated glybenclamide-sensitive Cl- secretion. These findings suggest that small concentrations of NO donors may prove beneficial in stimulating Cl- secretion across airway cells without promoting alveolar edema.

Alternate stimulation of apical CFTR by genistein in epithelia.

The cystic fibrosis transmembrane regulator (CFTR) is a Cl- channel regulated by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A. A cAMP-independent activation has been recently shown for the protein tyrosine kinase inhibitor genistein in CFTR-transfected NIH/3T3 fibroblasts. We further studied the role of genistein on Cl- secretion in HT-29/B6 and T84 colonic epithelial cells, which express native CFTR in their apical membranes. Transepithelial Cl- secretion was more effectively stimulated in T84 cells when compared with HT-29/B6 cells by mucosal perfusion with 50 microM genistein. Genistein, like the cAMP agonist forskolin, stimulated CFTR activity in cell-attached patches of single cells with similar slope conductances of 8.5 +/- 0.5 and 9.2 +/- 0.3 pS, respectively. Monolayers in Ussing chambers were basolaterally permeabilized with the pore former alpha-toxin, and gradient-driven Cl- current across the apical membrane (ICl) was measured. ICl was stimulated by serosal (i.e., cytosolic) cAMP (half-maximal stimulatory concentration = 9.8 +/- 1.9 microM). In the presence of cAMP (> 5 microM), subsequent mucosal, but not serosal, addition of genistein further increased Icl by approximately 16%; in the absence of cytosolic cAMP, genistein had no effect on ICl. The inactive analogue daidzein had no effect. When cAMP agonists were removed in the continued presence of genistein, ICl remained elevated in both permeabilized and intact monolayers as well as in cell-attached patches of single cells. In addition, genistein blocked K- currents across the basolateral membrane in apically amphotericin B-permeabilized monolayers (half maximal inhibitory concentration = 44.2 +/- 8.1 microM). Therefore, in intact epithelia, the overall secretory response to genistein is composed of stimulatory effects on the apical CFTR and inhibitory effects on the basolateral K+ conductance. We propose that genistein blocks a phosphatase, which regulates CFTR during cAMP-dependent stimulation.

Mechanism of inhibition of cAMP-dependent epithelial chloride secretion by phorbol esters.

In T84 cells, we investigated how stimulation of protein kinase C leads to an inhibition of cAMP-dependent chloride secretion. Specifically, we tested the hypothesis that the inhibition was caused by loss of the cystic fibrosis transmembrane regulator (CFTR), an apical membrane chloride channel. As described by others (Trapnell, B. C., Zeitlin, P. L., Chu, C.-S., Yoshimura, K., Nakamura, H., Guggino, W. B., Bargon, J., Banks, T. C., Dalemans, W., Pavirani, A., Lecocq, J.-P., and Crystal, R. G. (1991) J. Biol. Chem. 266, 10319-10323), we found that treatment with the phorbol ester, phorbol myristate acetate (PMA), reduced CFTR mRNA levels by approximately 80% with a t 1/2 of approximately 2 h. Chloride secretion, measured as forskolin-induced short circuit current, was also abolished by PMA with a t 1/2 of approximately 2 h. Levels of mature glycosylated CFTR measured by Western blotting also declined to 50 +/- 8% (n = 7) of control after a 12-h PMA treatment. However, a 12-h exposure to PMA did not affect the forskolin-stimulated efflux of 125I into high potassium medium, a measure of apical membrane CFTR activity. We conclude that increased turnover of apical membrane CFTR in PMA-treated cells compensates for the decline in anion channel numbers. By contrast to its lack of effect on 125I effluxes, PMA reduced the cAMP-induced increase in 86Rb efflux, suggesting that it inhibits chloride secretion mainly by an action on basolateral potassium channels.