Anthony M. Paradiso

Abnormal surface liquid pH regulation by cultured cystic fibrosis bronchial epithelium.

Cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-dependent airway epithelial bicarbonate transport is hypothesized to participate in airway surface liquid pH regulation and contribute to lung defense. We measured pH and ionic composition in apical surface liquid (ASL) on polarized normal (NL) and CF primary bronchial epithelial cell cultures under basal conditions, after cAMP stimulation, and after challenge with luminal acid loads. Under basal conditions, CF epithelia acidified ASL more rapidly than NL epithelia. Two ASL pH regulatory paths that contributed to basal pH were identified in the apical membrane of airway epithelia, and their activities were measured. We detected a ouabain-sensitive (nongastric) H+,K+-ATPase that acidified ASL, but its activity was not different in NL and CF cultures. We also detected the following evidence for a CFTR-dependent \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document} secretory pathway that was defective in CF: (i) ASL [\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document}] was higher in NL than CF ASL; (ii) activating CFTR with forskolin/3-isobutyl-1-methylxanthine alkalinized NL ASL but acidified CF ASL; and (iii) NL airway epithelia more rapidly and effectively alkalinized ASL in response to a luminal acid challenge than CF epithelia. We conclude that cultured human CF bronchial epithelial pHASL is abnormally regulated under basal conditions because of absent CFTR-dependent \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document} secretion and that this defect can lead to an impaired capacity to respond to airway conditions associated with acidification of ASL.

Chloride secretory response of cystic fibrosis human airway epithelia. Preservation of calcium but not protein kinase C- and A-dependent mechanisms.

Because the defect in Cl- secretion exhibited by cystic fibrosis (CF) epithelia reflects regulatory rather than conductive abnormalities of an apical membrane Cl- channel, we investigated the role of different regulatory pathways in the activation of Cl- secretion in freshly excised normal and CF nasal epithelia mounted in Ussing chambers. A beta agonist (isoproterenol [ISO]), a Ca2+ ionophore (A23187), and a phorbol ester (PMA) were all effective Cl- secretagogues in normal human nasal epithelia. Agonist addition studies indicated that ISO and PMA but not A23187 may share a common regulatory pathway. In contrast, only A23187 induced Cl- secretion in CF epithelia. Bradykinin raised cytosolic Ca2+ and induced Cl- secretion in both normal and CF tissues, indicating that receptor gated Ca2+ dependent Cl- secretory mechanisms were preserved in CF. The defective Cl- secretory response in CF epithelia to ISO and PMA did not reflect abnormalities in cAMP-dependent (A) and phospholipid Ca2+-dependent (C) kinase activities. We conclude that (a) a Ca2+-sensitive mechanism for regulating Cl- secretion is maintained in CF airway epithelia, and (b) a regulatory pathway shared by two distinct protein kinases is defective in CF, indicating that the CF genetic lesion is not tightly coupled to a single (e.g., cAMP dependent) regulatory mechanism.

Regulation of murine airway surface liquid volume by CFTR and Ca2+-activated Cl- conductances

Two Cl− conductances have been described in the apical membrane of both human and murine proximal airway epithelia that are thought to play predominant roles in airway hydration: (1) CFTR, which is cAMP regulated and (2) the Ca2+-activated Cl− conductance (CaCC) whose molecular identity is uncertain. In addition to second messenger regulation, cross talk between these two channels may also exist and, whereas CFTR is absent or defective in cystic fibrosis (CF) airways, CaCC is preserved, and may even be up-regulated. Increased CaCC activity in CF airways is controversial. Hence, we have investigated the effects of CFTR on CaCC activity and have also assessed the relative contributions of these two conductances to airway surface liquid (ASL) height (volume) in murine tracheal epithelia. We find that CaCC is up-regulated in intact murine CF tracheal epithelia, which leads to an increase in UTP-mediated Cl−/volume secretion. This up-regulation is dependent on cell polarity and is lost in nonpolarized epithelia. We find no role for an increased electrical driving force in CaCC up-regulation but do find an increased Ca2+ signal in response to mucosal nucleotides that may contribute to the increased Cl−/volume secretion seen in intact epithelia. CFTR plays a critical role in maintaining ASL height under basal conditions and accordingly, ASL height is reduced in CF epithelia. In contrast, CaCC does not appear to significantly affect basal ASL height, but does appear to be important in regulating ASL height in response to released agonists (e.g., mucosal nucleotides). We conclude that both CaCC and the Ca2+ signal are increased in CF airway epithelia, and that they contribute to acute but not basal regulation of ASL height.

Cystic fibrosis airway epithelial Ca2+ i signaling: the mechanism for the larger agonist-mediated Ca2+ i signals in human cystic fibrosis airway epithelia.

In cystic fibrosis (CF) airways, abnormal epithelial ion transport likely initiates mucus stasis, resulting in persistent airway infections and chronic inflammation. Mucus clearance is regulated, in part, by activation of apical membrane receptors coupled to intracellular calcium (Ca2+i) mobilization. We have shown that Ca2+i signals resulting from apical purinoceptor (P2Y2-R) activation are increased in CF compared with normal human airway epithelia. The present study addressed the mechanism for the larger apical P2Y2-R-dependent Ca2+i signals in CF human airway epithelia. We show that the increased Ca2+i mobilization in CF was not specific to P2Y2-Rs because it was mimicked by apical bradykinin receptor activation, and it did not result from a greater number of P2Y2-R or a more efficient coupling between P2Y2-Rs and phospholipase C-generated inositol 1,4,5-trisphosphate. Rather, the larger apical P2Y2-R activation-promoted Ca2+i signals in CF epithelia resulted from an increased density and Ca2+ storage capacity of apically confined endoplasmic reticulum (ER) Ca2+ stores. To address whether the ER up-regulation resulted from ER retention of misfolded ΔF508 CFTR or was an acquired response to chronic luminal airway infection/inflammation, three approaches were used. First, ER density was studied in normal and CF sweat duct human epithelia expressing high levels of ΔF508 CFTR, and it was found to be the same in normal and CF epithelia. Second, apical ER density was morphometrically analyzed in airway epithelia from normal subjects, ΔF508 homozygous CF patients, and a disease control, primary ciliary dyskinesia; it was found to be greater in both CF and primary ciliary dyskinesia. Third, apical ER density and P2Y2-R activation-mobilized Ca2+i, which were investigated in airway epithelia in a long term culture in the absence of luminal infection, were similar in normal and CF epithelia. To directly test whether luminal infection/inflammation triggers an up-regulation of the apically confined ER Ca2+ stores, normal airway epithelia were chronically exposed to supernatant from mucopurulent material from CF airways. Supernatant treatment expanded the apically confined ER, resulting in larger apical P2Y2-R activation-dependent Ca2+i responses, which reproduced the increased Ca2+i signals observed in CF epithelia. In conclusion, the mechanism for the larger Ca2+i signals elicited by apical P2Y2-R activation in CF airway epithelia is an expansion of the apical ER Ca2+ stores triggered by chronic luminal airway infection/inflammation. Greater ER-derived Ca2+i signals may provide a compensatory mechanism to restore, at least acutely, mucus clearance in CF airways.

The mitochondrial barriers segregate agonist-induced calcium-dependent functions in human airway epithelia.

In airway epithelia, purinergic receptor (P2Y2-R) stimulation of intracellular calcium (Ca2+ i)–regulated ion transport is restricted to the membrane domain ipsilateral to receptor activation, implying compartmentalization of Ca2+ i signaling. Because mitochondria can spatially restrict cellular Ca2+ i signals, immunocytochemical, electron microscopic, and fluorescent studies of mitochondria localization were performed in human airway epithelia. Although concentrated at the apical domain, mitochondria were found distributed at both the apical and the basolateral poles and in close association with the endoplasmic reticulum. The role of mitochondria in locally restricting P2Y2-R–induced Ca2+ i signals was investigated by measuring changes in mitochondrial Ca2+ (Ca2+ m) in human airway epithelial monolayers. P2Y2-R activation induced Ca2+ m accumulation in mitochondria confined to the domain ipsilateral to P2Y2-R stimulation, which was blocked by mitochondrial uncoupling with 1 μM CCCP and 2.5 μg/ml oligomycin. The role of mitochondria in restricting the cellular cross-talk between basolateral P2Y2-R–dependent Ca2+ i mobilization and apical membrane Ca2+-activated Cl− secretion was investigated in studies simultaneously measuring Ca2+ i and Cl− secretion in cystic fibrosis human airway epithelial monolayers. Activation of basolateral P2Y2-Rs produced similar increases in Ca2+ i in monolayers without and with pretreatment with uncouplers, whereas Ca2+ i-activated Cl− secretion was only efficiently triggered in mitochondria-uncoupled conditions. We conclude that (a) mitochondria function as a Ca2+ i-buffering system in airway epithelia, compartmentalizing Ca2+ i-dependent functions to the membrane ipsilateral to receptor stimulation; and (b) the mitochondria provide structural barriers that protect the airway epithelia against nonspecific activation of Ca2+ i-modulated functions associated with Ca2+ i signals emanating from the apical or the basolateral membrane domains.

Polarized distribution of HCO3− transport in human normal and cystic fibrosis nasal epithelia

The polarized distribution of HCO3− transport was investigated in human nasal epithelial cells from normal and cystic fibrosis (CF) tissues. To test for HCO3− transport via conductive versus electroneutral Cl−/HCO3− exchange (anion exchange, AE) pathways, nasal cells were loaded with the pH probe 2′,7′‐bis(carboxyethyl)‐5(6)‐carboxyfluorescein and mounted in a bilateral perfusion chamber. In normal, but not CF, epithelia, replacing mucosal Cl− with gluconate caused intracellular pH (pHi) to increase, and the initial rates (ΔpH min−1) of this increase were modestly augmented (∼26 %) when normal cells were pretreated with forskolin (10 μm). Recovery from this alkaline shift was dependent on mucosal Cl−, was insensitive to the AE inhibitor 4,4′‐diisothiocyanatodihydrostilbene‐2,2′‐disulfonic acid (H2DIDS; 1.5 mm), but was sensitive to the cystic fibrosis transmembrane conductance regulator (CFTR) channel inhibitor diphenylamine‐2‐carboxylate (DPC; 100 μm). In contrast, removal of serosal Cl− caused pHi to alkalinize in both normal and CF epithelia. Recovery from this alkaline challenge was dependent on serosal Cl− and blocked by H2DIDS. Additional studies showed that serosally applied Ba2+ (5.0 mm) in normal, but not CF, cells induced influx of HCO3− across the apical membrane that was reversibly blocked by mucosal DPC. In a final series of studies, normal and CF cells acutely alkaline loaded by replacing bilateral Krebs bicarbonate Ringer (KBR) with Hepes‐buffered Ringer solution exhibited basolateral, but not apical, recovery from an alkaline challenge that was dependent on Cl−, independent of Na+ and blocked by H2DIDS. We conclude that: (1) normal, but not CF, nasal epithelia have a constitutively active DPC‐sensitive HCO3− influx/efflux pathway across the apical membrane of cells, consistent with the movement of HCO3− via CFTR; and (2) both normal and CF nasal epithelia have Na+‐independent, H2DIDS‐sensitive AE at their basolateral domain.

Erratum: Cloning and expression of a human P(2U) nucleotide receptor, a target for cystic fibrosis pharmacotherapy (Proceedings of the National Academy of Sciences of the United States of America (April 12, 1994) 91:8 (3275-3279))

The Cl- secretory pathway that is defective in cystic fibrosis (CF) can be bypassed by an alternative pathway for Cl- transport that is activated by extracellular nucleotides. Accordingly, the P2 receptor that mediates this effect is a therapeutic target for improving Cl- secretion in CF patients. In this paper, we report the sequence and functional expression of a cDNA cloned from human airway epithelial (CF/T43) cells that encodes a protein with properties of a P2U nucleotide receptor. With a retrovirus system, the human airway clone was stably expressed in 1321N1 astrocytoma cells, a human cell line unresponsive to extracellular nucleotides. Studies of inositol phosphate accumulation and intracellular Ca2+ mobilization induced by extracellular nucleotides in 1321N1 cells expressing the receptor identified this clone as the target receptor in human airway epithelia. In addition, we independently isolated an identical cDNA from human colonic epithelial (HT-29) cells, indicating that this is the same P2U receptor that has been functionally identified in other human tissues. Expression of the human P2U receptor (HP2U) in 1321N1 cells revealed evidence for autocrine ATP release and stimulation of transduced receptors. Thus, HP2U expression in the 1321N1 cell line will be useful for studying autocrine regulatory mechanisms and in screening of potential therapeutic drugs.