Polarized signaling via purinoceptors in normal and cystic fibrosis airway epithelia.

Airway epithelia are confronted with distinct signals emanating from the luminal and/or serosal environments. This study tested whether airway epithelia exhibit polarized intracellular free calcium (Ca(2+)(i)) and anion secretory responses to 5' triphosphate nucleotides (ATP/UTP), which may be released across both barriers of these epithelia. In both normal and cystic fibrosis (CF) airway epithelia, mucosal exposure to ATP/UTP increased Ca(2+)(i) and anion secretion, but both responses were greater in magnitude for CF epithelia. In CF epithelia, the mucosal nucleotide-induced response was mediated exclusively via Ca(2+)(i) interacting with a Ca(2+)-activated Cl(-) channel (CaCC). In normal airway epithelia (but not CF), nucleotides stimulated a component of anion secretion via a chelerythrine-sensitive, Ca(2+)-independent PKC activation of cystic fibrosis transmembrane conductance regulator. In normal and CF airway epithelia, serosally applied ATP or UTP were equally effective in mobilizing Ca(2+)(i). However, serosally applied nucleotides failed to induce anion transport in CF epithelia, whereas a PKC-regulated anion secretory response was detected in normal airway epithelia. We conclude that (1) in normal nasal epithelium, apical/basolateral purinergic receptor activation by ATP/UTP regulates separate Ca(2+)-sensitive and Ca(2+)-insensitive (PKC-mediated) anion conductances; (2) in CF airway epithelia, the mucosal ATP/UTP-dependent anion secretory response is mediated exclusively via Ca(2+)(i); and (3) Ca(2+)(i) regulation of the Ca(2+)-sensitive anion conductance (via CaCC) is compartmentalized in both CF and normal airway epithelia, with basolaterally released Ca(2+)(i) failing to activate CaCC in both epithelia.

Heterogeneous responses of cell Ca2+ in human airway epithelium.

The Ca(2+)-mobilizing actions of adenosine 5'-triphosphate (ATP), bradykinin, and histamine were compared in phenotypically distinct human nasal epithelial (HNE) cell types and as a function of time in cell culture. Single-cell measurements of intracellular free Ca2+ (Ca2+i, Fura-2 fluorescence) were recorded in ciliated cells 1-2 days in primary culture, and in nonciliated cells 1-2 days (keratin 14-positive) or 4-5 days (keratin 18-positive) after seeding. No difference in basal Ca2+i was noted between ciliated and nonciliated cell preparations. For ciliated and nonciliated cells studied 1-2 days in culture, ATP, bradykinin, and histamine elicited a cytosolic Ca2+ response in 100% of the cells examined. For nonciliated HNE cells maintained 4-5 days in culture, ATP (10(-4) M) increased cytosolic Ca2+ in all cells tested, but only 85% of the cells responded to bradykinin (10(-5) M) addition, and 65% to histamine (10(-4) M) stimulation. In terms of the absolute change of Ca2+i (delta Ca2+i, peak-basal value), the efficacy was ATP > bradykinin > histamine for the 3 HNE cell preparations. However, the delta Ca2+i in response to agonists was smaller in nonciliated HNE cells studied 1-2 days or 4-5 days in culture as compared to the ciliated cell preparation. Thapsigargin (300 nM), an agent that mobilizes Ca2+i, was equally effective in raising cytosolic Ca2+ in nonciliated (1-2 days and 4-5 days in culture) and ciliated HNE cells. These data show that ciliated cells consistently respond to all agonists, whereas the cytosolic Ca2+ response to ATP, bradykinin, and histamine in nonciliated cells was quantitatively reduced at a comparable time period (1-2 days) and became smaller and less frequent in nonciliated cell preparations maintained 4-5 days in culture. These results demonstrate time-dependent differences in the magnitude and frequency of cytosolic Ca2+ responses to certain agonists, strongly indicating that measurements of Ca2+i in HNE cells must account for the heterogeneity of the cell types and the time cells are maintained in primary culture.

ATP-activated basolateral Na+/H+ exchange in human normal and cystic fibrosis airway epithelium.

This study investigated the distribution (apical/basolateral membrane) of Na+/H+ exchange in human nasal epithelial cell monolayers from cystic fibrosis (CF) and non-CF individuals. Monolayers of non-CF and CF preparations were loaded with the pH probe 2',7'-bis(carboxyethyl)-5(6)- carboxyfluorescein, and intracellular pH (pH1) was measured with a microfluorimeter. In nominally HCO3(-)-free NaCl Ringer solution, basal pHi values in non-CF and CF monolayers were essentially identical (approximately 7.1). When cells were acid loaded (NH4+ prepulse), subsequent alkalinization of non-CF and CF cell monolayers required serosal Na+ and was blocked by serosal, but not mucosal, amiloride (500 microM). In the presence of extracellular Na+, initial rates (delta pHi/min) of recovery from an acid load in non-CF and CF preparations were statistically identical (approximately 0.08). Pretreatment of monolayers with ATP (100 microM) or phorbol 12-myristate 13-acetate (PMA; 100 nM) caused initial rates of recovery to increase by twofold over control values in both preparations. These data provide evidence in support for an ATP- and PMA-activated Na+/H+ exchanger located at the basolateral surface of airway epithelia.

UDP activates a mucosal-restricted receptor on human nasal epithelial cells that is distinct from the P2Y2 receptor.

The presence of the P2Y2 (P(2U)-purinergic) receptor on the apical surface of airway tissue raises the possibility that aerosolized UTP might be used therapeutically to induce Cl- secretion in individuals with cystic fibrosis. However, the duration of the effects of UTP may be limited by enzymatic degradation. We therefore have analyzed the metabolism of UTP and its metabolite UDP on polarized human nasal epithelium (HNE), and have compared the pharmacological activities of these two uridine nucleotides. HPLC analysis of medium bathing the mucosal surface of HNE cells revealed the presence of an ecto-nucleotidase(s) that hydrolyzed [3H]UTP and [3H]UDP with t1/2 values (at 1 microM nucleotide) of 14 and 27 min, respectively. An ecto-nucleoside diphosphokinase activity also was observed, which promoted conversion of [3H]UDP into [3H]UTP in the presence of ATP. The effects of UDP on [3H]inositol phosphate accumulation, intracellular calcium levels ([Ca2+]i), and Cl- secretory rates (I(Cl-)) were quantitated in HNE cells in the presence of hexokinase and glucose to ensure that no UTP (or ATP) contaminated UDP solutions during the assays. Although UDP does not activate the human P2Y2 receptor, mucosal addition of UDP promoted [3H]inositol phosphate accumulation with an EC50 of 190 nM. Mucosal addition of UTP stimulated [3H]inositol phosphate accumulation with an EC50 of 280 nM. The maximal effects of mucosal UDP on [3H]inositol phosphate, [Ca2+]i, and I(Cl-) responses were approximately one-half of those observed with mucosal UTP. Serosal application of UTP promoted a 50% greater [3H]inositol phosphate and calcium response than did mucosal application of UTP. In contrast, UDP had no effect when added to the serosal medium. Repetitive mucosal applications of UDP to HNE cells resulted in a progressive loss, i.e., desensitization, of the [Ca2+]i and I(Cl-) response to UDP, whereas the corresponding responses to UTP remained unchanged. Our results provide evidence for the existence of a UDP receptor on HNE cells that is pharmacologically distinct from the P2Y2 receptor. The relative stability of UDP on the airway surface and the apparent predominant mucosal expression of this putative UDP receptor make it a potential target for cystic fibrosis treatment.

Membrane-restricted regulation of Ca2+ release and influx in polarized epithelia.

Epithelial cells exist in a complex setting in which responses to mucosal or serosal environments are mediated by receptors expressed on specialized cellular domains, such as apical versus basolateral cell membranes. We investigated whether airway epithelia can react selectively through G-protein-coupled receptors to stimuli in the mucosal or serosal environments by measuring inositol phosphate and intracellular Ca2+ responses in polarized human nasal epithelial monolayers. We report here that unilateral ATP (10(-4) M) administration stimulated P2 purinoceptors and tapped pools of intracellular Ca2+ associated with the plasma membrane ipsilateral but not contralateral to stimulated receptors. Similarly, activation of plasma membrane Ca2+ influx by ATP was confined to the membrane ipsilateral to receptor stimulation. These findings demonstrate that polarized epithelia restrict P2 receptor-mediated responses to a single domain of the cell, reflecting membrane-specific generation and catabolism of inositol phosphates and confinement of calcium influx regulation to the membrane ipsilateral to the stimulated receptors.

Cl- secretion by trachea of CFTR (+/-) and (-/-) fetal mouse.

The absence of pathologic changes in newborn cystic fibrosis (CF) lung suggests that the fetal CF lung is inflated with a normal volume of liquid and that Cl- is secreted through paths other than the cystic fibrosis transmembrane conductance regulator (CFTR)-associated Cl- channel. We studied liquid content of distal lung and transepithelial electrical potential difference (PD) of cultured cystic tracheal explants from 16 to 19 day gestation fetal mice of CFTR (+/-)(heterozygous) females that were mated with CFTR (-/-) "knockout" males. Distal lung water content was not affected by fetal genotype. Basal PDs were not different (CFTR (+/-), 8.6 mV, and CFTR (-/-), 9.1 mV), and PDs of both groups were inhibited by intraluminal injection of amiloride (10(-4) M) (-25%) and after addition of bumetanide (10(-4) M) to the bath (-40%). Terbutaline (3 x 10(-5) M) induced a similar increase in PD (about 65%) in both groups. Intraluminal injection of ionomycin (2 x 10(-5) and 5 x 10(-6) M) raised PD in both groups (CFTR (+/-) by 32 and 27% and CFTR (-/-) by 41 and 11%). All of the increase in PD induced by terbutaline and ionomycin was inhibited by bumetanide. The PD response to terbutaline was not attenuated by pretreatment with ionomycin or the Ca2+ chelator BAPTA (10(-4) M). Ionomycin or ATP, but not terbutaline, increased intracellular Ca2+ concentration of isolated cultured tracheal epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Purinergic regulation of ion transport across nonciliated bronchiolar epithelial (Clara) cells.

Previous studies demonstrated that elevation of intracellular calcium concentration ([Ca2+]i) increased electrogenic anion transport by bronchiolar epithelia. Extracellular nucleotides were shown to elevate [Ca2+]i and transepithelial short-circuit current (Isc) in proximal airways epithelia. In this study purine and pyrimidine nucleotides were investigated for their ability to regulate ion transport by rabbit nonciliated bronchiolar epithelial (Clara) cells in culture. ATP in the apical bath induced a concentration-dependent transient increase in [Ca2+]i and Isc. Mean effective doses (ED50) of the responses were 10(-7) M and 10(-6) M, respectively. Transepithelial resistance (Rt) decreased. The peak changes in Isc and Rt were 7.8 +/- 1.2 microA/cm2 and -59 +/- 14 omega.cm2 (n = 26, basal Isc = 47.4 +/- 4.3 microA/cm2 and Rt = 428 +/- 40 omega.cm2). Some preparations exhibited a small residual increase in Isc after the initial response, but the change was not statistically significant (delta Isc = 1.7 +/- 1.2 microA/cm2, n = 18). Addition of ATP to the basolateral bath had no detectable effects. Purinoceptor agonists were used to characterize the receptors mediating the change in Isc. UTP and ATP gamma S increased Isc and inhibited subsequent stimulation by ATP. ADP, ADP beta S, 2-methylthio-ATP, and alpha, beta-methylene-ATP had negligible effects on the peak delta Isc and subsequent stimulation by ATP. The ionic mechanism underlying the ATP-induced increase in Isc was investigated with the use of specific ion-transport inhibitors and by ion substitution.(ABSTRACT TRUNCATED AT 250 WORDS)

Alteration of sulfation of glycoconjugates, but not sulfate transport and intracellular inorganic sulfate content in cystic fibrosis airway epithelial cells.

The secreted and cell surface high molecular weight glyco-conjugates (HMG) generated by primary cultures of airway epithelial cells from cystic fibrosis (CF) patients are oversulfated. To determine whether this abnormality is maintained in transformed CF airway epithelial cells and whether differences in transport or intracellular accumulation of sulfate can explain this alteration, we assessed sulfate metabolism in paired CF and normal cell lines as well as primary cultures of CF and normal cells. Both 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid-inhibitable and -resistant [35S]sulfate efflux and influx were identical for each pair of CF and normal cell lines. Furthermore, cell content of inorganic sulfate was not significantly different in CF and normal cells. However, compared with primary CF cells that oversulfate HMG transformed CF cells oversulfated cell surface HMG but not HMG released into culture medium. Our results suggest that plasma membrane sulfate transport is not altered in CF airway epithelial cells and the abnormal sulfation of HMG may be due to perturbation in intracellular sulfate activation or transfer of activated sulfate to HMG. The relationship of this abnormality to CF transmembrane conductance regulator mutations remains to be determined.

Role of CNP in human airways: cGMP-mediated stimulation of ciliary beat frequency.

Ciliated airway epithelial cells contribute to mucociliary transport systems via ciliary beating and electrolyte transport mechanisms. Both of these activities are regulated by agonists acting through intracellular calcium- and adenosine 3',5'-cyclic monophosphate (cAMP)-dependent processes (5, 15, 18, 27). This study examines the role of guanosine 3',5'-cyclic monophosphate (cGMP) in the regulation of both ciliary beat frequency (CBF) and electrolyte transport in human airway epithelia (HAE). In a previous report, cGMP production in HAE was observed after stimulation with either C-type natriuretic peptide (CNP) or sodium nitroprusside (SNP) (6). In this study, CNP was found to increase CBF by 30 +/- 6.9%, and this effect was mimicked by the cGMP analogue, 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), but not by sodium nitroprusside. CNP-induced increases in CBF do not appear to be mediated by changes in either intracellular calcium or cAMP levels. Using modified Ussing chambers, we also investigated CNP's potential modulation of sodium and chloride transport rates. Neither CNP, nor SNP, nor 8-BrcGMP altered active ion transport rates. We conclude that CNP regulates ciliary beat via cGMP-dependent mechanisms, whereas no effect of CNP or cGMP on ion transport was detected.

In vivo nasal potential difference: techniques and protocols for assessing efficacy of gene transfer in cystic fibrosis.

Cystic fibrosis (CF) is a monogenetic disease that is associated with chronic airways disease and early death. The pulmonary disease reflects mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and associated abnormal epithelial ion transport, including defective cAMP-mediated (CFTR) Cl- secretion and an accelerated rate of basal Na+ transport. With the development of vectors for gene therapy, the airway epithelium of CF patients has been targeted for studies of gene transfer. The biological efficacy of gene transfer of the normal CFTR cDNA into CF respiratory epithelia can be assessed by in vivo measurements of the transepithelial potential difference (PD), a parameter of ion transport that reflects the expression and function of CFTR. This paper describes techniques that can be used to discriminate in vivo between the ion transport phenotype of normal subjects and patients with cystic fibrosis. Protocols are outlined to allow assessment of individual components of the electrolyte transport phenotype, i.e., the magnitude of the basal and cAMP-mediated (CFTR) Cl- secretion, and the rate of Na+ transport. The physiologic basis of the protocols and important technical features of these measurements are defined. If performed properly, the in vivo nasal PD technique clearly discriminates between normal subjects and cystic fibrosis patients, and can yield estimates of the biological efficacy of gene transfer to achieve correction of the electrolyte transport defects in CF patients.

Activation of CFTR Cl- conductance in polarized T84 cells by luminal extracellular ATP.

Luminal extracellular ATP evoked a bumetanide-sensitive short-circuit current in cultured T84 cell epithelia (90.2 +/- 18.2 microA/cm2 at 100 microM ATP, apparent 50% effective concentration, 11.5 microM). ATP appeared to increase the Cl- conductance of the apical membrane but not the driving force for Cl- secretion determined by basolateral membrane K+ conductance. Specifically, the magnitude of Cl- secretion stimulated by ATP was independent of basal current, and forskolin pretreatment abolished subsequent stimulation of Cl- secretion by ATP. Whereas ATP stimulated modest production of adenosine 3',5'-cyclic monophosphate (cAMP) by T84 cells, ATP caused smaller increases in intracellular Ca2+ and inositol phosphate activities than the Ca(2+)-signaling Cl- secretagogue carbachol. An inhibitor of 5'-nucleotidase, alpha,beta-methyleneadenosine 5'-diphosphate, blocked most of the response to luminal ATP. The adenosine receptor antagonist 8-(p-sulfophenyl)theophylline blocked both the luminal ATP-dependent generation of cAMP and Cl- secretion when administered to the luminal but not submucosal bath. These results demonstrate that the Cl- secretion stimulated by luminal ATP is mediated by a A2-adenosine receptor located on the apical cell membrane. Thus metabolism of extracellular ATP to adenosine regulates the activity of cystic fibrosis transmembrane conductor regulator (CFTR) in the apical membrane of polarized T84 cells.

Multiple modes of regulation of airway epithelial chloride secretion by extracellular ATP.

Cultured normal and cystic fibrosis (CF) airway epithelia were exposed to 5'-(N-ethylcarboxamido)-adenosine (NECA), ATP, or ionomycin. NECA activated a sustained, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-insensitive Cl- secretory response in normal but not CF, consistent with stimulation of the CF transmembrane conductance regulator (CFTR). In normal and CF, ionomycin or ATP induced Cl- secretion with an initial peak that was inhibited > 50% by DIDS, but in normals there was a prolonged current that was not inhibited by DIDS. The ATP and ionomycin responses in CF were of greater magnitude, and the prolonged phase was inhibited by DIDS. Although we expected ATP to regulate Cl- conductance through intracellular Ca2+ activity, ATP further stimulated Cl- secretion in tissues pretreated to maximally elevate intracellular Ca2+ activity. ATP also activated whole cell Cl- currents in cells dialyzed with 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Thus ATP and ionomycin regulate a Cl- conductance that is distinct from CFTR, but the regulation by ATP is not tightly coupled to intracellular Ca2+ activity. Alternatively, ATP regulates separate Ca(2+)-sensitive and Ca(2+)-insensitive Cl- conductances. Furthermore, extracellular ATP activates DIDS-resistant Cl- secretion in normal but not CF cultured epithelia, consistent with activation of CFTR by extracellular ATP.

Anomalies in ion transport in CF mouse tracheal epithelium.

The cystic fibrosis (CF) mouse trachea has become a model for gene transfer. To characterize ion transport properties of tracheal epithelium from normal and CF mice, tracheas were excised, mounted in Ussing chambers, and basal properties and responses to pharmacological agents and/or ion substitution protocols measured. No difference in basal short-circuit (Isc) was observed between normal (29.1 +/- 3.8 muA/cm2, n = 21) and CF (34.7 +/- 4.5 muA/cm2, n = 16) tracheas. The relative contribution of Na+ transport to basal Isc was small (30-40%). Ionomycin stimulated large increases in Isc in both normal and CF murine tracheas [change in Isc (delta Isc) with ionomycin: 30.5 +/- 8.8 muA/cm2, n = 11, normal; 27.3 +/- 6.7 muA/cm2, n = 6, CF]. Unexpectedly, forskolin increased Isc in both CF and normal amiloride-pretreated tracheas (delta Isc: 10.5 +/- 2.1 muA/cm2, n = 21, normal; 13 +/- 2.3 muA/cm2, n = 16, CF). Forskolin was observed to increase intracellular Ca2+ in both normal and CF tracheal cells, suggesting this as a mechanism to induce Cl- secretion. These similarities in ion transport, in part reflecting the dominance of Ca(2+)-regulated Cl- conductance, suggest that the murine trachea is not an ideal target for assessment of CF correction by gene transfer.

L-glutamine and L-asparagine stimulate Na+ -H+ exchange in porcine jejunal enterocytes.

L-Glutamine (Gln) is a major respiratory fuel and substrate for nucleic acid synthesis in mammalian intestinal cells. The structurally related amino acid, L-asparagine (Asn), stimulates the proliferative enzyme ornithine decarboxylase in colonocytes, an effect that is blocked by the Na+-H+ exchange inhibitor amiloride. In an epithelial cell line derived from newborn piglet jejunum (IPEC-J2 cells), we determined intracellular pH (pHi) by computer-assisted microfluorimetry in single cells loaded with pH-sensitive dye 2',7'-bis(2-carboxyethyl)5-(6)- carboxyfluorescein. Resting pHi in N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid-buffered NaCl Ringer was 7.06 +/- 0.02. Removal of external Na+ caused reversible acidification; recovery of pHi from NH+4-induced acid load was Na+ dependent, amiloride inhibitable, and Cl-independent. Asn and Gln had no measurable effect on resting pHi, but pretreatment with Asn or Gln induced a consistent twofold increase in pHi recovery from an acid challenge that was not seen with L-proline, D-glutamine, or L-phenylalanine. Inhibition of Gln metabolism by aminooxyacetate abolished the stimulatory effect of Gln on the exchanger. The tumor promotor phorbol 12-myristate 13-acetate (PMA) stimulated recovery rate from acid load and also increased resting pHi. The effects of PMA and Gln on Na+-H+ exchange from acid load were additive. Stimulation of Na+-H+ exchange by PMA, but not by Gln, was inhibited by protein kinase C (PKC) inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpeperazine. We conclude that Gln metabolism stimulates Na+-H+ exchange of acid-loaded porcine enterocytes by a mechanism not requiring activation of PKC.

Cloning and expression of a human P2U nucleotide receptor, a target for cystic fibrosis pharmacotherapy.

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.

Sulfate concentrations and transport in human bronchial epithelial cells.

Inorganic sulfate concentrations in the cytoplasm of human bronchial epithelial cells exceeded levels in the bathing medium under all circumstances tested. Cell sulfate concentrations were directly related to medium sulfate concentrations and inversely related to medium chloride concentrations. In physiological media there was a sulfate compartment of approximately 0.3 mM that exchanged very slowly with extracellular sulfate. In media lacking chloride, sulfate was accumulated by the cells to a level as high as 2 mM. Sulfate uptake was markedly inhibited by external chloride and by stilbene sulfonic acid derivatives but was not affected by sodium in the medium. Efflux of 35SO4(2-) was stimulated by both chloride and sulfate in the bathing medium but inhibited by stilbenes. The following compounds had no effect on sulfate movements: phorbol esters, adenosine 3',5'-cyclic monophosphate derivatives, and okadaic acid. Changes in medium tonicity were likewise without effect. Our results suggest that human bronchial epithelial cells maintain a steady-state disequilibrium for inorganic sulfate. Furthermore, sulfate appears to exist in at least two compartments in the cells: one that is slowly exchangeable with sulfate in the medium and another exchangeable compartment that is of negligible size in physiological media but that becomes very large in media lacking chloride. Sulfate is transported by an anion exchanger of broad specificity that is not influenced by substances known to modulate chloride channels.

Papilloma virus immortalized tracheal epithelial cells retain a well-differentiated phenotype.

Human airway epithelial cell lines that retain phenotypic properties representative of the native tissue will be useful physiological models. Human papilloma viral (HPV) genes can immortalize human genital keratinocytes and breast and bronchial epithelia. We transfected cystic fibrosis (CF) and normal tracheobronchial epithelial cell cultures with DNA encoding the HPV-18 E6 and E7 genes and characterized phenotypic properties of resultant cell lines. Of the 11 CF clones isolated, 6 developed a polarized phenotype with vectorial ion transport and membrane-specific expression of histamine and purinergic receptors. The ion transport properties of these lines differed from the normal lines and approximated those of primary CF airway epithelial cell cultures more closely than do those of cell lines transformed with the simian virus 40 large T gene. When transplanted into denuded tracheal grafts, these cells can differentiate into ciliated and secretory phenotypes. We conclude that HPV-18 E6 and E7 genes are sufficient to transform human airway epithelial cells and that the resultant cell lines express differentiated phenotypic properties that approximate those of the native epithelium.

Histamine-induced Cl- secretion in human nasal epithelium: responses of apical and basolateral membranes.

The mechanism by which receptors coupled to phospholipase C (PLC) induce Cl- secretion in amiloride-pretreated cultures of human nasal epithelial (HNE) cultures was investigated. Histamine (10(-4) M, basolateral administration) stimulated a rapid increase in equivalent short-circuit current, an index of Cl- secretion, that returned to baseline within 5 min. Intracellular recordings with double-barreled Cl(-)-selective microelectrodes showed that the apical and basolateral membrane potentials rapidly hyperpolarized, the fractional resistance of the apical membrane increased, and the transepithelial resistance decreased in response to histamine. Intracellular Cl- activity remained constant. Equivalent circuit analysis revealed that the early portion (< 0.9 min) of the Cl- secretory response was driven by an activation of a hyperpolarizing basolateral conductance, likely K+, whereas the later (> 0.9 min) phase of Cl- secretion reflects activation of the apical membrane Cl- conductance. Histamine raised intracellular Ca2+ (Ca2+i) measured by fura-2 in HNE with a potency similar to that observed for induction of Cl- secretion. Both intracellular release and plasma membrane influx pathways were identified, typical of receptor-mediated activation of PLC. The intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (15 microM), coupled with reduced bathing solution Ca2+, blunted the rise in Ca2+i and the net transepithelial Cl- secretory response to histamine. We conclude that 1) histamine induced Cl- secretion in HNE by a sequential mechanism: the rapid initial component reflects activation of the basolateral K+ conductance, and the later component reflects activation of an apical Cl- conductance; and 2) the level of Ca2+i may participate in the activation of both the basolateral and apical conductances.

Intracellular Ca2+ and regulation of ion transport across rabbit Clara cells.

We investigated whether Ca2+ was involved in regulation of ion transport across rabbit distal airway epithelial cells by studying the effects that elevation of intracellular Ca2+ (Cai) had on the bioelectric properties of nonciliated bronchiolar (Clara) cell epithelia in culture. Exposure of Clara cells to 5 x 10(-7) M ionomycin increased Cai concentration and transepithelial short-circuit current (Isc). Changing extracellular Ca2+ concentration in the presence of ionomycin demonstrated that changes in Isc paralleled changes in Cai. Another ionophore, 4-bromo-A23187, also increased Cai and Isc. Ionomycin-induced changes in Isc were insensitive to amiloride and were inhibited greater than 50% by pretreating the cells with bumetanide or substituting gluconate for Cl- in the bathing solution. Bradykinin and carbachol, which increased Cai and caused an increase in Isc across tracheal cell cultures, had no effect on Cai or Isc in Clara cell preparations. These results support the hypothesis that changes in Cai are linked to regulation of Cl- secretion across bronchiolar epithelial cells, but physiological regulators of Cai in Clara cells remain to be defined.