Pharmacology of INS37217 [P(1)-(uridine 5')-P(4)- (2'-deoxycytidine 5')tetraphosphate, tetrasodium salt], a next-generation P2Y(2) receptor agonist for the treatment of cystic fibrosis.

INS37217 [P(1)-(uridine 5')-P(4)-(2'-deoxycytidine 5')tetraphosphate, tetrasodium salt] is a deoxycytidine-uridine dinucleotide with agonist activity at the P2Y(2) receptor. In primate lung tissues, the P2Y(2) receptor mRNA was located by in situ hybridization predominantly in epithelial cells and not in smooth muscle or stromal tissue. The pharmacologic profile of INS37217 parallels that of UTP, leading to increased chloride and water secretion, increased cilia beat frequency, and increased mucin release. The combined effect of these actions was confirmed in an animal model of tracheal mucus velocity that showed that a single administration of INS37217 significantly enhanced mucus transport for at least 8 h after dosing. This extended duration of action is consistent with the ability of INS37217 to resist metabolism by airway cells and sputum enzymes. The enhanced metabolic stability and resultant increased duration of improved mucociliary clearance may confer significant advantages to INS37217 over other P2Y(2) agonists in the treatment of diseases such as cystic fibrosis.

Compartmentalized autocrine signaling to cystic fibrosis transmembrane conductance regulator at the apical membrane of airway epithelial cells.

Physical stimulation of airway surfaces evokes liquid secretion, but the events that mediate this vital protective function are not understood. When cystic fibrosis transmembrane conductance regulator (CFTR) channel activity was used as a functional readout, we found signaling elements compartmentalized at both extracellular and intracellular surfaces of the apical cell membrane that activate apical Cl(-) conductance in Calu-3 cells. At the outer surface, ATP was released by physical stimuli, locally converted to adenosine, and sensed by A(2B) adenosine receptors. These receptors couple to G proteins, adenylyl cyclase, and protein kinase A, at the intracellular face of the apical membrane to activate colocalized CFTR. Thus, airways have evolved highly efficient mechanisms to "flush" noxious stimuli from airway surfaces by selective activation of apical membrane signal transduction and effector systems.

SRC family kinases mediate epithelial Na+ channel inhibition by endothelin.

The epithelial Na(+) channel (ENaC) is implicated in the pathogenesis of salt-sensitive hypertension. Recent evidence from animal models suggests that the vasoactive peptide, endothelin (ET-1), may be an important negative regulator of ENaC in vivo. We investigated the signaling pathway involved in endothelin-mediated ENaC inhibition. Experiments were performed in NIH 3T3 cells stably expressing genes for the three (alpha, beta, and gamma) ENaC subunits. In whole cell patch clamp experiments, we found that ET-1 treatment induced a dose-dependent decrease in amiloride-sensitive currents. Using receptor-specific antagonists, we determined that the effects of ET-1 were attributed to activation of the ET(B) receptor. Moreover, the inhibitory effect of ET-1 on ENaC could be completely blocked when cells were pretreated with the selective Src family kinase inhibitor, PP2. Further studies revealed that basal Src family kinase activity strongly regulates ENaC whole cell currents and single channel gating. These results suggest that Src family kinases lie in a signaling pathway activated by ET-1 and are components of a novel negative regulatory cascade resulting in ENaC inhibition.

PKA holoenzyme is functionally coupled to CFTR by AKAPs.

Cystic fibrosis transmembrane regulator (CFTR) is reported to be preferentially regulated by membrane-bound protein kinase A (PKAII). We tested for close physical and functional association of PKA with CFTR in inside-out membrane patches excised from Calu-3 cells. In the presence of MgATP, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) increased the product of CFTR channel number and open probability (from 0.36 +/- 0.12 to 1.23 +/- 0.57, n = 20, P < 0.0025), and this stimulation was abolished by PKI. Thus Calu-3 membrane isolated from cells retains PKA holoenzyme that is functionally coupled to CFTR. PKAII is anchored at specific subcellular sites by A kinase anchoring proteins (AKAPs). Exposure of excised patches to HT-31, a peptide that disrupts the association of PKAII and AKAPs, prevented CPT-cAMP stimulation of CFTR. Therefore, PKA holoenzyme in isolated membrane patches is bound to AKAPs. In whole cell voltage-clamp studies, intracellular dialysis of Calu-3 cells with HT-31 blocked the activation of CFTR by extracellular adenosine. These results suggest that AKAPs mediate PKA compartmentalization with CFTR and are required for activation of CFTR by physiological regulators.

Basal nucleotide levels, release, and metabolism in normal and cystic fibrosis airways.

BACKGROUND: Cystic fibrosis (CF) is a syndrome caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. Despite advances in our understanding of the molecular pathogenesis of CF, the link between CFTR gene mutations and the pathogenesis of CF lung disease remains poorly defined. CFTR has been assigned a number of putative functions that may contribute to innate airway defense, including the regulation of adenosine 5'-triphosphate (ATP) release into the extracellular environment. Because extracellular ATP and uridine 5'-triphosphate (UTP) may regulate airway mucociliary clearance via interaction with luminal P2Y2 receptors, the loss of CFTR-mediated nucleotide release could explain the defect in CF airway defense. MATERIALS AND METHODS: We tested the physiologic importance of CFTR-mediated nucleotide release in vivo by directly measuring levels of ATP and UTP in nasal airway surface liquid from normal and CF subjects. Because these basal nucleotide levels reflect the net activities of nucleotide release and metabolic pathways, we also measured constitutive rates of nucleotide release and metabolism on well-differentiated normal and CF airway cultures in vitro. The measurement of ATP release rates were paralleled by in vivo studies employing continuous nasal perfusion in normal and CF subjects. Finally, the regulation of ATP release by isoproterenol and methacholine-stimulated submucosal gland secretion was tested. RESULTS: These studies revealed that steady-state ATP and UTP levels were similar in normal (470 +/- 131 nM and 37 +/- 7 nM, respectively) and CF (911 +/- 199 nM and 33 +/- 12 nM, respectively) subjects. The rates of both ATP release and metabolism were also similar in normal and CF airway epithelia both in vitro and in vivo. Airway submucosal glands did not secrete nucleotides, but rather, secreted a soluble nucleotidase in response to cholinergic stimuli. CONCLUSION: The concentration of ATP in airway surface liquid is in a range that is relevant for the activation of airway nucleotide receptors. However, despite this finding that suggests endogenous nucleotides may be important for the regulation of mucociliary clearance, our data do not support a role for CFTR in regulating extracellular nucleotide concentrations on airway surfaces.

Alternative splicing regulates the subcellular localization of A-kinase anchoring protein 18 isoforms.

The cAMP-dependent protein kinase (PKA) is localized to specific subcellular compartments by association with A-kinase anchoring proteins (AKAPs). AKAPs are a family of functionally related proteins that bind the regulatory (R) subunit of PKA with high affinity and target the kinase to specific subcellular organelles. Recently, AKAP18, a low molecular weight plasma membrane AKAP that facilitates PKA-mediated phosphorylation of the L-type Ca(2+) channel, was cloned. We now report the cloning of two additional isoforms of AKAP18, which we have designated AKAP18beta and AKAP18gamma, that arise from alternative mRNA splicing. The AKAP18 isoforms share a common R subunit binding site, but have distinct targeting domains. The original AKAP18 (renamed AKAP18alpha) and AKAP18beta target the plasma membrane when expressed in HEK-293 cells, while AKAP18gamma is cytosolic. When expressed in epithelial cells, AKAP18alpha is targeted to lateral membranes, whereas AKAP18beta is accumulated at the apical membrane. A 23-amino acid insert, following the plasma membrane targeting domain, facilitates the association of AKAP18beta with the apical membrane. The data suggest that AKAP18 isoforms are differentially targeted to modulate distinct intracellular signaling events. Furthermore, the data suggest that plasma membrane AKAPs may be targeted to subdomains of the cell surface, adding additional specificity in intracellular signaling.

Yes-associated protein 65 localizes p62(c-Yes) to the apical compartment of airway epithelia by association with EBP50.

We recently showed that the COOH terminus of the cystic fibrosis transmembrane conductance regulator associates with the submembranous scaffolding protein EBP50 (ERM-binding phosphoprotein 50 kD; also called Na(+)/H(+) exchanger regulatory factor). Since EBP50 associates with ezrin, this interaction links the cystic fibrosis transmembrane conductance regulator (CFTR) to the cortical actin cytoskeleton. EBP50 has two PDZ domains, and CFTR binds with high affinity to the first PDZ domain. Here, we report that Yes-associated protein 65 (YAP65) binds with high affinity to the second EBP50 PDZ domain. YAP65 is concentrated at the apical membrane in airway epithelia and interacts with EBP50 in cells. The COOH terminus of YAP65 is necessary and sufficient to mediate association with EBP50. The EBP50-YAP65 interaction is involved in the compartmentalization of YAP65 at the apical membrane since mutant YAP65 proteins lacking the EBP50 interaction motif are mislocalized when expressed in airway epithelial cells. In addition, we show that the nonreceptor tyrosine kinase c-Yes is contained within EBP50 protein complexes by association with YAP65. Subapical EBP50 protein complexes, containing the nonreceptor tyrosine kinase c-Yes, may regulate apical signal transduction pathways leading to changes in ion transport, cytoskeletal organization, or gene expression in epithelial cells.

CFTR is a conductance regulator as well as a chloride channel.

CFTR Is a Conductance Regulator as well as a Chloride Channel. Physiol. Rev. 79, Suppl.: S145-S166, 1999. - Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter gene family. Although CFTR has the structure of a transporter that transports substrates across the membrane in a nonconductive manner, CFTR also has the intrinsic ability to conduct Cl- at much higher rates, a function unique to CFTR among this family of ABC transporters. Because Cl- transport was shown to be lost in cystic fibrosis (CF) epithelia long before the cloning of the CF gene and CFTR, CFTR Cl- channel function was considered to be paramount. Another equally valid perspective of CFTR, however, derives from its membership in a family of transporters that transports a multitude of different substances from chemotherapeutic drugs, to amino acids, to glutathione conjugates, to small peptides in a nonconductive manner. Moreover, at least two members of this ABC transporter family (mdr-1, SUR) can regulate other ion channels in the membrane. More simply, ABC transporters can regulate somehow the function of other cellular proteins or cellular functions. This review focuses on a plethora of studies showing that CFTR also regulates other ion channel proteins. It is the hope of the authors that the reader will take with him or her the message that CFTR is a conductance regulator as well as a Cl- channel.

An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton.

The function of the cystic fibrosis transmembrane conductance regulator (CFTR) as a Cl- channel in the apical membrane of epithelial cells is extensively documented. However, less is known about the molecular determinants of CFTR residence in the apical membrane, basal regulation of its Cl- channel activity, and its reported effects on the function of other transporters. These aspects of CFTR function likely require specific interactions between CFTR and unknown proteins in the apical compartment of epithelial cells. Here we report that CFTR interacts with the recently discovered protein, EBP50 (ERM-binding phosphoprotein 50). EBP50 is concentrated at the apical membrane in human airway epithelial cells, in vivo, and CFTR and EBP50 associate in in vitro binding assays. The CFTR-EBP50 interaction requires the COOH-terminal DTRL sequence of CFTR and utilizes either PDZ1 or PDZ2 of EBP50, although binding to PDZ1 is of greater affinity. Through formation of a complex, the interaction between CFTR and EBP50 may influence the stability and/or regulation of CFTR Cl- channel function in the cell membrane and provides a potential mechanism through which CFTR can affect the activity of other apical membrane proteins.

Effects of topically delivered benzamil and amiloride on nasal potential difference in cystic fibrosis.

The raised nasal transepithelial potential difference (PD) in cystic fibrosis (CF) reflects accelerated active transport of Na+, and is inhibited by topical administration of the Na+ channel blocker, amiloride. The aim of this study was to investigate the dose-effect and time course of topically administered Na+ conductance inhibitors to inhibit nasal PD, including benzamil, an analog of amiloride. We measured the magnitude of drug inhibition of Na+ transport [percent inhibition of baseline PD (DeltaPD%)] and duration of inhibition of PD, defined as the time when drug inhibition of PD had recovered by 50% (effective time = ET50). Amiloride [10(-)3 M (n = 16), 3 x 10(-)3 M (n = 9), 6 x 10(-)3 M (n = 7), 10(-)2 M (n = 3)] or benzamil [1.7 x 10(-)3 M (n = 7), and 7 x 10(-)3 M (n = 5)] were administered to the nasal surface via an aerosol generated by a jet nebulizer and a nasal mask. The concentration-dependent magnitude (DeltaPD%) of inhibition was similar for amiloride and benzamil ( approximately 67- 77%), whereas the duration of inhibition (ET50) was about two-and-a-half times longer after benzamil administration as compared with equivalent concentrations of amiloride [1.6 +/- 0. 06 versus 4.5 +/- 0.6 h (ET50 +/- SEM), at 6-7 x 10(-)3 M]. In vitro studies of cultured normal nasal epithelia demonstrated directly that benzamil induced an approximately 2-fold more prolonged inhibition of active Na+ transport than amiloride. These data suggest aerosolized benzamil is a candidate long-duration Na+ channel blocker for CF.

Cystic fibrosis transmembrane conductance regulator inverts protein kinase A-mediated regulation of epithelial sodium channel single channel kinetics.

Abnormal regulation of ion channels by members of the ABC transport protein superfamily has been implicated in hyperinsulinemic hypoglycemia and in excessive Na+ absorption by airway epithelia in cystic fibrosis (CF). How ABC proteins regulate ion conductances is unknown, but must generally involve either the number or activity of specific ion channels. Here we report that the cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in CF, reverses the regulation of the activity of single epithelial sodium channels (ENaC) by cAMP. ENaC expressed alone in fibroblasts responded to activation of cAMP-dependent protein kinase with increased open probability (Po) and mean open time, whereas ENaC co-expressed with CFTR exhibited decreased Po and mean open time under conditions optimal for PKA-mediated protein phosphorylation. Thus, CFTR regulates ENaC at the level of single channel gating, by switching the response of single channel Po to cAMP from an increase to a decrease.

CFTR-like chloride channels in non-ciliated bronchiolar epithelial (Clara) cells.

Distal airways are believed to be a major site of disease in cystic fibrosis. The product of the CF gene, CFTR, is expressed in non-ciliated bronchiolar epithelial (Clara) cells. Clara cells in primary culture are capable of Cl- secretion which can be stimulated by cAMP and extracellular nucleotides. We used the patch clamp technique to look for Cl- channels in the apical membrane of rabbit Clara cells. In cell-attached patches, we recorded Cl- channels with a conductance for outward currents of 7.5 +/- 0.4 pS (n = 10) and for inward currents of 3.2 +/- 0.5 pS (n = 10); these channels typically exhibited slow kinetics and were not inhibited by the Cl- channel blockers NPPB and DIDS. Channel activity was not noticeably dependent on pipette potential. Addition of chlorophenylthio-cyclic AMP (cpt-cAMP) to the bath increased the percentage of cell-attached patches with active channels (33.8% vs 56.7%; p< 0.05) and the channel open probability (0.49 +/- 0.03 vs 0.84 +/- 0.02; p< 0.05). Extracellular ATP increased the percentage of cell-attached patches with active channels (28.7% to 50.0%; p< 0.05) but had no significant effect on the channel open probability (0.62 +/- 0.07 vs 0.60 +/- 0.06). In conclusion, rabbit non-ciliated bronchiolar epithelial cells express low-conductance Cl- channels that share many similarities with the CFTR-related Cl- channel and are regulated by cAMP and extracellular ATP.

Clusters of Cl- channels in CFTR-expressing Sf9 cells switch spontaneously between slow and fast gating modes.

The Sf9 insect Spodoptora frugiperda cell line was used for heterologous expression of the cloned human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, or the cloned beta-galactosidase gene, using the baculovirus Autographa califonica as the infection vector. Using application of the patch-clamp technique, evidence for functional expression of CFTR was obtained according to the following three criteria. Firstly, whole-cell currents recorded 2 days after infection with CFTR revealed a statistically significant increase of membrane conductance, approximately 25 times above that of mock-infected control cells, with the reversal potential of the major current component being governed by the chloride equilibrium potential (ECl). Secondly, in contrast to uninfected cells and cells infected with beta-galactosidase, the membrane conductance to chloride of CFTR-injected cells was stimulated by cytosolic adenosine 3',5'-cyclic monophosphate (cAMP), which was raised by exposing the cells to 10 microM forskolin. Thirdly, recordings of currents through single channels in excised outside-out membrane patches of CFTR-infected cells revealed channels which were clearly different from the native insect chloride channel. Excised outside-out patches of CFTR-infected and forskolin-stimulated cells exhibited wave-like gating kinetics of well-resolved current transitions. All-point Gaussian distributions revealed contributions from several (five to nine) identical channels. Such channels, in excised outside-out patches, studied with a pipette [Cl-] = 40 mM and a bath [Cl-] = 150 mM, rectified the current in agreement with simple electrodiffusion and with a single-channel Goldman-Hodgkin-Katz permeability, PCl = 1. 34 x 10(-14) +/- 0.23 x 10(-14 )cm3/s (n = 5), corresponding to a physiological single-channel conductance of 2.8 +/- 0.5 pS (VM = ECl) and a limiting conductance, gamma150/150, = 7.7 +/- 1.3 pS ([Cl-]Bath = [Cl-]Cell = 150 mM). Currents recorded from multichannel excised outside-out patches could shift from the above mode of resolvable unitary conductance transitions to one which was too fast to reveal the dwell-times of closed and open states. During periods characterized by noisy currents, the variance (sigma2) of current fluctuations about their stationary mean value depicted a U-shaped function of membrane potential, with a minimum value at a pipette potential where the chloride current was shown to be zero. Thus, it can be concluded that the current fluctuations are caused by fast gating of channels specific for chloride ions. Switching back and forth between the two gating modes of clusters of chloride channels occurred from moment to moment in excised patches when the membrane potential was held at a constant value indicating cooperative gating as a result of interaction between neighbouring chloride channels.

Endogenous chloride channels of insect sf9 cells. Evidence for coordinated activity of small elementary channel units.

The endogenous Cl- conductance of Spodoptera frugiperda (Sf9) cells was studied 20-35 h after plating out of either uninfected cells or cells infected by a baculovirus vector carrying the cloned beta-galactosidase gene (beta-Gal cells). With the cation Tris+ in the pipette and Na+ in the bath, the reversal potential of whole-cell currents was governed by the prevailing Cl- equilibrium potential and could be fitted by the Goldman-Hodgkin-Katz equation with similar permeabilities for uninfected and beta-Gal cells. In the frequency range 0.12 < f < 300 Hz, the power density spectrum of whole-cell Cl- currents could be fitted by three Lorentzians. Independent of membrane potential, >50% of the total variance of whole-cell current fluctuations was accounted for by the low frequency Lorentzian (fc = 0.40 +/- 0.03 Hz, n = 6). Single-Cl- channels showed complex gating kinetics with long lasting (seconds) openings interrupted by similar long closures. In the open state, channels exhibited fast burst-like closures. Since the patches normally contained more than a single channel, it was not possible to measure open and closed dwell-time distributions for comparing single-Cl- channel activity with the kinetic features of whole-cell currents. However, the power density spectrum of Cl- currents of cell-attached and excised outside-out patches contained both high and low frequency Lorentzian components, with the corner frequency of the slow component (fc = 0.40 +/- 0.02 Hz, n = 4) similar to that of whole-cell current fluctuations. Chloride channels exhibited multiple conductance states with similar Goldman-Hodgkin-Katz-type rectification. Single-channel permeabilities covered the range from approximately 0.6.10(-14) cm5/s to approximately 6.10(-14) cm3/s, corresponding to a limiting conductance (gamma 150/150) of approximately 3.5 pS and approximately 35 pS, respectively. All states reversed near the same membrane potential, and they exhibited similar halide ion selectivity, P1 > PCl approximately PBr. Accordingly, Cl- current amplitudes larger than current flow through the smallest channel unit resolved seem to result from simultaneous open/shut events of two or more channel units.

Enzymatic synthesis of UTP gamma S, a potent hydrolysis resistant agonist of P2U-purinoceptors.

1. The defective Cl- secretion characteristic of cystic fibrosis airway epithelial cells can be bypassed by an alternative Ca2+ dependent Cl- secretory pathway that is activated by extracellular nucleotides, e.g. uridine-5'triphosphate (UTP), acting on P2U purinoceptors. Since UTP is susceptible to hydrolysis by nucleotidases and phosphatases present in the airways, the identification of stable P2U-purinoceptor agonists would be of therapeutic relevance. 2. Uridine-5'-O-(3-thiotriphosphate) (UTP gamma S) was synthesized by nucleoside diphosphate kinase-catalyzed transfer of the gamma-phosphorothioate from guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) or adenosine-5' = O-(3-thiotriphosphate) (ATP gamma S) to UDP. Formation of UTP gamma S was illustrated by observation of transfer of 35S from [35S]-GTP gamma S and transfer of 3H from [3H]-UDP. The chemical identity of high performance liquid chromatography (h.p.l.c.)-purified UTP gamma S was confirmed by nuclear magnetic resonance analysis. 3. Human 1321N1 astrocytoma cells stably expressing the phospholipase C-coupled human P2U-purinoceptor were utilized to test the activity of UTP gamma S. UTP gamma S (EC50 = 240 nM) was essentially equipotent to UTP and ATP for stimulation of inositol phosphate formation. 4. Unlike [3H]-UTP, [3H]-UTP gamma S was not hydrolyzed by alkaline phosphatase, acid phosphatase, or apyrase. Moreover, no hydrolysis was detected during a 1 h incubation with human nasal epithelial cells. 5. UTP gamma S was equally potent and efficacious with UTP for stimulation of Cl- secretion by human nasal epithelium from both normal donors and cystic fibrosis patients. Based on its high potency and resistance to hydrolysis, UTP gamma S represents a promising compound for treatment of cystic fibrosis.

Pharmacological selectivity of the cloned human P2U-purinoceptor: potent activation by diadenosine tetraphosphate.

1. The human P2U-purinoceptor was stably expressed in 1321N1 human astrocytoma cells and the pharmacological selectivity of the expressed receptor was studied by measurement of inositol lipid hydrolysis. 2. High basal levels of inositol phosphates occurred in P2U-purinoceptor-expressing cells. This phenomenon was shown to be due to release of large amounts of ATP from 1321N1 cells, and could be circumvented by adoption of an assay protocol that did not involve medium changes. 3. UTP, ATP and ATP gamma S were full and potent agonists for activation of phospholipase C with EC50 values of 140 nM, 230 nM, and 1.72 microM, respectively. 5BrUTP, 2C1ATP and 8BrATP were also full agonists although less potent than their natural congeners. Little or no effect was observed with the selective P2Y-, P2X-, and P2T-purinoceptor agonists, 2MeSATP, alpha,beta-MeATP, and 2MeSADP, respectively. 4. Diadenosine tetraphosphate, Ap4A, was a surprisingly potent agonist at the expressed P2U-purinoceptor with an EC50 (720 nM) in the range of the most potent P2U-purinoceptor agonists. Ap4A may be a physiologically important activator of P2U-purinoceptors.

Overexpression of the cystic fibrosis transmembrane conductance regulator in NIH 3T3 cells lowers membrane potential and intracellular pH and confers a multidrug resistance phenotype.

Because of the similarities between the cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance (MDR) proteins, recent observations of decreased plasma membrane electrical potential (delta psi) in cells overexpressing either MDR protein or the CFTR, and the effects of delta psi on passive diffusion of chemotherapeutic drugs, we have analyzed chemotherapeutic drug resistance for NIH 3T3 cells overexpressing different levels of functional CFTR. Three separate clones not previously exposed to chemotherapeutic drugs exhibit resistance to doxorubicin, vincristine, and colchicine that is similar to MDR transfectants not previously exposed to chemotherapeutic drugs. Two other clones expressing lower levels of CFTR are less resistant. As shown previously these clones exhibit decreased plasma membrane delta psi similar to MDR transfectants, but four of five exhibit mildly acidified intracellular pH in contrast to MDR transfectants, which are in general alkaline. Thus the MDR protein and CFTR-mediated MDR phenotypes are distinctly different. Selection of two separate CFTR clones on either doxorubicin or vincristine substantially increases the observed MDR and leads to increased CFTR (but not measurable MDR or MRP) mRNA expression. CFTR overexpressors also exhibit a decreased rate of 3H -vinblastine uptake. These data reveal a new and previously unrecognized consequence of CFTR expression, and are consistent with the hypothesis that membrane depolarization is an important determinant of tumor cell MDR.

CFTR as a cAMP-dependent regulator of sodium channels.

Cystic fibrosis transmembrane regulator (CFTR), the gene product that is mutated in cystic fibrosis (CF) patients, has a well-recognized function as a cyclic adenosine 3',5'-monophosphate (cAMP)-regulated chloride channel, but this property does not account for the abnormally high basal rate and cAMP sensitivity of sodium ion absorption in CF airway epithelia. Expression of complementary DNAs for rat epithelial Na+ channel (rENaC) alone in Madin Darby canine kidney (MDCK) epithelial cells generated large amiloride-sensitive sodium currents that were stimulated by cAMP, whereas coexpression of human CFTR with rENaC generated smaller basal sodium currents that were inhibited by cAMP. Parallel studies that measured regulation of sodium permeability in fibroblasts showed similar results. In CF airway epithelia, the absence of this second function of CFTR as a cAMP-dependent regulator likely accounts for abnormal sodium transport.

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.