beta 3-Adrenoceptor Control the Cystic Fibrosis Transmembrane Conductance Regulator through a cAMP/Protein Kinase A-independent Pathway*

Véronique LeblaisDagger , Sophie DemolombeDagger , Geneviève Vallette§, Dominique Langin, Isabelle BaróDagger , Denis EscandeDagger , and Chantal GauthierDagger parallel **

From the Dagger  Laboratoire de Physiopathologie et de Pharmacologie Cellulaires et Moléculaires, INSERM CJF 96-01 and § INSERM CJF 94-04, Hotel-Dieu, 44093 Nantes,  INSERM U317, CHU Rangueil, 31056 Toulouse, and parallel  Faculté des Sciences et Techniques, Université de Nantes, 44322 Nantes, France

    ABSTRACT
Top
Abstract
Introduction
References

In human cardiac myocytes, we have previously identified a functional beta 3-adrenoceptor in which stimulation reduces action potential duration. Surprisingly, in cardiac biopsies obtained from cystic fibrosis patients, beta 3-adrenoceptor agonists produced no effects on action potential duration. This result suggests the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) chloride current in the electrophysiological effects of beta 3-adrenoceptor stimulation in non-cystic fibrosis tissues. We therefore investigated the control of CFTR activity by human beta 3-adrenoceptors in a recombinant system: A549 human cells were intranuclearly injected with plasmids encoding CFTR and beta 3-adrenoceptors. CFTR activity was functionally assayed using the 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescent probe and the patch-clamp technique. Injection of CFTR-cDNA alone led to the expression of a functional CFTR protein activated by cAMP or cGMP. Co-expression of CFTR (but not of mutated Delta F508-CFTR) with high levels of beta 3-adrenoceptor produced an increased halide permeability under base-line conditions that was not further sensitive to cAMP or beta 3-adrenoceptor stimulation. Patch-clamp experiments confirmed that CFTR channels were permanently activated in cells co-expressing CFTR and a high level of beta 3-adrenoceptor. Permanent CFTR activation was not associated with elevated intracellular cAMP or cGMP levels. When the expression level of beta 3-adrenoceptor was lowered, CFTR was not activated under base-line conditions but became sensitive to beta 3-adrenoceptor stimulation (isoproterenol plus nadolol, SR 58611, or CGP 12177). This later effect was not prevented by protein kinase A inhibitors. Our results provide molecular evidence that CFTR but not mutated Delta F508-CFTR is regulated by beta 3-adrenoceptors expression through a protein kinase A-independent pathway.

    INTRODUCTION
Top
Abstract
Introduction
References

beta 3-Adrenoceptors differ from beta 1- and beta 2-adrenoceptor subtypes by their molecular structure and pharmacological profile (for review see Ref. 1). The beta 3-adrenoceptor gene contains two introns (2, 3) leading to alternative splice isoforms, whereas beta 1- and beta 2-adrenoceptor genes are intronless. beta 3-Adrenoceptors are G protein-coupled receptors that interact with either Gs or Gi proteins (4, 5). Depending on the tissue, beta 3-adrenoceptor stimulation produces functional effects that are either comparable with or opposite to those produced by beta 1- and beta 2-adrenoceptor stimulation. For instance, in adipose tissue, beta 3-adrenoceptor stimulation increases lipolysis through an elevation in intracellular cAMP concentration (6, 7) as does beta 1- or beta 2-adrenoceptor stimulation. In the human heart, we have previously demonstrated that beta 3-adrenoceptors mediate negative inotropic effects (5) in stark contrast to the classical positive inotropic effects caused by beta 1- and beta 2-adrenoceptor stimulation. Negative inotropy as produced by beta 3-adrenoceptors stimulation is unlikely to be related to stimulation of the cAMP pathway but rather to stimulation of the cGMP pathway (8) and is associated with an acceleration in the relaxation phase of the twitch and with a shortening of the action potential duration (5).

The present study issued from the observation that in myocardial samples from cystic fibrosis patients, beta 3-adrenoceptor stimulation produced negative inotropic effects but remarkably did not shorten the action potential. Cystic fibrosis is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR)1 gene encoding an ATP-binding cassette protein (9) with anionic channel properties (10) and expressed in the human heart (11). Therefore, the simplest explanation accounting for our observation was that beta 3-adrenoceptors in non-cystic fibrosis tissues control a repolarizing CFTR chloride conductance lacking in cystic fibrosis tissues. We confirmed this hypothesis using various techniques in a heterologous mammalian expression system.

    EXPERIMENTAL PROCEDURES

Action Potential Recordings-- Human ventricular biopsies were obtained from three cystic fibrosis (CF) patients homozygous for the Delta F508 mutation who underwent cardiopulmonary transplantation and from five non-CF patients used as control. Heart and tissues were placed in a transport solution and conveyed rapidly to the laboratory. Preparations were dissected and placed in an experimental chamber. They were superfused at a flow rate of 5 ml/min with oxygenated (95% O2, 5% CO2) Tyrode solution (37 ± 0.5 °C) composed as follows: 120 mM NaCl, 5 mM KCl, 2.7 mM CaCl2, 1.1 mM MgCl2, 0.33 mM NaH2PO4, 5 mM glucose, and 20 mM NaHCO3. Tissues were equilibrated for 60 min and then subjected to field stimulation at a pacing cycle length of 1,700 ms. Action potentials were recorded as described previously (12) using conventional 3 M KCl-filled microelectrodes coupled to an Ag-AgCl electrode connected to an amplifier (Biologic VF 102; Claix, France). The tissue chamber was grounded through an Ag-AgCl electrode. The action potentials were recorded on a digital tape recorder (Biologic DTR-1200) for off-line analysis.

Cell Cultures-- The human lung epithelial-derived cell line A549, the African green monkey kidney-derived cell line COS-7, and the human colonic carcinoma cell line T84 were obtained from the American Type Culture Collection (Manassas, VA). A549 and COS-7 cells were cultured as previously reported (13). T84 cells were grown in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, and antibiotics (100 IU/ml penicillin and 100 µg/ml streptomycin; all from Life Technologies, Inc., Paisley, Scotland), maintained in a humidified incubator (95% air, 5% CO2) at 37 °C and passaged weekly.

Plasmids-- Transgene cDNAs were subcloned into pECE or pcDNA3 mammalian expression vectors under the control of a SV40 enhancer/promoter or a cytomegalovirus promoter, respectively. pECE-CFTR (a kind gift from P. Fanen, INSERM, Créteil, France) and pcDNA3-CFTR (a gift from J. Ricardo, Lisbon, Portugal) plasmids encoded the wild-type CFTR protein. pcDNA3-Delta F508-CFTR plasmid (also from J. Ricardo) encoded the mutated Delta F508-CFTR protein. Alternative splicing of the beta 3-adrenoceptor mRNA could generate three isoforms with different C-terminal regions: (i) the A isoform is the shortest protein; (ii) the B isoform, which corresponds to the A isoform extended by 12 amino acids in the C-terminal region, is the only isoform expressed in rat and is supposedly not expressed in human; and (iii) the C isoform corresponds to the A isoform extended by 6 amino acids in the C-terminal region and is the prevalent isoform in humans (2, 14). As the C-terminal region of the beta 3-adrenoceptor is involved in the coupling with G proteins (1), plasmids encoding either for the A (pcDNA3-beta 3A) and C (pcDNA3-beta 3C) isoforms present in human tissues were generated and expressed in mammalian cells. For control experiments, we used a pECE plasmid lacking the insert and a pcDNA3-ROMK2 plasmid encoding a renal outer medulla potassium channel (a gift from S. C. Hebert, Vanderbilt University, Nashville, TN). For microcytofluorometry and patch-clamp experiments, cells settled on glass coverslips (Nunclon; InterMed Nunc, Roskilde, Denmark) were microinjected with plasmids at day 1 or 2 after plating. Our protocol to intranuclearly microinject individual cells using the Eppendorf ECET microinjector 5246 system and the ECET micromanipulator 5171 system has been reported in detail elsewhere (13). Plasmids were diluted at various final concentrations (0.01-130 µg/ml) in an injection buffer made of 50 mM NaOH, 40 mM NaCl, 50 mM HEPES, pH 7.4, with NaOH, supplemented with 0.5% fluorescein isothiocyanate-dextran to visualize successfully injected cells. The method we used for cell transfection with the 22-kDa polyethylenimine synthetic vector (a gift from J. P. Behr, CNRS, Illkirch, France) has been reported in detail elsewhere (15). Cells were seeded either on glass coverslips for microcytofluorometry experiments or in 6-well plates (Poly Labo, Strasbourg, France) for cAMP and cGMP assays. The area of coverslips and wells was identical. COS-7 cells were transfected at a polyethylenimine/DNA ratio of 2 equivalents with 4 µg of plasmids/well. pcDNA3-CFTR was used at 2 µg/well, pcDNA3-beta 3A at 1 µg/well, and pcDNA3-beta 3C at 1 µg/well. To identify successfully transfected cells, we also added a plasmid encoding a green fluorescent protein (pTR-UF2-green fluorescent protein; a gift from P. Lory, CNRS, Montpellier, France) to obtain a final amount of 4 µg of plasmid/well.

SPQ Fluorescence Assay-- The Cl- channel activity of CFTR was assessed using the halide-sensitive fluorescent probe SPQ (Molecular Probes, Eugene, OR) as described previously (13). 24 h post-injection or transfection, cells placed on glass coverslips were loaded with SPQ using a hypotonic shock procedure. The coverslip was mounted on the stage of an inverted microscope (Diaphot; Nikon, Japan) equipped for fluorescence and illuminated at 360 nm. The emitted light was collected at 456 ± 33 nm by a high resolution image intensifier coupled to a video camera (Extended ISIS camera system; Photonic Science, Robertsbridge, United Kingdom) connected to a digital image processing board controlled by FLUO software (Imstar, France). Cells were maintained at 37 °C and continuously superfused with the extracellular solution containing: 145 mM NaCl, 4 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM HEPES, 5 mM glucose, pH 7.4, with NaOH. A microperfusion system allowed local application and rapid change of the different experimental mediums. I- and NO3- mediums were identical to the extracellular solution except that I- and NO3- replaced Cl- as the dominant extracellular anion. I- and NO3- mediums also contained 10 µM bumetamide (Sigma) to inhibit the Cl-/Na+/K+ co-transporter. To standardize fluorescence intensity, the initial fluorescence level in the presence of I- was taken as zero. The membrane permeability to halides (p) was determined as the rate of SPQ dequenching upon perfusion with NO3- medium (see Fig. 2A).

Current Recordings-- Whole cell currents were recorded with the ruptured patch configuration of the patch-clamp technique as described previously (16). Cells were placed on the stage of an inverted microscope and bathed at 37 °C in the same extracellular solution as used in SPQ experiments. Patch pipettes with a tip resistance of 2.5-5 MOmega were electrically connected to a patch-clamp amplifier (Axopatch 200A; Axon Instruments, Foster City, CA). Stimulation, data recording and analysis were performed by Acquis I software made by Gérard Sadoc (distributed by Biologic) through an analog-to-digital converter (Tecmar TM100 Labmaster; Scientific Solution, Solon, OH). The pipette solution contained 74.5 mM CsCl, 70.5 mM aspartic acid, 5 mM HEPES, 1 mM BAPTA, 5 mM MgATP, pH 7.2, with CsOH. During Cl- current recording, the cell was locally perfused with a Cl- free solution containing 149 mM CsCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM glucose, 5 mM HEPES, pH 7.4, with CsOH. Two stimulation protocols were used: voltage ramps applied at a frequency of 0.2 Hz from -80 to +60 mV (depolarization rate, 46.7 mV/s; holding potential, -60 mV) and voltage steps imposed for 500 ms every 2 s from -60 mV to various potentials between -100 and +60 mV.

cAMP and cGMP Assays-- Determination of intracellular cAMP and cGMP contents was performed 24 h after transfection. COS-7 cells were incubated in the extracellular solution supplemented with 100 µM 3-isobutyl-1-methylxanthine (Sigma) for 1 h at 37 °C. For the cAMP assay, cells were incubated at 37 °C with the same medium containing either 0 or 10 µM forskolin (Sigma) for 5 min. For the cGMP assay, cells were incubated in the presence or absence of 500 µM cGMP analog for 20 min. At the end of the incubation period, cells were lysed by three cycles of freezing and thawing. All samples were subsequently boiled at 90 °C for 5 min and then centrifuged at 12,000 × g for 15 min at 4 °C. The supernatants were assayed for cAMP or cGMP by using a cAMP or cGMP enzyme immunoassay kit (Cayman Chemical Company, Ann Arbor, MI). Data are the means ± S.E. of duplicate assays and normalized to total cell protein content determined by the method of Lowry et al. (17).

Drugs-- The cAMP-stimulating mixture contained 10 µM forskolin plus 100 µM 3-isobutyl-1-methylxanthine. A cGMP analog, CPT-cGMP (8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphate) and two PKA inhibitors, Rp-8-Br-cAMPS (Rp-8-bromoadenosine-3',5'-cyclic monophosphorothiorate), and Rp-8-CPT-cAMPS (Rp-8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphorothiorate) were obtained from Biolog (Bremen, Germany). (-)-Isoproterenol and nadolol were from Sigma. BRL 37344 (4-[-[2-hydroxy-(3-chlorophenyl)ethyl-amino]propyl] phenoxyacetate) was a gift from SmithKline Beecham Pharmaceuticals (Surrey, UK), SR 58611 ((R,S)-N-[(25)-7-ethoxycarbonylmethoxy-1,2,3,4-tetrahydronapht-2-yl]-(2R)-2-(3-chlorophenyl)-2-hydroxyethanamine hydrochloride) was from Sanofi Recherche (Montpellier, France) and CGP 12177 (4-[3-t-butylamino-2-hydroxypropoxy]benzimidazol-2-1) was from Ciba Geigy (Basel, Switzerland). For SPQ experiments, drugs were dissolved in Me2SO (Sigma) so that the final concentration of the solvent was less than 0.1%.

Statistics-- Data are expressed as the means ± S.E. of n number of experiments. Statistical significance of the observed effects was assessed by a Student's t test.

    RESULTS

beta 3-Adrenergic Stimulation Does Not Shorten Action Potential in CF Cardiac Myocytes-- In control human endomyocardial tissues from non-CF patients (Fig. 1), the specific beta 3-adrenoceptor agonist BRL 37344 (0.3 µM) reduced the action potential duration by -13.8 ± 3.8% (n = 5; p < 0.01) and also slightly reduced the action potential amplitude (-3.7 ± 0.4%; p < 0.05). These effects were not observed in eight myocardial preparations obtained from Delta F508/Delta F508 CF patients (Fig. 1), suggesting that the electrophysiological effects of beta 3-adrenoceptor stimulation in non-CF tissues were mediated by activation of a chloride repolarizing current flowing through CFTR channels that are not functional in CF cardiac muscle.


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Fig. 1.   Effect of beta 3-adrenoceptor stimulation on human myocardial action potentials from non-CF and CF biopsies. Superimposed action potentials recorded before (Ctr) and after perfusion of the beta 3-adrenoceptor agonist, BRL 37344 (0.3 µM; beta 3-agonist).

Activation of CFTR by Recombinant beta 3-Adrenoceptors-- CFTR channels are known to activate under intracellular cAMP (18, 19) or cGMP elevation (20, 21). The sensitivity of recombinant CFTR protein to intracellular cAMP and cGMP was investigated in A549 cells intranuclearly injected with a CFTR encoding plasmid (100 µg/ml) and monitored with the SPQ fluorescence assay. A549 cells were chosen because this cell line was previously demonstrated to lack endogenous CFTR protein (22). Under base-line conditions, cells injected with plasmids alone or containing the CFTR expression cassette exhibited a low permeability to halide (Fig. 2A and Fig. 3, left panel). Cells expressing CFTR but not cells injected with the plasmid lacking the insert displayed a approx 6-fold increase in the rate of SPQ dequenching upon application of the cAMP-stimulating mixture (Fig. 2A and Fig. 3, left panel). Similarly, in cells expressing CFTR, the rate of SPQ dequenching was increased approximately 3-fold by preincubation with 500 µM CPT-cGMP (Fig. 2, B and C). As shown in Fig. 2C, the effect of cGMP stimulation was partially reversible upon CPT-cGMP washout. From this first set of experiments, we concluded that recombinant CFTR proteins produced by intranuclear injection of CFTR plasmid were sensitive to stimulation through both the cAMP- and cGMP-dependent pathways.


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Fig. 2.   Regulation of recombinant CFTR protein by cAMP (A) and cGMP (B and C) pathways assayed using the SPQ fluorescent probe. A549 cells were intranuclearly injected with CFTR cDNA (100 µg/ml pECE-CFTR; shaded circles) or a control plasmid lacking the insert (130 µg/ml pECE; mock; open circles). A and B illustrate two SPQ experiments. A, relative SPQ fluorescence plotted against time. Cells were sequentially perfused with I- (white horizontal bars) and NO3- (black horizontal bar) mediums. cAMP-stimulating mixture was applied as indicated by the filled inverted triangle. The slopes of the two lines correspond to the rate of SPQ dequenching in NO3- medium under base-line conditions (pbase line) and under cAMP stimulation (pcAMP). Data are the means ± S.E. of three mock injected cells and three CFTR injected cells. B, relative SPQ fluorescence plotted against time and measured after a 20-min incubation with 500 µM CPT-cGMP. Data are the means ± S.E. of three mock injected cells and three CFTR injected cells. C, the response to CPT-cGMP stimulation expressed as the ratio between the actual membrane permeability (p) to the base-line membrane permeability (pbase line) and plotted against time. CPT-cGMP applied as indicated by the horizontal bar. Data are the means ± S.E. of seven mock injected cells and twenty CFTR injected cells. A paired Student's t test determines the statistical significance between the ratio p/pbase line and the 100% control ratio. *, p < 0.05; **, p < 0.01; ***, p < 0.001.


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Fig. 3.   CFTR activity in the presence of recombinant beta 3-adrenoceptor in A549, COS-7, and T84 cells assayed using the SPQ fluorescent probe. Cells were either injected (A549 cells, left panel; T84 cells, right panel) or transfected (COS-7 cells, middle panel) with various mixtures of plasmids as indicated on the abscissa: CFTR cDNA (CFTR +), mutated Delta F508-CFTR cDNA (Delta F508-CFTR), plasmid lacking the insert (mock), the A or C isoform of beta 3-adrenoceptor cDNAs (beta 3A and beta 3C), and K+ channel cDNA (ROMK2). For cDNA injection experiments, each plasmid was used at 100 µg/ml, except the plasmid lacking the insert (mock; 130 µg/ml). For transfection, CFTR cDNA was used at 2 µg/coverslip and beta 3-adrenoceptor cDNAs at 1 µg/coverslip. A green fluorescent protein plasmid was also added so as to obtain a final amount of 4 µg of plasmid/coverslip. 24 h post-injection or post-transfection, the membrane permeability to halide (p in min-1) was measured under base line (open columns) and under application of cAMP-stimulating mixture (filled columns). Data are the means ± S.E. of 3-42 different A549 cells, 24-44 COS-7 cells, and 14-27 T84 cells. A paired Student's t test compares the p value in the presence of cAMP to the p value under base-line conditions. An unpaired Student's t test compares the pbase line value to the pbase line value in cells expressing CFTR alone. **, p < 0.01; ***, p < 0.001.

In a second set of experiments, various cell lines co-expressing either recombinant or endogenous CFTR and recombinant beta 3-adrenoceptors were investigated. In A549 cells injected with beta 3-adrenoceptor isoform A alone, the base-line membrane permeability to halide was not different from cells injected with the plasmid lacking the insert (Fig. 3, left panel) either in the absence or presence of cAMP. Surprisingly, cells co-injected with CFTR plus the A or the C isoform of beta 3-adrenoceptor exhibited an 8-10-fold increase in pbase line that was not further increased upon cAMP stimulation (Fig. 3, left panel). To ensure that this effect was related to beta 3-adrenoceptor expression, cells were co-injected with CFTR cDNA and with a cDNA encoding a K+ channel (ROMK2). In these cells, pbase line was similar to that in control cells and markedly increased in response to cAMP elevation. In cells expressing the mutated Delta F508-CFTR protein, there was no increase in pbase line related to co-expression with beta 3-adrenoceptor A or C isoforms. As expected, Delta F508-CFTR proteins were not sensitive to cAMP even in the presence of the beta 3-adrenoceptor (Fig. 3, left panel). Similar results were also obtained in COS-7 cells injected (not illustrated) or transfected (Fig. 3, middle panel) with CFTR and beta 3-adrenoceptor plasmids. In transfected COS-7 cells, pbase line increased about 3-fold in cells co-expressing CFTR and either the A or C isoform of beta 3-adrenoceptor. This effect was not observed in cells expressing CFTR or beta 3-adrenoceptor alone. In COS-7 cells co-transfected with CFTR and the A or C isoform of beta 3-adrenoceptor, cAMP stimulation produced a small albeit significant increase in halide permeability (Fig. 3, middle panel). T84 cells endogenously express the CFTR protein (23). Again, T84 cells injected with the A isoform of beta 3-adrenoceptor cDNA exhibited a 2-fold increase in pbase line as compared with noninjected cells (Fig. 3, right panel). This set of experiments shows that recombinant beta 3-adrenoceptor activates both endogenous and recombinant CFTR irrespectively to the transfection method used.

Patch-clamp experiments were performed in cells co-expressing beta 3-adrenoceptor and CFTR to further characterize the halide conductance responsible for the increased membrane permeability. A549 cells co-expressing CFTR and the A isoform of beta 3-adrenoceptor displayed a high amplitude time-independent Cl- current in the absence of cAMP stimulation (Fig. 4). This Cl- current possessed characteristics reminiscent to the CFTR Cl- current recorded under cAMP stimulation in cells expressing CFTR alone. On average, in the absence of cAMP stimulation, the current amplitude at +60 mV was 25.2 ± 5.4 pA/pF (n = 12) in cells injected with CFTR plus beta 3-adrenoceptor but only 8.1 ± 3.1 pA/pF (n = 11) in cells injected with CFTR alone (p < 0.05). These results confirmed that the increase in pbase line in cells co-expressing CFTR and beta 3-adrenoceptors was related to the activation of CFTR chloride current in the absence of cAMP stimulation.


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Fig. 4.   Whole cell chloride currents in the presence of recombinant beta 3-adrenoceptor in A549. A549 cells were injected with 100 µg/ml CFTR cDNA in the absence (top; CFTR+) or presence of beta 3-adrenoceptor isoform A cDNA (100 µg/ml; bottom; CFTR+/beta 3A+). 24 h post-injection, whole cell Cl- currents were recorded in the absence (baseline) and presence of the cAMP-stimulating mixture (cAMP). Superimposed current traces obtained by voltage steps, 20 mV increment, applied from -100 to +60 mV. Holding potential, -80 mV.

Variable Expression Levels of CFTR and beta 3-Adrenoceptors-- To modulate the effects of beta 3-adrenoceptor on CFTR activity, the levels of expression of CFTR and beta 3-adrenoceptor were varied independently. In A549 cells, varying the level of CFTR expression in the absence of beta 3-adrenoceptor expression did not modify the base-line permeability to halide (Fig. 5, left panel). In contrast, in the presence of a constant expression level of beta 3-adrenoceptor isoform A (100 µg/ml), pbase line increased as the CFTR plasmid concentration in the injected medium increased from 3 to 100 µg/ml (Fig. 5, right panel).


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Fig. 5.   CFTR activity as a function of CFTR expression level in A549 cells. Membrane permeability to halide (p in min-1) expressed as a function of the injected CFTR cDNA concentration (3-100 µg/ml) as indicated on the abscissa. The left panel (beta 3A -) shows membrane permeability values in cells not expressing recombinant beta 3-adrenoceptor isoform A whereas the right panel (beta 3A +) shows membrane permeability values in cells co-injected with beta 3-adrenoceptor isoform A cDNA (100 µg/ml). p values were measured under base line (open columns) and under application of the cAMP-stimulating mixture (filled columns). Data are the means ± S.E. of 13-42 different cells. A paired Student's t test compares the p value in the presence of cAMP to the p value under base-line conditions. An unpaired Student's t test compares the pbase line values between two experimental conditions. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

In another set of experiments, we varied the injected concentration of beta 3-adrenoceptor isoform A plasmid from 0.03 to 3 µg/ml in the presence of a constant CFTR concentration (30 µg/ml). In these cells, beta 3-adrenoceptors were selectively activated using 10 µM isoproterenol, a nonselective beta -adrenoceptor agonist, in the presence of 10 µM nadolol, a beta 1- and beta 2-adrenoceptor antagonist (24). It is noteworthy that cells expressing CFTR alone were not responsive to isoproterenol in the presence of nadolol (data not shown), suggesting that A549 cells lack endogenous beta 3-adrenoceptors. As the expression level of beta 3-adrenoceptor isoform A was increased, pbase line increased, the effects of beta 3-adrenoceptor stimulation with isoproterenol plus nadolol were progressively reduced, and the effects of the cAMP-activating mixture were also reduced (Fig. 6). So, at a low injected beta 3-adrenoceptor cDNA concentration (i.e. 0.1 µg/ml), the base-line halide permeability was not different from control cells but increased 3-4-fold upon beta 3-adrenoceptor stimulation. Inversely, at the highest injected beta 3-adrenoceptor cDNA concentration (i.e. 3 µg/ml), the increase in pbase line was such that the effects of beta 3-adrenoceptor stimulation or direct cAMP elevation were abolished.


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Fig. 6.   CFTR activity as a function of beta 3-adrenoceptor expression level in A549 cells. Membrane permeability to halide (p in min-1) expressed as a function of the injected concentration of beta 3-adrenoceptor isoform A cDNA (0.03-3 µg/ml) in the presence of CFTR cDNA (30 µg/ml). p was measured under base line (open columns), under beta 3-adrenoceptor stimulation with 10 µM isoproterenol after a 10-min preincubation with 10 µM nadolol (iso+nado; hatched columns), and under application of cAMP-stimulating mixture (filled columns). Data are the means ± S.E. of 9-56 different cells. A paired Student's t test compares p values in the presence of beta 3-adrenoceptor stimulation or cAMP to the p value under base-line conditions. An unpaired Student's t test compares the pbase line values between two experimental conditions. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

To further assess the CFTR regulation by beta 3-adrenoceptor stimulation, we tested a full beta 3-adrenoceptor agonist, SR 58611, and a partial beta 3-adrenoceptor agonist that possesses also beta 1- and beta 2-adrenoceptor antagonistic properties, CGP 12177 (25). In cells co-expressing CFTR (30 µg/ml) and a low level of beta 3-adrenoceptor isoform A (0.1 µg/ml), a 15-min application of SR 58611 (1 µM) or CGP 12177 (1 µM) increased p values 2- or 3-fold, respectively (Fig. 7, middle and right panels). This effect was not observed in cells expressing CFTR alone. We concluded that the effects produced by beta 3-adrenoceptor expression on the CFTR protein depend on the expression level of the beta 3-adrenoceptors: (i) at a low level of beta 3-adrenoceptor expression, the CFTR channels remained in the close state and were sensitive to beta 3-adrenoceptor pharmacological stimulation and (ii) at a high level of beta 3-adrenoceptor expression, CFTR channels were permanently activated and lost their sensitivity to either beta 3-adrenoceptor agonists or direct cAMP stimulation.


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Fig. 7.   Effect of PKA inhibitors on CFTR regulation by beta -adrenoceptor stimulation in A549 cells. Cells were injected with CFTR cDNA (30 µg/ml) in the absence or presence of beta 3-adrenoceptor isoform A cDNA (0.1 µg/ml; +beta 3A). Membrane permeability to halide (p in min-1) measured under base line (open columns) and under beta -adrenoceptor stimulation (hatched columns) with a nonselective beta -adrenoceptor agonist (isoproterenol 0.1 µM; Iso; left panel) or with a full beta 3-adrenoceptor agonist (SR 58611 1 µM; SR; middle panel) or with a partial beta 3-adrenoceptor agonist (CGP 12177 1 µM; CGP; right panel), in the absence or presence of PKA inhibitors (100 µM Rp-8-Br-cAMPS and 100 µM Rp-8-CPT-cAMPS). Data are the means ± S.E. of 21-109 different cells. A paired Student's t test compares the p value in the presence of beta -adrenoceptor stimulation to the p value under base-line conditions. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Lack of PKA Involvement in CFTR Activation by beta 3-Adrenoreceptors-- In an attempt to determine whether the PKA activation was involved in CFTR regulation by beta 3-adrenoceptor stimulation, A549 cells were incubated for 20 min with a mixture of two PKA inhibitors, Rp-8-Br-cAMPS (100 µM) and Rp-8-CPT-cAMPS (100 µM). Rp-8-Br-cAMPS and Rp-8-CPT-cAMPS are specific for PKA type I and II, respectively (26). PKA-dependent CFTR stimulation by isoproterenol (for review see Ref. 27) was used as a control experiment to check efficient PKA inhibition under our experimental conditions. In A549 cells injected with CFTR cDNA alone, isoproterenol, through a beta 1- and/or beta 2-adrenoceptor stimulation, increased the p value 3-fold (Fig. 7, left panel). After incubation with PKA inhibitors, this effect was abolished (Fig. 7, left panel). In contrast, in cells co-expressing CFTR and beta 3-adrenoceptor, pre-treatment with PKA inhibitors had no effects on CFTR activation by beta 3-adrenoceptor agonists (SR 58611 or CGP 12177; Fig. 7, middle and right panels). These results demonstrated that the regulation of CFTR conductance by beta 3-adrenoceptor stimulation was independent from the PKA pathway.

In another set of experiments, we determined whether the basal activation of CFTR by a high level beta 3-adrenoceptor expression was dependent on an increase in intracellular cyclic nucleotides. To test this hypothesis, intracellular cAMP and cGMP levels were measured in COS-7 cells co-transfected with CFTR and beta 3-adrenoceptor isoform A. COS-7 cells were used for these experiments because transfection of A549 cells using various synthetic vectors led to a low number of cells expressing the transgene as assessed with a green fluorescent protein reporter. COS-7 cells were transfected under the same experimental conditions as used for functional CFTR tests (Fig. 3, middle panel). 24 h post-transfection, intracellular cAMP levels were determined under base line and after 5 min-incubation with 10 µM forskolin. As illustrated in Fig. 8A, base-line cAMP levels were not significantly modified by expression of either the A or C isoforms of beta 3-adrenoceptor in cells expressing CFTR or not. As expected, pre-incubation with forskolin induced an elevation in cAMP level in every experimental situation. The level of intracellular cGMP was also determined under base line and after a 20-min incubation with a cGMP analog, CPT-cGMP (500 µM; Fig. 8B). In cells expressing the CFTR protein and either the A or C isoforms of beta 3-adrenoceptor, base-line cGMP concentration was not different from that of control cells. Comparison between results shown on Fig. 3 (middle panel) and Fig. 8, which were both obtained under the same transfection conditions, shows that beta 3-adrenoceptor-mediated CFTR activation was not related to the activation of either the cAMP or cGMP pathways.


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Fig. 8.   Intracellular cAMP (A) and cGMP (B) concentrations in COS-7 cells expressing CFTR and beta 3-adrenoceptor isoform A or C. COS-7 cells were transfected with various mixtures of plasmids as indicated on the abscissa. CFTR cDNA (CFTR +) was used at 2 µg/well, the plasmid lacking insert (mock) at 2 µg/well, A or C isoforms of beta 3-adrenoceptor cDNA (beta 3A and beta 3C) at 1 µg/well. Green fluorescent protein cDNA was also used to obtain a final amount of 4 µg of plasmid/well. The transfection conditions were the same as those used for functional assay of CFTR activity illustrated in Fig. 3 (middle panel). 24 h post-transfection, cells were pre-incubated with 3-isobutyl-1-methylxanthine (100 µM). A, cAMP intracellular level (pmol/mg protein) measured in the absence of stimulation (baseline) and after a 5-min stimulation with 10 µM forskolin (Fsk). Data are the means ± S.E. of 3-13 experiments. An unpaired t test compares the cAMP level in stimulated cells to cAMP level in unstimulated cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001. B, cGMP intracellular level (pmol/mg protein) measured in the absence of stimulation (baseline) or after a 20-min stimulation with 500 µM CPT-cGMP (CPT-cGMP). Data are the means ± S.E. of 8-11 experiments. An unpaired t test compares cGMP level in stimulated cells to cGMP level in unstimulated cells. ***, p < 0.001.


    DISCUSSION

The present study suggests that beta 3-adrenoceptors are functionally coupled to the CFTR protein. Interestingly, coupling between beta 3-adrenoceptors and CFTR depends on the expression level of beta 3-adrenoceptors: (i) expression of a low level of beta 3-adrenoceptors induces CFTR activation in response to beta 3-adrenoceptor agonists and (ii) expression of a high level of beta 3-adrenoceptors induces permanent CFTR activation independently from beta 3-adrenoceptor stimulation. In addition, we also show that functional coupling between CFTR and beta 3-adrenoceptors occurs irrespectively to the beta 3-adrenoceptor splice variants expressed in human tissues. Finally, our results show that regulation of CFTR by beta 3-adrenoceptors does not imply activation of the cAMP/PKA pathway.

We have previously reported that intranuclear injection of large quantities of CFTR cDNA into mammalian cells produces hyper-expression of CFTR proteins with altered physiological properties inasmuch as hyper-expressed recombinant CFTR channels are permanently opened and not susceptible to cAMP stimulation (13). This phenomenon, which we attributed to CFTR clustering within the cell membrane, appears for various CFTR cDNA concentrations depending on the cell line. Accordingly, it may be argued that the increase in base-line permeability that we observed in cells co-expressing CFTR and beta 3-adrenoceptors was caused by a comparable phenomenon. We judge this explanation unlikely for several reasons: (i) in A549 cells, permanent activation of hyper-expressed CFTR protein usually occurs for plasmid concentrations greater than 350 µg/ml (13), whereas in the present study, the injected concentration never exceeded 100 µg/ml, a concentration that did not lead to permanently activated CFTR channels in the absence of beta 3-adrenoceptor co-expression; (ii) base-line activation of CFTR channels by recombinant beta 3-adrenoceptors was observed in COS-7 cells co-transfected with CFTR and beta 3-adrenoceptors, although permanent activation of CFTR channels was never observed in cells transfected with CFTR alone irrespectively to the quantity of cDNA used for cell transfection; thus, base-line activation of CFTR in the presence of beta 3-adrenoceptors was unlikely to be caused by our intranuclear injection technique; (iii) furthermore, activation of CFTR at base line was not observed when beta 3-adrenoceptor cDNA was replaced by a K+ channel cDNA inserted into the same plasmid and injected at the same concentration as beta 3-adrenoceptor cDNA; and (iv) finally and most importantly, CFTR endogenously expressed in T84 cells was also susceptible to activation by beta 3-adrenoceptors, suggesting that agonist-independent activation of CFTR can be observed for level of CFTR expression close to physiological ones.

Our results show that the effects of beta 3-adrenoceptor expression on CFTR activity can be modulated by the density of the receptors within the cell membrane. Increasing the expression of beta 3-adrenoceptor in the presence of a constant CFTR expression level dose-dependently increased base-line permeability and also concomitantly reduced the activating effects of beta 3-agonists. The beta 3-adrenoceptor belongs to the superfamily of G protein-coupled receptors (28). These receptors are known to exist in the cell membrane in two subpopulations: (i) an inactive subpopulation that requires agonist occupancy for coupling to G protein and (ii) a constitutively active subpopulation that can couple to G protein in the absence of agonist (29-31). An increase in the constitutively active subpopulation has been reported for mutated G protein-coupled receptors in which mutations induce a conformational change in the receptor that normally requires the binding of an agonist to occur (29, 32). Similarly, an increase in the constitutively active receptor subpopulation has been reported for receptors overexpressed at a high level. For example, overexpression of the wild-type beta 2-adrenoceptor either in Chinese hamster ovary cells or in myocardial cells of transgenic mice produced an elevation of base-line adenylyl cyclase activity (29, 30, 33). Comparable behavior has been reported with the beta 3-adrenoceptor so that basal adenylyl cyclase activity was raised with beta 3-adrenoceptor density in Chinese hamster ovary cells (34). In the present study, when the expression level of beta 3-adrenoceptors was high, CFTR was activated in the absence of beta 3-adrenoceptor stimulation. In such conditions, intracellular cAMP and cGMP levels were close to normal and in any case much lower than the level required for CFTR activation as shown in Fig. 3 (middle panel). It could be hypothesized that overexpression of beta 3-adrenoceptors led to a greater number of receptors in the active state, resulting in saturation in the beta 3-adrenoceptor signaling capacity in the absence of agonist. Inversely, when the expression of beta 3-adrenoceptors was lowered, CFTR was not activated under base-line conditions and became sensitive to agonists. Activation of CFTR by beta 3-adrenoceptor agonists was not sensitive to PKA inhibitors, ruling out the involvement of the cAMP/PKA pathway. Activation of CFTR independently of the cAMP/PKA pathway has previously been reported for other receptors such as P2x-subtype of purinergic receptors (35) or mu-opioid receptors (36). Our results suggest that either another second messenger is implied in the coupling pathway between beta 3-adrenoceptor and CFTR or a direct interaction exists between the membrane receptor (or an associated G protein) and CFTR. Although direct regulation of CFTR channel protein by G proteins has not yet been reported, it has been shown that Galpha i2 protein modulates its vesicle trafficking and its delivery to the plasma membrane (37). As yet, two coupling pathways have been ascribed to beta 3-adrenoceptors: (i) in adipose tissue and gastrointestinal tract, their stimulation produces an increase in cAMP levels (6, 7) and (ii) in human ventricle, the negative inotropic effects mediated by beta 3-adrenoceptor agonists are associated with an increase in NO production and cGMP levels (8). Clearly, the involvement of another mechanism leading to activation of CFTR by beta 3-adrenoceptors needs clarification.

The physiological relevance of our findings should be found in the various organs where beta 3-adrenoceptors and CFTR are co-expressed. In the human heart, beta 3-adrenoceptor agonists produce a negative inotropic effect and a shortening in the action potential duration (5). Our results suggest that the action potential shortening produced by beta 3-adrenoceptor agonists is caused by the activation of a CFTR-related repolarizing chloride current that is nonfunctional in CF patients. beta 3-Adrenoceptors and CFTR are also endogenously co-expressed in other tissues such as airways (38, 39) and gallbladder (40) epithelia. In these tissues, beta 3-adrenoceptors may modulate water and salt secretion through apical CFTR channels.

    ACKNOWLEDGEMENTS

We thank Béatrice Leray, Karine Laurent, and Marie-Joseph Louerat for expert technical assistance with cell cultures, cyclic nucleotide assays, and plasmid amplifications.

    FOOTNOTES

* This work was supported by grants from the Association Française de Lutte contre la Mucoviscidose, the Institut National de la Santé et de la Recherche Médicale (INSERM), and the Fédération Française de Cardiologie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: Lab. de Physiopathologie et de Pharmacologie Cellulaires et Moléculaires, INSERM CJF 96.01, Bât. HBN, Hotel-Dieu, BP 1005, 44093 Nantes, France. Tel.: 33-240-08-75-18; Fax: 33-240-08-75-23; E-mail: chantal.gauthier{at}sante.univ-nantes.fr.

    ABBREVIATIONS

The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; CF, cystic fibrosis; PKA, cAMP-dependent protein kinase; SPQ, 6-methoxy-N-(3-sulfopropyl)quinolinium; CPT-cGMP, 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphate; Rp-8-Br-cAMPS, Rp-8-bromoadenosine-3',5'-cyclic monophosphorothiorate; Rp-8-CPT-cAMPS, Rp-8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphorothiorate; BRL 37344, 4-[-[2-hydroxy-(3-chlorophenyl)ethyl-amino]propyl] phenoxyacetate; SR 58611, (R,S)-N-[(25)-7-ethoxycarbonylmethoxy-1,2,3,4-tetrahydronapht-2-yl]-(2R)-2-(3-chloro-phenyl)-2-hydroxyethanamine hydrochloride; CGP 12177, 4-[3-t- butylamino-2-hydroxypropoxy]benzimidazol-2-1.

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