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Am J Physiol Lung Cell Mol Physiol 273: L1065-L1072, 1997;
1040-0605/97 $5.00
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Vol. 273, Issue 5, L1065-L1072, November 1997

In vivo activation of CFTR-dependent chloride transport in murine airway epithelium by CNP

Thomas J. Kelley1, Calvin U. Cotton1, and Mitchell L. Drumm1,2

Departments of 1 Pediatrics and 2 Genetics and Center for Human Genetics, Case Western Reserve University, Cleveland, Ohio 44106-4948

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Inhibitors of guanosine 3',5'-cyclic monophosphate (cGMP)-inhibited phosphodiesterases stimulate Cl- transport across the nasal epithelia of cystic fibrosis mice carrying the Delta F508 mutation [cystic fibrosis transmembrane conductance regulator (CFTR) (Delta F/Delta F)], suggesting a role for cGMP in regulation of epithelial ion transport. Here we show that activation of membrane-bound guanylate cyclases by C-type natriuretic peptide (CNP) stimulates hyperpolarization of nasal epithelium in both wild-type and Delta F508 CFTR mice in vivo but not in nasal epithelium of mice lacking CFTR [CFTR(-/-)]. With the use of a nasal transepithelial potential difference (TEPD) assay, CNP was found to hyperpolarize lumen negative TEPD by 6.1 ± 0.6 mV in mice carrying wild-type CFTR. This value is consistent with that obtained with 8-bromoguanosine 3',5'-cyclic monophosphate (6.2 ± 0.9 mV). A combination of the adenylate cyclase agonist forskolin and CNP demonstrated a synergistic ability to induce Cl- secretion across the nasal epithelium of CFTR(Delta F/Delta F) mice. No effect on TEPD was seen with this combination when used on CFTR(-/-) mice, implying that the CNP-induced change in TEPD in CFTR(Delta F/Delta F) mice is CFTR dependent.

guanylate cyclase; Delta F508 cystic fibrosis transmembrane conductance regulator; pharmacological activation; C-type natriuretic peptide

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

NATRIURETIC PEPTIDES are known regulators of fluid and ion transport in several systems (reviewed in Ref. 25) and have been shown to regulate Cl- transport in shark rectal gland cells (28) and in porcine colon via a guanosine 3',5'-cyclic monophosphate (cGMP)-dependent mechanism (1). Similarly, both heat-stable enterotoxin (Sta) and guanylin, peptides structurally and functionally related to the natriuretic peptides, have been shown to stimulate Cl- transport in the human colonic cell lines T84 and Caco-2 (3, 5) through a cystic fibrosis transmembrane conductance regulator (CFTR)-dependent pathway.

The regulatory actions of the natriuretic peptides are thought to be mediated predominantly through cGMP. The natriuretic peptides bind to and activate particulate, transmembrane receptors that contain intracellular guanylate cyclase (GC) domains. Each of the natriuretic peptides, atrial (ANP), brain (BNP), and C-type (CNP), have varied affinities for specific receptors. ANP and BNP both bind to GC-A, with ANP having the greater affinity, and CNP binds to the GC-B receptor (reviewed in Ref. 32).

Although there is an apparent specificity of function for ANP and BNP in cardiac tissue and in the pulmonary vascular system compared with CNP (9, 20, 21, 29), CNP binding and function predominates in airway epithelial cells (6, 7, 30). Specific binding of CNP to human airway epithelial cells has been demonstrated by the functional ability of CNP to elevate cGMP levels in these cells. ANP had no significant effect on cGMP levels, although sodium nitroprusside (SNP), an activator of soluble GCs, did elevate cGMP concentrations in this system. Additionally, CNP was shown to increase ciliary beat frequency in primary airway epithelial cells, implying a role for CNP in the regulation of mucociliary clearance (6) and raising the possibility that CNP may play a role in the regulation of other nonrespiratory functions in the airway.

We have previously shown that cGMP-inhibited phosphodiesterases (cGI-PDEs) represent a key step in the regulatory pathway of the CFTR in human airway epithelial cells (12). Inhibition of cGI-PDEs with the cardiotonic drug milrinone stimulates CFTR activity in cells that express either wild-type or Delta F508 forms of CFTR (11, 14). The specificity of cGI-PDEs compared with other PDE classes in the regulation of CFTR activity clearly implies an important role for cGMP in this process. To establish this regulatory role for cGMP, we have also shown that the addition CNP can mimic the effects of milrinone and can lead to the stimulation of CFTR activity in both Calu-3 and CF-T43 cells (13). These results are completely consistent with the previously mentioned reports that demonstrate CFTR activity in colonic cells in response to guanylin and Sta (3, 5).

Although we have shown that CNP is capable of regulating Cl- secretion in cultured airway epithelial cells, the possible paracrine or autocrine regulation of CFTR activity by natriuretic peptides has not been established. The first step in this process is to demonstrate stimulation of CFTR in intact tissue by CNP. We have chosen the mouse as a model in which to study this system because the CNP gene is conserved between mice and humans (22, 23) and because CFTR null [CFTR(-/-)] mice are available to serve as controls for CNP-mediated CFTR activation (2, 8, 17, 26). In this paper, in vivo mouse nasal transepithelial potential difference (TEPD) measurements as well as in vitro mouse tracheal TEPD measurements were made in response to apically applied CNP and other GC agonists to demonstrate their ability to regulate Cl- transport across murine respiratory epithelium.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Peptides. Both CNP and vasonatrin were purchased from Bachem Bioscience.

Measurement of mouse nasal TEPD values. Mouse nasal TEPD was measured by the protocol of Grubb et al. (8). Briefly, mice were anesthetized with 200 µl/20 g body wt of 0.4 mg acepromazine, 11 mg ketamine, and 2 mg xylazine/ml phosphate-buffered saline. PE-10 tubing drawn out to approximately one-half of its original diameter was inserted 2-3 mm into the nostril of the mouse. Solutions were perfused at room temperature with the use of a Razel A-99 (Razel Scientific Instruments, Stamford, CT) syringe pump at a rate of ~7 µl/min. Each syringe was bridged to a Calomel electrode through a 4% agar bridge made up in either Cl--replete or Cl--free Ringer solution. An ~10-mV junction potential in the negative direction was instantly seen upon switching to Cl--free solutions. This junctional potential is not shown in Figs. 1-9 and is not corrected for in the values given. A series of valves was used to change solutions, with a delay time of ~45 s between solution change and solution contact with the nasal epithelia. Ringer solutions consisted of Cl--replete N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered Ringer solution [HBR; containing (in mM) 10 HEPES (pH 7.4), 138 NaCl, 5 KCl, 2.5 Na2HPO4, 1.8 CaCl2, and 1.0 MgSO4] and Cl--free HBR [containing (in mM) 10 HEPES (pH 7.4), 138 sodium gluconate, 5 potassium gluconate, 2.5 Na2HPO4, 3.6 hemicalcium gluconate, and 1.0 MgSO4; all chemicals were from Sigma Chemical, St. Louis, MO]. Some mice were studied on multiple occasions. Mice that were retested were given at least 48 h between assays.

Measurement of mouse tracheal potential difference values. Excised tracheae were mounted on hold pipettes (30-ml micropipettes; Drummond Scientific) inserted into each end of the trachea, and the luminal and bath compartments were perfused independently. All experiments were performed at 37°C by placing all solutions and the trachea-mounting chamber in an Isolette infant incubator (Narco, Hatboro, PA). HBR was perfused to both luminal and basolateral sides by gravity. Luminal perfusion rates were between 3 and 5 ml/min. TEPD was measured via 4% agar bridges in HBR placed on both luminal and basal sides and was connected through calomel electrodes to a DVC 1000 voltage-current clamp (WPI). Data were collected on a MacLab/4e from Advanced Instruments.

cGMP measurements. Mouse tracheal tissue was excised and cut into three equal sections for each experiment. The epithelial layer was not isolated for these experiments, so various cell types were present, including neuronal cells. The tissue was incubated for 5 min at 37°C in physiological salt solution buffered by 10 mM tris(hydroxymethyl)aminomethane (Tris; pH 7.5) containing either 1 µM CNP or 300 µM SNP or in the absence of any GC agonist as a control. Samples were then placed in 0.5 ml of 10 mM Tris (pH 7.5) and 100 µM EDTA at 90°C for 10 min. Samples were then placed on ice, homogenized, and spun down in a microfuge at 14,000 revolutions/min for 5 min, and the supernatant was collected for analysis. Measurements of cGMP levels were performed using an enzyme-linked immunoassay kit, according to the manufacturer's instructions (Cayman Chemical, Ann Arbor, MI).

Mice. Mice were genotyped from tail clip DNA. Delta F508 mice were a generous gift from Kirk Thomas of the University of Utah School of Medicine and were genotyped by the procedures described previously (33). CFTR(-/-) mice (27) were obtained from Jackson Laboratories and were genotyped as described by Koller et al. (16). To increase survival of cystic fibrosis animals, mice were fed a liquid diet as described by Eckman et al. (4). Mice were cared for in accordance with Case Western Reserve University guidelines.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effects of cGMP agonists on mouse nasal TEPD. Three GC agonists and a cGMP analog, 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), were tested by nasal TEPD assay for their ability to stimulate Cl- secretion in the nasal epithelia of mice when perfused onto murine nasal epithelia in Cl--free HBR (Fig. 1). An agonist of cytosolic GCs, SNP (300 µM), had very little effect on TEPD values, hyperpolarizing lumen negative TEPD only 2.4 ± 0.9 mV (n = 7). In contrast, 1 µM CNP, which activates GC-B, a membrane-bound GC, stimulates a change in TEPD (Delta TEPD) of 6.1 ± 0.6 mV (n = 8), representing lumen negative hyperpolarization. Similarly, the GC-B-specific peptide vasonatrin (1 µM), a chimeric peptide that contains the bioactive loop region of CNP, and the carboxy-terminus of ANP hyperpolarized lumen negative TEPD 6.5 ± 1.1 mV (n = 4). Consistent with these effects being mediated by increased cGMP production, addition of the analog 8-BrcGMP (100 µM) resulted in a Delta TEPD of 6.2 ± 0.9 mV (n = 3).


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  		Fig. 1.   Changes in nasal transepithelial potential difference (Delta TEPD) in response to guanylate cyclase agonists and 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). Plots show the Delta TEPD in individual cystic fibrosis transmembrane conductance regulator (CFTR) (+/+), CFTR(+/-), and CFTR(+/Delta F) mice to 1 µM C-type natriuretic peptide (CNP) (A), 1 µM vasonatrin (B), 100 µM 8-BrcGMP (C), and 300 µM sodium nitroprusside (SNP) (D). All traces are performed in the presence of 100 µM amiloride in Cl--free HEPES-buffered Ringer solution (HBR). Before refers to the TEPD 30 s before drug addition. After shows the maximal TEPD obtained within 2.5 min after drug addition. Average TEPDs of each experiment are shown by the black bars, and average Delta TEPDs ± SE are given at bottom. * P < 0.05; ** P < 0.005; *** P < 0.0001. Paired t-tests were performed to determine significance.

Pharmacological characterization of CNP-mediated Cl- secretion in mouse nasal epithelia. Previously, we reported that CNP stimulated CFTR-mediated Cl- current in Calu-3 cells as determined by whole cell patch-clamp recordings (13). To determine if CFTR has a role in CNP-stimulated Cl- secretion in vivo, the Cl- channel blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and diphenylamine-2-carboxylate (DPC) were used to pharmacologically characterize responses to CNP (18, 31). DPC (1 mM) reduced CNP-stimulated changes in mouse nasal TEPD ~70%, whereas 500 µM DIDS did not have a significant effect (Fig. 2). This inhibitor sensitivity profile is consistent with CFTR activation. For comparison, forskolin-stimulated nasal TEPD changes were also tested in the presence of DPC and DIDS. Forskolin-stimulated Delta TEPDs were reduced ~55% by DPC and were unaffected by the presence of DIDS (Fig. 2B).


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  		Fig. 2.   Effect of Cl- channel blockers on CNP-mediated Delta TEPDs. A: averaged traces of nasal TEPD assays performed on CFTR(+/+), CFTR(+/-), and CFTR(+/Delta F) mice not treated with channel blockers or in the presence of either 500 µM DIDS or 1 mM diphenylamine-2-carboxylate (DPC). Values for changes in TEPD were taken at 15-s intervals and were plotted as Delta TEPD. Addition of amiloride and CNP in the assay is indicated by the black bars. Zero Delta TEPD and time 0 correspond to the point at which 1 µM CNP was added. All traces were performed in the presence of 100 µM amiloride in Cl--free HBR. Positive Delta TEPD values indicate a hyperpolarization of lumen negative potential difference. Error bars represent SE. B: bar graph comparing the effects of DIDS and DPC treatment on Delta TEPD values stimulated by either 1 µM CNP or 10 µM forskolin (Forsk) after 2 min. Error bars represent SE, and the number of experiments (n) is shown in parentheses. Comparisons were made with each condition to Forsk. Significance was determined by Duncan's multiple-range test with alpha  = 0.05 with a Bonferroni correction for multiple comparisons. Significance corresponds to P < 0.01. * P < 0.001; ** P < 0.0001.

To more directly test for CFTR activity in response to CNP, CFTR(-/-) mice were compared with wild-type mice in their ability to respond to CNP. CNP (1 µM), in Cl--free HBR and in the presence of amiloride, was perfused into the nasal cavity of CFTR(-/-) mice as described for the wild-type mice. The CFTR(-/-) mice failed to show any hyperpolarization of lumen negative potential in response to either Cl--free HBR alone or with the agonist CNP (Fig. 3). These data, coupled with the inhibitor profile of CNP-induced changes in TEPDs, are strong evidence that CNP stimulates epithelial Cl- transport by regulating CFTR activity.


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  		Fig. 3.   CNP increases TEPD in CFTR(+/+) and CFTR(+/-) mice. Averaged traces of nasal TEPD assays performed on CFTR(+/+) and CFTR(+/-) mice (n = 3) and on CFTR(-/-) mice (n = 4). Values were taken at 15-s intervals and were plotted as Delta TEPD. The addition of amiloride and CNP in the assay is indicated by the black bars. Zero Delta TEPD and time 0 correspond to the point at which 1 µM CNP was added. All traces are performed in the presence of 100 µM amiloride in Cl--free HBR. Positive Delta TEPD values indicate a hyperpolarization of lumen negative potential difference. Error bars represent SE.

To show that CNP was acting through its receptor GC-B, two inhibitors of GC activity were used (Fig. 4). First, perfusion with the compound LY-83583 (50 µM), which directly inhibits GC activity (15, 19), completely inhibited any response to CNP. Second, perfusion with 250 nM phorbol 12,13-dibutyrate (PDBu) inhibited CNP-mediated changes in TEPD by ~90%, consistent with reports that phorbol esters cause protein kinase (PK) C-dependent desensitization of natriuretic peptide receptors (24). For comparison, PDBu had no effect on forskolin-stimulated changes in TEPD, indicating that inhibition of the CNP effect was not due to PDBu interfering the ability of CFTR to activate.


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  		Fig. 4.   CNP-mediated Delta TEPDs in the presence of guanylate cyclase antagonists. A: averaged traces of nasal TEPD assays performed on CFTR(+/+), CFTR(+/-), and CFTR(+/Delta F) mice not treated with guanylate cyclase inhibitors or in the presence of either 250 nM phorbol 12,13-dibutyrate (PDBu) or 50 µM LY-83583. Values were taken at 15-s intervals and were plotted as Delta TEPD. Addition of 100 µM amiloride and 1 µM CNP in the assay is indicated by the black bars. Zero Delta TEPD and time 0 correspond to the point at which CNP was added. Positive Delta TEPD values indicate a hyperpolarization of lumen negative potential difference. Error bars represent SE. B: bar graph comparing the effects of PDBu treatment on Delta TEPD values stimulated by either 1 µM CNP or 10 µM Forsk. Error bars represent SE, and n is shown in parentheses. Statistical analysis is a comparison of the responses to the indicated agents with the responses to Forsk. Duncan's multiple range test with a Bonferroni correction for multiple comparisons was used. Significance at alpha  = 0.05 corresponds to P < 0.013. * P < 0.0001.

Stimulation of Cl- secretion in mouse trachea by CNP. To determine if CNP acts on epithelia in other portions of the airway, the effects of CNP on Cl- transport were tested by measuring TEPD values in excised mouse trachea. We first established that the CNP receptor was present and functional in tracheal tissue by measuring cGMP production in response to 1 µM CNP and 300 µM SNP (Fig. 5). Compared with tissues not incubated with GC agonists, SNP increased cGMP levels ~2-fold, whereas CNP increased cGMP levels ~50-fold.


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  		Fig. 5.   Elevation of cGMP values in response to either 300 µM SNP or 1 µM CNP in trachea of CFTR(+/+) or CFTR(+/-) mice. Determinations of cGMP content were performed as described in MATERIALS AND METHODS. Error bars represent SE, and n is shown in parentheses.

Whereas the nasal TEPD assays were performed by perfusing Cl--free Ringer solution onto the surface of murine nasal epithelium to increase the electrochemical driving force for Cl- secretion, the tracheal TEPD assays were performed with Cl--replete Ringer solution on both the basolateral and luminal sides. Under these conditions, CNP alone was not sufficient as a stimulus to generate hyperpolarization in tracheal epithelium and resulted in further depolarization of TEPD. Forskolin alone did generate a detectable hyperpolarization of TEPD in the trachea. However, a combination of forskolin plus CNP stimulated a twofold greater Delta TEPD than forskolin alone (Fig. 6). This synergistic relationship between forskolin and CNP suggests an enhancement of the adenosine 3',5'-cyclic monophosphate-PKA pathway rather than additive effects of two separate pathways.


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  		Fig. 6.   In vitro TEPD changes of a wild-type mouse trachea in response to Forsk (Fsk) and CNP. A: raw trace of tracheal TEPD assay showing responses to luminal applications of 100 µM amiloride (A), 10 µM Fsk, and 1 µM CNP. HBR represents perfusion with Cl--replete HEPES-buffered Ringer solution. Reduction (depolarization) in TEPD caused by amiloride is partially restored by the addition of Fsk. After washout with HBR, the TEPD returns (hyperpolarizes) to baseline. Treatment with amiloride again depolarizes the TEPD, and CNP has no appreciable effect. After a second washout and depolarization with amiloride, Fsk is added a second time. Basal TEPD values for these experiments were -2.8 ± 0.3 mV (n = 4). B: bar graph showing Delta TEPD in response to the first addition of Fsk alone (Fsk1; n = 3), CNP alone (CNP; n = 4), Fsk plus CNP (Fsk + CNP; n = 3), and a second addition of Fsk (Fsk2; n = 3). Significance is measured against responses to Fsk. Positive Delta TEPD values indicate a hyperpolarization of lumen negative potential difference. Error bars represent SE. Statistical analysis is a comparison of the responses to the indicated agents with the responses to Fsk. Duncan's multiple range test with a Bonferroni correction for multiple comparisons was used. Significance at alpha  = 0.05 corresponds to P < 0.017. * P < 0.0005.

In vivo activation of Delta F508 CFTR by forskolin and CNP. We have previously shown that Cl- transport can be stimulated in the nasal epithelium of Delta F508/Delta F508 mice with the combination of forskolin and the cGI-PDE inhibitor milrinone but not with either compound alone (14). Activation of Cl- secretion was not observed when nasal epithelium of CFTR(-/-) mice was exposed to these compounds. Our hypothesis from these data is that both CNP and milrinone are acting to stimulate CFTR activity by inhibition of cGI-PDEs. If true, the combination of forskolin and CNP, like forskolin and milrinone, should stimulate Cl- secretion in Delta F508/Delta F508 mice.

Mouse nasal TEPD assays with Delta F508/Delta F508 mice showed that neither forskolin nor CNP alone stimulates a hyperpolarization of TEPD, as we had found with milrinone alone. Consistent with our hypothesis, after a 2-min exposure to forskolin plus CNP, TEPD was hyperpolarized 3.4 ± 1.0 mV (n = 8) compared with a depolarization of 2.1 ± 0.6 mV (n = 3) induced by forskolin or a 2.1 ± 0.9 mV (n = 3) depolarization induced by CNP alone (Fig. 7). This 3.4 ± 1.0-mV hyperpolarization of TEPD in CFTR(Delta F/Delta F) mice is ~35% of the amount of activation achieved with the combination of forskolin and CNP in CFTR(+/Delta F) mice (9.7 ± 1.3 mV; n = 3), whereas forskolin plus CNP induced a 2.3 ± 0.8-mV (n = 4) depolarization of TEPD in CFTR(-/-), implicating a role for CFTR in the response to this combination of compounds. One differential response of CFTR(-/-) mice and CFTR(Delta F/Delta F) mice is illustrated in Fig. 8. These data show that CFTR is involved in the mechanism of CNP-induced Cl- secretion in airway epithelia of mice.


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  		Fig. 7.   Stimulation of CFTR-dependent Delta TEPDs in mouse nasal epithelia. A: averaged traces of nasal TEPD assays performed on CFTR(Delta F/Delta F) mice treated with either 10 µM Forsk (n = 3), 1 µM CNP (n = 3), or 10 µM Forsk plus 1 µM CNP (n = 8). Zero Delta TEPD and time 0 correspond to the point at which agonists were added. All traces were performed in the presence of 100 µM amiloride in Cl--free HBR. Positive Delta TEPD values indicate a hyperpolarization of lumen negative potential difference. Error bars represent SE. B-D: raw traces of Forsk alone (B), CNP alone (C), and Forsk plus CNP (D). Agonists were added at the 2-min time point (arrows).


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  		Fig. 8.   Averaged traces of nasal TEPD assays performed on CFTR (Delta F/Delta F) mice (n = 8) or CFTR(-/-) mice (n = 4) treated with 10 µM Forsk plus 1 µM CNP. Values were taken at 15-s intervals and were plotted as a Delta TEPD. Zero Delta TEPD and time 0 correspond to the point at which agonists were added. All traces were performed in the presence of 100 µM amiloride in Cl--free HBR. Positive Delta TEPD values indicate a hyperpolarization of lumen negative potential difference. Error bars represent SE. B and C: raw traces of CFTR(Delta F/Delta F) mice and CFTR(-/-) mice treated with Forsk plus CNP, respectively. Agonists were added at the 2-min time point.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have previously shown that cGI-PDEs are an important part of the CFTR regulatory pathway (11, 12, 14). The involvement of this PDE class suggests an important role for cGMP in the normal regulation of Cl- transport across CFTR-expressing epithelium. This notion is supported by other reports that show CFTR activation in human colonic cells in response to both Sta and guanylin, both of which are peptides that activate membrane-bound GCs (3, 5). A similar peptide, CNP, has been shown to stimulate cGMP production in primary cultures of human airway epithelial cells (7). We have demonstrated in cultured lung epithelial cells (Calu-3) that CNP is capable of stimulating CFTR activity as shown by whole cell patch-clamping experiments (13). This stimulation of activity was mediated by cGMP but via a PKA-dependent pathway, consistent with our previous results from the cGI-PDE inhibitor milrinone.

Natriuretic peptides have been shown to regulate fluid and electrolyte transport in several systems (1, 19, 25, 28). The ability of CNP to stimulate cGMP production in human airway epithelial cells suggests a role for CNP in the regulation of airway electrolyte transport. Our data show that CNP can stimulate Cl- secretion when applied to the apical membrane of mouse nasal epithelia in vivo. This stimulated Cl- transport has the pharmacological characteristics of CFTR and is not present when CNP is applied to the nasal epithelium of CFTR(-/-) mice.

It has also been reported, however, that CNP has no effect on Cl- transport when applied to cultured monolayers of ciliated human nasal epithelial cells obtained from nasal scrapings (6). A possible reason for this discrepancy is that culture conditions for primary cells are not optimal for facilitating responses to CNP, thus reducing reproducibility and magnitude of CNP-mediated stimulation. Thus our in vivo measurements of nasal TEPD and our in vitro studies with freshly excised trachea may provide a more appropriate model than studies with cultured cells. A methodological difference between the studies is that our nasal TEPD measurements were made with the luminal side of the epithelium perfused with Cl--free HBR, whereas the short-circuit current measurements made in the previous report were in symmetrical Cl--replete Krebs-buffered Ringer (KBR). The short-circuit data are consistent with our findings in freshly excised trachea in KBR (Fig. 6) in which we were unable to see a response with CNP in the presence of amiloride. We could demonstrate, however, a synergistic increase in forskolin-mediated hyperpolarization of TEPD by CNP. Another possible difference between systems may lie in the fact that CNP and its receptor GC-B have been identified in brain tissue and nerve fibers (reviewed in Ref. 10). It is possible that in our in vivo system CNP is interacting with nerve fibers within the nasal passage and stimulating the release of other factors that may be initiating CFTR activation in the epithelium. This pathway would not be available in cultured monolayers of harvested primary epithelial cells. We think this is unlikely to explain the difference seen here, as our previous study in Calu-3 and CF-T43 cells showed that cultured epithelial cells exposed to CNP are sufficient to stimulate CFTR-dependent Cl- transport (13).

The demonstrated ability of natriuretic peptides to generically regulate salt and fluid transport in several systems opens the possibility that an in vivo role of CNP may be to regulate Cl- transport in the airways through CFTR. The similarity of effects that both cGI-PDE inhibitors and the GC agonist CNP have on CFTR activity implies a regulatory role for cGMP in ion transport regulation across airway epithelial cells. The specificity of CNP-mediated regulation of CFTR activity extends to its ability to stimulate Cl- transport across nasal epithelia of CFTR(Delta F/Delta F) mice when used in combination with the adenylate cyclase agonist forskolin but has no effect on Cl- secretion in CFTR(-/-) mice. These results are consistent with our previous data demonstrating that the combination of forskolin and the cGI-PDE inhibitor milrinone stimulates Delta F508 CFTR activity in the nasal epithelia of Delta F/Delta F mice and support the hypothesis that milrinone is acting via a cGMP-dependent pathway (Fig. 9). The ability to stimulate Cl- secretion in the nasal epithelium of Delta F/Delta F mice in an in vivo system demonstrates the therapeutic potential of CNP as a pharmacological agent and the possible importance of CNP as an in vivo regulator of CFTR function.


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  		Fig. 9.   Schematic diagram showing the proposed mechanism of CNP-mediated CFTR activation. PKA, protein kinase A; PDE, phosphodiesterase; GC, guanylate cyclase. (-) Inhibitory effect.

    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants HL-50160 and DK-51878 and by grants from the Cystic Fibrosis Foundation.

    FOOTNOTES

Address for reprint requests: M. L. Drumm, Dept. of Pediatrics, Case Western Reserve Univ., 8th floor BRB, 10900 Euclid Ave., Cleveland, OH 44106-4948.

Received 21 February 1997; accepted in final form 7 August 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Argenzio, R. A., and M. Armstrong. ANP inhibits NaCl absorption and elicits Cl secretion in porcine colon: evidence of cGMP and Ca mediation. Am. J. Physiol. 265 (Regulatory Integrative Comp. Physiol. 34): R605-R613, 1993.

2.   Barker, P. M., K. K. Brigman, A. M. Paradiso, R. C. Boucher, and J. T. Gatzy. Cl- secretion by trachea of CFTR (+/-) and (-/-) fetal mouse. Am. J. Respir. Cell Mol. Biol. 13: 307-313, 1995[Abstract].

3.   Chao, A. C., F. J. deSauvage, Y.-J. Dong, J. A. Wagner, D. V. Goeddel, and P. Gardner. Activation of intestinal CFTR Cl- channel by heat stable enterotoxin and guanylin via cAMP-dependent protein kinase. EMBO J. 13: 1065-1072, 1994[Medline].

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AJP Lung Cell Mol Physiol 273(5):L1065-L1072
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