1 Center for Human Genetics, Department of Genetics, and 2 Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio 44106-4948
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ABSTRACT |
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Epithelia of humans and mice with cystic fibrosis are unable to secrete chloride in response to a chloride gradient or to cAMP-elevating agents. Bioelectrical properties measured using the nasal transepithelial potential difference (TEPD) assay are believed to reflect these cystic fibrosis transmembrane conductance regulator (CFTR)-dependent chloride transport defects. Although the response to forskolin is CFTR mediated, the mechanisms responsible for the response to a chloride gradient are unknown. TEPD measurements performed on inbred mice were used to compare the responses to low chloride and forskolin in vivo. Both responses show little correlation between or within inbred strains of mice, suggesting they are mediated through partially distinct mechanisms. In addition, these responses were assayed in the presence of several chloride channel inhibitors, including DIDS, diphenylamine-2-carboxylate, glibenclamide, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and a protein kinase A inhibitor, the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS). The responses to low chloride and forskolin demonstrate significantly different pharmacological profiles to both DIDS and Rp-cAMPS, indicating that channels in addition to CFTR contribute to the low chloride response.
cystic fibrosis; cystic fibrosis transmembrane conductance regulator; ion channel inhibitors; protein kinase A
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INTRODUCTION |
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CYSTIC FIBROSIS (CF) is an autosomal recessive disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR; see Refs. 17, 25, 26). CFTR can function as a cAMP-regulated chloride channel (2, 3) and also appears to affect the function of other ion channels, including the epithelial sodium channel (12, 33), the outward rectifying chloride channel (ORCC; see Refs. 7 and 27), and the renal inward rectifying potassium channel (22). Most of the characterization of CFTR as an ion channel or regulator of other channels has been restricted to cell lines and heterologous expression systems in part because of the limitations of in vivo assays. An exception is utilization of the transepithelial potential difference (TEPD) measurement in vivo as a means to monitor electrophysiological changes across an intact tissue.
The nasal TEPD measurement was originally described (18, 19) as a means to discriminate between CF and non-CF individuals based on bioelectrical potential difference (PD) properties that differ between the two groups. With the use of this assay, it was observed that the unstimulated basal PD measurement is hyperpolarized to a greater degree in CF compared with non-CF tissue. This difference was shown to be predominantly the result of amiloride-sensitive sodium absorption. In the presence of amiloride, the PD can be hyperpolarized by imposing a chloride gradient on the epithelium such that the luminal chloride concentration is low relative to the serosal fluid. The PD can be further hyperpolarized by stimulation with agents that raise cAMP levels. Both of these hyperpolarizing responses are absent or dramatically reduced in CF (20), indicating that they are CFTR dependent at some level.
It has been inferred from in vitro experiments that the chloride permeability increase that occurs in response to increased cAMP levels is the result of activation of CFTR channels (6, 24). However, the mechanism responsible for the hyperpolarization in response to a chloride gradient is more questionable. A reasonable hypothesis is that the low chloride response reflects the basal level of CFTR activity. Unfortunately, genetic and nongenetic heterogeneity in humans makes characterization of factors involved in the electrophysiological gradients measured by the PD difficult. However, with slight modification, this assay can be applied to the mouse (8-10, 14-16, 23, 29, 30, 32) in which both types of heterogeneity can be minimized. We have found that the assay is very reproducible when animals are maintained in a consistent manner and matched for age and gender when assayed (unpublished observations). Our aim in using the PD assay with a mouse model is to investigate the mechanisms responsible for the low chloride response. Because this response is absent in patients with CF, understanding its physiological basis may aid in a greater understanding of the pathophysiology in CF. If channels other than CFTR are involved, their identification could potentially provide additional therapeutic targets.
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MATERIALS AND METHODS |
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Animals.
Female mice of the A/J, C57BL/6J, DBA/2J, BALB/cJ, C3H/HeJ, and
129/SvEms-+Ter?/J strains were obtained from Jackson Laboratory (Bar
Harbor, ME). Mice with the Cftr mutation F508 on a mixed 129/SvEv and C57BL/6J background (37) were a generous gift
from Dr. Kirk Thomas at the University of Utah School of Medicine. As
noted, in some experiments, mice used had the
F508 mutation backcrossed onto the C57BL/6J background. Experiments were performed when the mice were between 12 and 31 wk of age. The project was approved by the Case Western Reserve University Institutional Animal
Care and Use Committee.
Nasal PD measurements.
The PD across the nasal epithelium of the mice was measured according
to procedures previously described elsewhere (11, 16),
with some modifications. Briefly, mice were anesthetized with 11 mg/ml
ketamine, 2 mg/ml xylazine, and 0.4 mg/ml acepromazine in PBS. Animals
were dosed intramuscularly with ~0.003 ml/g mouse weight for baseline
measurements and 0.0075 ml/g for the assay in the presence of
pharmacological agents requiring longer periods of anesthesia. A PE-10
tube stretched to one-half the original diameter was inserted 2 mm into
the mouse nostril and placed against the septum. With the use of a
syringe pump (model A-99; Razel Scientific Instruments) and 3-ml
syringes, Ringer solution warmed to 37°C was perfused into the
nostril at a rate of ~7 µl/min. Valves controlled which solution
entered the nostril, with a 30-s delay until the new solution reached
the nasal epithelium. Readings were taken until a steady-state value
was reached before perfusing the nasal epithelium with a new solution.
Filter paper wicks were used to absorb excess liquid from the mouth and
opposite nostril of the mouse being tested. Bridges (4% agar)
connected the tubing to calomel electrodes, and each bridge was made in
the same solution as the perfusate it measured (HEPES-buffered Ringer
or chloride-free HEPES-buffered Ringer). A needle containing 4% agar
in HEPES-buffered Ringer was placed subcutaneously in the mouse's back
and connected to another calomel electrode to serve as a reference. The
measuring and reference calomel electrodes were situated in a KCl bath
in which one end of the agar bridge was also immersed. The other end of
the measuring electrode's bridge was in contact with the perfusate,
~20 cm from the mouse nose. The transepithelial difference was
measured with a voltmeter (model ISO-DAM-D; World Precision Instruments), and the signal was recorded on a chart recorder (model
BD112; Kipp and Zonen). Solution resistance from the measuring electrode's agar bridge to the mouse nose was calculated to be ~1.2 × 106 , whereas input resistance of the
amplifier was >1012
. The PD measurements were not
corrected for junction potentials, and changes in PD values were
calculated by subtracting the final steady-state value from the first
value taken after switching solutions. Before measurements, the system
was zeroed by adjusting the voltmeter after connecting the reference
needle to the tube normally placed in the mouse nose to complete the circuit.
Solutions and drugs.
The Ringer solutions used to perfuse the nasal epithelium included
HEPES-buffered Ringer with (in mM) 10 HEPES, pH 7.4, 138 NaCl, 5 KCl, 5 Na2HPO4, 1.8 CaCl2, and 1 MgSO4 and chloride-free HEPES-buffered Ringer with (in mM)
10 HEPES, pH 7.4, 138 sodium gluconate, 5 potassium gluconate, 2.5 Na2HPO4, 3.6 hemicalcium gluconate, and 0.5 MgSO4 where chloride ions were replaced with gluconate.
Drugs added to the Ringer solutions included amiloride (104 M; Sigma, St. Louis, MO), forskolin
(10
5 M; Calbiochem, La Jolla, CA), glibenclamide (5 × 10
4 M; Calbiochem),
5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 10
4 M;
Calbiochem), DIDS (5 × 10
4 M; Calbiochem),
diphenylamine-2-carboxylate (DPC; 10
3 M; RBI, Natick,
MA), and the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS; 10
4 M; BioLog,
La Jolla, CA). Except for forskolin, stock solutions of the drugs were
prepared on the day of the experiment.
Statistics. Data are presented as means ± SE. If an individual mouse was tested multiple times, the data were averaged and presented as a single data point in the group mean. Statistics were performed using Statview (versions 4.5 and 5.0; SAS Institute, Cary, NC) using either an unpaired Student's t-test, ANOVA followed by Dunnett's post hoc test, or Fisher's r-to-z covariance analysis as indicated. P < 0.05 was considered statistically significant.
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RESULTS |
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Effect of CFTR(F508/
F508) mutation on the nasal TEPD.
The TEPD measurement predominantly reflects the level of sodium and
chloride transport across the airway epithelium in vivo. For this work,
the TEPD assay was separated into two separate traces to increase mouse
survival. First, a baseline TEPD was measured while the epithelium was
perfused with an isotonic Ringer solution until a stable value was
determined. A second trace was made up of three distinct phases.
Initially, amiloride-sensitive sodium absorption was inhibited by
perfusion with Ringer solution containing amiloride. Once a
steady-state value was reached, a chloride gradient was created across
the epithelium by perfusion with chloride-free Ringer solution in which
chloride ions were replaced with impermeant gluconate ions to stimulate
chloride secretion into the lumen. Once a new steady state was reached and the change in potential difference (
TEPD) was determined, forskolin was added to the chloride-free Ringer solution to stimulate adenylate cyclase and raise cAMP. The response to cAMP was measured again until a steady state was reached, and the
TEPD was determined again.
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The nasal TEPD chloride-free and forskolin responses show little correlation. The lack of chloride transport in CF in response to low chloride challenge or to stimulation by forskolin suggested that both transport mechanisms may occur through CFTR. It has been suggested that, in non-CF humans, the hyperpolarization that occurs in response to a chloride gradient is the result of basal levels of chloride secretion and that the hyperpolarization with the further addition of isoproterenol is a result of stimulated chloride secretion, both occurring through CFTR (20). Therefore, it was hypothesized that because of limiting amounts of CFTR and its regulatory compounds, a larger CFTR basal chloride response would result in a reduced CFTR-stimulated chloride response and vice versa.
We tested this hypothesis in mice by comparing the changes in PD caused by chloride-free Ringer solution and those caused by the further addition of forskolin to stimulate CFTR chloride secretion. We found no correlation for the two responses between several inbred strains (Fig. 2) or within an individual inbred strain (Fig. 3). We found that the forskolin response measured in six inbred strains of mice varies little between the strains. This contrasts with large variations in the low chloride response measured in the same strains (Fig. 2). Specifically, the C57BL/6J strain had the most negative low chloride response (
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Effect of chloride channel inhibitors on the chloride-free and
forskolin responses.
The lack of correlation between the low chloride and forskolin
responses raised the possibility that the low chloride response may
involve channels other than CFTR. To test this possibility further, we
examined the effect of several chloride channel inhibitors that have
been characterized as to their effect on CFTR on the low chloride and
forskolin responses in C57BL/6J mice. Figure 4 shows the effect of
DPC, which is known to inhibit CFTR and other chloride channels
(4, 21, 35, 36, 38), and DIDS, which is known to inhibit
chloride channels other than CFTR (5, 13, 28, 35). DIDS
and DPC have little effect on the response to a low chloride gradient
compared with that in control mice, as shown in Fig. 4A
(TEPD =
19.5 mV for control,
17.5 mV for DPC, and
17.5 mV
for DIDS). In contrast, DIDS and DPC had the expected results on the
forskolin response (Fig. 4B), presumed to be mediated by
CFTR. DPC reduced the forskolin response to
1.9 mV compared with the
control (
TEPD =
7.0 mV), whereas DIDS (
TEPD =
7.0
mV) had little effect.
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Effect of a protein kinase A inhibitor, Rp-cAMPS, on the
chloride-free and forskolin responses.
CFTR is regulated by cAMP-dependent phosphorylation and by
intracellular ATP (1). By inhibiting protein kinase A
(PKA) with Rp-cAMPS, we investigated the effect of acute PKA
inhibition on both the low chloride and forskolin responses. Figure
5 demonstrates that inhibiting PKA
eliminates the forskolin response (TEPD =
6.8 ± 1.6 mV
for control and
0.4 ± 1.4 mV for Rp-cAMPS,
P = 0.015). In contrast, Rp-cAMPS had no
significant effect on the response to low chloride challenge
(
TEPD =
18.4 ± 1.4 mV for control and
17.4 ± 1.7 mV for Rp-cAMPS). There was no effect of
Rp-cAMPS on the low chloride response when the duration of low chloride exposure was extended (data not shown), suggesting the
lack of effect is not an issue of drug access to the cell.
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DISCUSSION |
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The absence or reduction of functional CFTR is clearly the fundamental defect in CF. However, it is not clear how this deficit is involved in the pathophysiology of the disease. Loss of CFTR results in a failure of the airway epithelium to secrete chloride not only in response to cAMP but also in response to a chloride gradient. Concomitantly, there is an increase in sodium absorption across the airway epithelium. The loss of response to cAMP appears to be the result of the loss of both CFTR and a channel (ORCC) whose physiological role is unknown, at least in some airway cells. The ORCC activity appears to be distinct from CFTR in channel pharmacology as well as in activation and conductance properties. It is nonetheless CFTR dependent, since CF airway cells heterologously expressing functional CFTR display ORCC activity in response to cAMP (27).
The chloride-free response, also absent from CF epithelia, is clearly CFTR dependent at some level. The mechanism responsible for this basal conductance has not been identified. A reasonable assumption would be that it represents basally active CFTR, possibly because of resting levels of cAMP and PKA activities. In human nasal TEPD studies, it is the combination of the low chloride and cAMP-stimulated responses that discriminates between CF and non-CF individuals, leading to the postulation that the responses reflect two activity states of CFTR, a basal state and a stimulated state (20). If so, one might predict an inverse relationship between the two responses.
The results presented here show that the chloride-free and cAMP responses are distinct. Our murine studies found a lack of correlation between the chloride-free and cAMP-stimulated responses either within or between inbred strains of mice. Additionally, we found that each response had a characteristic pharmacology both to a panel of chloride channel inhibitors and to a PKA inhibitor that differentiated the two responses. Although it cannot be excluded that there are channel activities common to both responses, there must be activities distinct to each. This suggests that the response to low chloride challenge may reflect channels or regulatory processes that are CFTR dependent, analogous to the ORCC properties of single cells, where CFTR is required for an ORCC-mediated chloride conductance, but neither channel is the sole carrier of the apical epithelial chloride conductance.
In trying to identify the mechanism involved in the low chloride response, we have attempted to reconcile with data on known channels. A caveat to that approach is that most channels are characterized at the channel or cell level to remove the complexities of intact tissue from the analysis. Tarran et al. (34) have shown that, even within ciliated cells of mouse nasal epithelium, there are at least two cell types with regard to unstimulated chloride transport. When one considers that there are even more epithelial cell types and that some ion permeation occurs paracellularly, the intact epithelium is certain to display characteristics not found in cultured or individual primary cells. So, although the TEPD assay is limited in the detail with which electrophysiological responses can be described, it does allow for the intact epithelium to be monitored in vivo.
We propose that the simplest and most consistent explanation of our data is that the low chloride response is transcellular and involves channels distinct from CFTR. Furthermore, the same response is virtually absent in CF mice, establishing a requirement of the low chloride response for CFTR. Although the nature of the requirement for CFTR in the chloride-free response is unclear, we can propose several modes of interaction. It is possible that CFTR may dimerize with other chloride channels such that mutations in CFTR alter chloride transport through these partner channel(s). Alternatively, the observation that different subsets of airway epithelial cells express different chloride conductances raises the possibility that the stoichiometry of cell types is altered in CF and the proportion of cell types responsible for the chloride-free response are reduced or absent in CF mice. Finally, CFTR may play a role in regulating signaling in cells of the epithelium, or its lack may trigger signals that downregulate the low chloride participants. Previous studies support this last possibility. We have shown that nitric oxide synthase-2 levels are decreased in CF airway epithelia (32). Furthermore, we have demonstrated that decreasing nitric oxide synthase-2 activity either acutely by pharmacological means or constitutively by knocking out the Nos2 gene reduces the chloride-free response to levels observed in CF mice (8). The causal link between reduced CFTR and nitric oxide synthase-2 is not clear but could explain the CFTR dependence of the low chloride response as one of the aberrant signaling processes. These and other possibilities need to be explored further.
The difference in the low chloride response observed between several inbred mouse strains in this study permits the genetic dissection of the low chloride response. Studies are thus currently underway to genetically identify the regulatory mechanisms responsible for the low chloride response. Regardless of the mechanism, if ion transport is contributing to the pathophysiology in CF patients, there is no reason to expect any one of the processes, lack of cAMP response, lack of low chloride response, or increased sodium absorption, to be more important than the other. Consequently, we need to better understand each of the processes so that they can be manipulated singularly and jointly to determine their relative roles in the disease process.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Specialized Center of Research Grant HL-60293, Core Center Grant DK-27651, and Training Grant HL-07415.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. L. Drumm, Dept. of Pediatrics, Case Western Reserve Univ., 8th floor BRB, 10900 Euclid Ave., Cleveland, OH 44106-4948 (E-mail: mxd34{at}po.cwru.edu).
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.
Received 5 February 2001; accepted in final form 11 July 2001.
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