CFTR is functionally active in GnRH-expressing GT1-7 hypothalamic neurons

Richard T. Weyler1, Karin A. Yurko-Mauro2, Ronald Rubenstein3, Wouter J. W. Kollen3, William Reenstra2,4, Steven M. Altschuler1, Marie Egan5, and Andrew E. Mulberg1

Divisions of 1 Gastroenterology and Nutrition and 3 Pulmonary Medicine, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104; Departments of 2 Clinical Research and 4 Pediatrics, A. I. duPont Hospital for Children, Thomas Jefferson University, Wilmington, Delaware 19803; and 5 Division of Pulmonary Medicine, Yale University School of Medicine, New Haven, Connecticut 06520


    ABSTRACT
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ABSTRACT
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We have demonstrated the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, mRNA, and protein within the rat and human brains, in areas regulating sexual differentiation and function. We have found that GT1-7, a gonadotropin-releasing hormone (GnRH)-secreting hypothalamic neuronal cell line, expresses the CFTR gene, mRNA, and protein and cAMP-dependent 36Cl efflux. A linear 7-pS Cl- conductance, which is stimulated by ATP and cAMP analogs and inhibited by glibenclamide, consistent with CFTR activity, has been identified in GT1-7 cells. Antisense oligo(dN) generated against exon 10 of the CFTR gene transcript (mRNA) inhibit GnRH secretion into media [312 ± 73, 850 ± 150, 963 ± 304, and 912 ± 74 pg GnRH/4 × 106 cells for antisense, sense, missense, and no oligo(dN), respectively; P < 0.029 for antisense oligo(dN)-treated vs. normal cells]. No changes in intracellular synthesis of GnRH were noted [1,400 ± 371 and 1,395 ± 384 pg GnRH/4 × 106 cells for antisense and sense oligo(dN), respectively]. Antisense oligo(dN), but not sense or missense oligo(dN), inhibited cAMP-dependent 36Cl efflux. The expression of CFTR protein, detected by Western blotting, was also inhibited 68% by preincubation of cells with antisense oligo(dN). GT1-7 hypothalamic neurons express the CFTR gene, mRNA, and protein, which modulate neurosecretion. Abnormal neuropeptide vesicle trafficking by mutant CFTR may help to explain some of the diverse manifestations of cystic fibrosis.

neuropeptide secretion; vesicle trafficking; infertility; congenital bilateral absence of the vas deferens; cystic fibrosis transmembrane conductance regulator; gonadotropin-releasing hormone


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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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CYSTIC FIBROSIS (CF) is characterized by defective electrolyte transport of epithelial cells in several organ systems including the lung, pancreas, and genital organs; other tissues, including bovine brain-derived clathrin-coated vesicles (CCV) and rat brain, also express the 1,480-amino acid protein termed the CF transmembrane conductance regulator (CFTR) and the associated mRNA (2, 29-31, 35). CFTR functions as a cAMP-dependent Cl- channel in epithelia, a cAMP-dependent water and bicarbonate channel, a regulator of membrane recycling, a modulator of the outwardly rectified Cl- and Na+ channels, and putatively as a transporter of ATP (3, 4, 8, 14, 32, 38, 39). The exact function of CFTR in the brain remains elusive, but it may regulate membrane trafficking events and secretion of neuropeptides.

CFTR gene and protein expression in brain may correlate to other commonly described defects in children and adults with CF. Infertility results from congenital bilateral absence of the vas deferens (CBAVD) and occurs in 95% of male adults with CF (10, 12, 33). This condition is associated with azoospermia and defects in the anatomic transport of sperm cells, due mainly to the presence of CBAVD. Investigators have identified the 5T genetic polymorphism in which 80% of males with CBAVD express a detectable mutation (5, 10). The pathogenic basis, however, for this anatomic defect remains elusive and unsubstantiated. There are reports of surgical and autopsy specimens of 35 males with CF, ranging in age from 38 days to 25 years, with deficient epididymides and absent vasa deferentia (25). An alternative hypothesis to explain these defects involves the regulation of gonadotropin secretion and secondary abnormal developmental differentiation of the vas deferens and epididymis by mutant CFTR. Others, earlier, have reported the possibility of a developmental defect in Wolffian duct derivatization leading to the absence of the vas deferens (18).

In addition, the hypothalamic-pituitary-gonadal axis matures later in children with CF, which may be unrelated to nutritional state, as suggested by a recent report (22). Female adolescents who were homozygous for the Delta F508 mutation of the CFTR gene and exhibited delays in pubertal development had normal nutritional status (22). Johannesson and colleagues (21) have also demonstrated that the CFTR mRNA is expressed within the cerebral cortex and medial preoptic area, which regulate the visceral and endocrine functions associated with sexual differentiation. The exact nature of the pathogenesis of infertility and delayed sexual maturation could relate to changes in gonadotropin-releasing hormone (GnRH) secretion in a developmentally sensitive manner.

To elucidate the regulation of GnRH secretion by effects on CFTR mRNA expression, the following study was designed. GT1-7 is an immortalized hypothalamic neuronal cell line developed by genetically targeted tumorigenesis by simian virus 40 (SV40) large T antigen coupled to the GnRH promoter. GT1-7 expresses the CFTR gene, mRNA, and protein. We hypothesized that decreased CFTR expression in GT1-7 cells would lead to decreased GnRH release. Our results indicate that antisense oligo(dN) against exon 10 of the CFTR mRNA inhibit CFTR protein expression leading to an inhibition of GnRH secretion. These data suggest that there is an effect of CFTR expression on the regulated GnRH secretory pathway (27, 28, 40).


    METHODS
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Cell culture and preparation. GT1-7 murine hypothalamic cells were a gift from Pamela Mellon (University of California at San Diego). 3T3 CFTR and 3T3 Mock cells were a gift from Michael Welsh (University of Iowa at Iowa City). Each cell type was grown in DMEM (Irvine Scientific, Santa Anna, CA) with 10% fetal bovine serum (Intergen, Purchase, NY) and 1% penicillin-streptomycin (GIBCO BRL, Gaithersburg, MD) in a 5% CO2 humidified atmosphere at 37°C. The GT1-7 cells were split or fed every 2-3 days and harvested with trypsin-EDTA (GIBCO BRL) for RNA isolation (see below for RNA isolation). For other experiments using in situ hybridization, GT1-7 cells were plated on coverslips (Fisher 12 × 12 × 0). After growth to confluency, cells were washed and coverslips were removed from the wells and fixed in 4% paraformaldehyde.

RNA extraction and RT-PCR from GT1-7 cells. Total RNA was isolated from GT1-7 hypothalamic neuronal cells by solubilization in 4 M guanidinium thiocyanate-0.1 M 2-mercaptoethanol and subsequently extracted with phenol-chloroform-2-propanol (6, 37). RNA was precipitated with 2-propanol and washed with 75% ethanol. The isolated RNA was analyzed by formamide gel electrophoresis and quantitated by ultraviolet spectrophotometry. The sequences of the CFTR primers were as follows: sense primer, 5'-GACTACATGGAACACATACCTTCG-3' (bases 2661-2684); antisense primer, 5'-ATAGCAAGCAAAGTGTCGGCTACTC-3' (bases 2918-2894). The primers were chosen from human CFTR exons 14 and 15, respectively (29, 30). Other primer pairs used were derived from exons 3 and 4, 6 and 7, 10 and 11, and 16 and 17 of the human CFTR sequence, shown in Table 1. The beta -tubulin sense and antisense primers were 5'-AAGAAGTCCAAGCTGGAGTTC-3' and 5'-GTTGGTCTGGAATTCTGTCAG-3', respectively.

                              
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Table 1.   Expected sizes of PCR-amplified products by using primer pairs from various exons of CFTR gene

Reverse transcription required 2 µg of total RNA, 5 mM MgCl2 (Promega, Madison, WI), 1 mM (each) dATP, dGTP, dTTP, and dCTP (Promega), 0.5 µg of oligo(dT) primer (Promega), 10 units of RNasin RNase inhibitor (Promega), 100 units of Superscript II RT (GIBCO BRL), 1× PCR buffer II (Perkin-Elmer, Norwalk, CT), and nuclease-free water (Promega) in a final reaction volume of 20 µl in thin-walled microcentrifuge tubes (Marsh Biomedical, Rochester, NY). Controls included water as the negative control, kanamycin RNA as the reverse transcription positive control (Promega), and plasmid pMT-CFTR containing the full-length human CFTR cDNA (generously provided by Genzyme, Framingham, MA). The reaction mixture was incubated for 1 h at 42°C in the OmniGene thermal cycling system (National Labnet, Woodbridge, NJ); this was followed by a heat inactivation step of 70°C for 15 min. The cDNA was amplified directly from the cDNA synthesis reaction mixture in a PCR mixture consisting of 2 mM MgCl2, 100 nM sense primer, 100 nM antisense primer, 2.5 units of AmpliTaq polymerase (Perkin-Elmer), 1× PCR buffer II (Perkin-Elmer), and nuclease-free water (Promega) in a 100-µl volume. The PCR began with a hot-start reaction for 10 min at 95°C and then proceeded for 45 cycles of 94°C for 1 min, 60°C for 45 s, and 72°C for 1 min (plus an incremental 1 s/cycle). The first PCR cycle was preceded by a 94°C incubation for 2 min; the final cycle was followed by a 72°C extension for 5 min. Each product was electrophoresed on a 1.5% agarose gel and stained with ethidium bromide with a 123-bp ladder (GIBCO BRL).

Nonisotopic in situ hybridization of CFTR mRNA in GT1-7 hypothalamic neuronal cells. All solutions were treated with 0.1% diethyl pyrocarbonate and sterilized by autoclave. Tris solutions were sterilized with filters. The CFTR/pGEM-T clone (RBCFTR14-1) was linearized by separate restriction digestion with Spe I and Sac II as previously described (29, 31). Digoxigenin-labeled sense and antisense RNA probes were made by in vitro transcription by T7 and SP6 RNA polymerase (Promega), with the Genius 4 RNA labeling kit (Boehringer Mannheim, Indianapolis, IN). The yield of digoxigenin-labeled RNA was quantitated with the Genius 3 nucleic acid detection kit (Boehringer Mannheim). GT1-7 hypothalamic neuronal cells grown on coverslips were washed twice for 5 min in 1× PBS (in mM: 140 NaCl, 2.7 KCl, 10 Na2HPO4, 1.8 KH2PO4) and in 1× PBS-0.1% Tween 20 (PTW). Sections were prehybridized at 52°C for 1 h in hybridization solution (HS) consisting of 50% formamide, 5× sodium chloride-sodium citrate, 100 µg/ml Torula yeast RNA, 100 µg/ml wheat germ tRNA (Sigma, St. Louis, MO), 50 µg/ml heparin, and 0.1% Tween 20. Coverslips were then incubated at 52°C for 3 h in HS with 1.5 µg/ml sense or antisense cRNA probes. After hybridization, coverslips were washed three times for 10 min in HS, for 5 min in PTW-HS, and twice in PTW for 5 min. All washes were at 60°C. Coverslips were then washed in solution containing 3× PBS, 0.1% acetylated BSA (GIBCO BRL), and 0.2% Triton X-100 solution (PBT). Coverslips were incubated at 27°C for 1 h in PBT containing 0.67 U/ml anti-digoxigenin antibody alkaline phosphatase conjugate (Boehringer Mannheim). After two 5-min washes in PBT and in a solution containing 100 mM NaCl, 50 mM MgCl2, 100 mM Tris (pH 9.5), and 0.1% Tween 20 (SMT), sections were incubated in the dark at room temperature for 60 min in SMT. Color was developed with the addition of 175 µg/ml 5-bromo-4-chloro-3-indolylphosphate and of 4-nitro blue tetrazolium chloride in 70% dimethylformamide (Boehringer Mannheim). Coverslips were processed through PBS, graded alcohols, and xylene. Slides were air-dried for 12 h. A sense riboprobe was used as the control for nonspecific hybridization (29, 31). No specific signal could be detected in these control hybridizations.

cAMP-dependent 36Cl in GT1-7 hypothalamic neuronal cells. For 36Cl experiments, GT1-7 hypothalamic neuronal cells were grown to confluency on 35-mm plates. Cl- channel activity was assayed by measuring the rate of efflux as previously described with 36Cl, which sensitively measures Cl- permeability across CFTR (34, 36). Briefly, cells were grown to 80-90% confluency and loaded with 3 µCi of 36NaCl/dish under equilibrium conditions with bicarbonate-free Ringer buffered with 10 mM HEPES, pH 7.4, for 2-4 h at 37°C. Cells were washed three times with 1 ml of ice-cold Ringer before the addition of 1 ml of Ringer with (stimulated) or without (basal) forskolin (12.5 µM) and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (500 µM; Sigma). The Ringer was replaced every 15 s and counted by liquid scintillation in CytoScint (ICN Research Products, Costa Mesa, CA). Cells were lysed after 3 min with 0.2 N NaOH, and residual 36Cl was determined by liquid scintillation. Apparent first-order rate constants for Cl- secretion were determined from three to four independent time courses for each condition by a nonlinear least-squares method. The overall rate law for this process is an algebraic sum of the rates for these individual paths: efflux (percent remaining) = e-k1t + e-k2t + · · · + e-knt where k is the rate constant and t is the time. At early times (initial rate conditions), the complex rate of efflux can be, for purposes of comparison, approximated by an apparent or pseudo-first-order rate constant as e-kappt e-k1t + e-k2t + · · · + e-knt.

The term kapp is the apparent pseudo-first-order rate constant, which can be determined from a semilog plot of the fraction of Cl- remaining in cells vs. time, where kapp is the slope of this plot (36). Statistical analysis of these rate constants was performed by using a two-tailed t-test. For the effects of various oligo(dN) on 36Cl efflux, experiments were conducted as described in Transfection of GT1-7 hypothalamic neuronal cells with CFTR oligo(dN) below. 36Cl efflux was measured as described above.

Western blotting of CFTR protein in GT1-7 hypothalamic neuronal cells. For protein extraction from transfected cell lines, including the 3T3 CFTR, 3T3 Mock, and GT1-7 hypothalamic neuronal cells, cell monolayers were washed three times with ice-cold PBS containing 0.1 mM phenylmethylsulfonyl chloride (Sigma). Cells were lysed by the addition of 3× Laemmli buffer; whole cell lysates (50 µg) were analyzed by SDS-PAGE (6% polyacrylamide) by the method of Laemmli (26). Western blotting was performed as described in previously published protocols used for CCV membranes, whole cell lysates of CFTR-overexpressing cell lines including 3T3 CFTR, and rat brain homogenates (30). Subsequent to SDS-PAGE, proteins were electroblotted to nitrocellulose 45-µm membranes (Bio-Rad) for 1 h at 100 V and 4°C. CFTR was detected by Western blotting using affinity-purified anti-CFTR antibody pAb3145 at a dilution of 1:250. Buffer contained 5% nonfat dry milk in 150 mM NaCl-10 mM Tris (pH 7.5) containing 0.1% Triton X-100 and 1 µg/ml protease inhibitors including aprotinin, chymostatin, pepstatin A, leupeptin, and antipain (TTBS buffer; Sigma). The blots were incubated at 4°C overnight for 16 h with gentle rocking. Blots were subsequently rinsed twice in TTBS buffer. Secondary donkey anti-rabbit horseradish peroxidase-conjugated antibody (Jackson Immunobiologicals, Westgrove, PA) at a dilution of 1:6,000 in TTBS buffer was added to blots for 40 min. The subsequent wash protocol included three 10-min washes, first in TTBS and then in Tris-buffered saline (TBS). Immunodetection was through enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL). For CFTR peptide-inhibited pAb3145, 1 mg of peptide was incubated with antibody overnight at 4°C as previously described (30). Western blotting was performed as described above for non-peptide-inhibited conditions. Under the conditions of the antisense oligo(dN) preincubation experiments, Western blotting of CFTR protein from whole cell lysates was performed as described above.

Electrophysiological characterization of CFTR functional activity in GT1-7 hypothalamic neurons. Egan and colleagues (8, 36) have previously published the experimental protocol for whole cell membrane current determination. Cells were visualized with a Nikon Diaphot phase-contrast microscope, which was mounted on an air table, and patch pipettes were positioned with a Newport micromanipulator. A cell-attached configuration was achieved by having the patch-electrode press against the cell membrane of the intact cell, and then, by applying negative pressure, a high-resistance seal (gigaseal) was formed. Inside-outside patches were subsequently formed. Solutions used included standard NaCl bath [(in mM) 141 NaCl, 1 EGTA, 0.5 CaCl2 (free Ca2+ was 110 nM as measured with fura 2), 5 HEPES, and 2 MgCl2] and standard NaCl pipette solution [(in mM) 141 NaCl, 5 HEPES, and 2 CaCl2]. The pH of both solutions was 7.3. For experiments under stimulated conditions, the cAMP stimulation cocktail in the bath consisted of 1 mM ATP, 10 µM forskolin, and 100 µM IBMX. Glibenclamide was added as an inhibitor of CFTR-dependent Cl- channel activity.

Transfection of GT1-7 hypothalamic neuronal cells with CFTR oligo(dN). Cells at 70% confluency were grown in routine fashion as described in Cell culture and preparation. For these experiments, GT1-7 neuronal cells were incubated overnight in serum-free medium with 1.25 µM sense, missense, or antisense oligo(dN) generated against exon 10 of the mouse CFTR sequence developed at the Department of Genetics Core Facility of the University of Pennsylvania School of Medicine. Sequences chosen were as follows: antisense, 5'-GGCAAGCTTTGACAACACTC-3'; missense, 5'-TATGACCGAGTCATCGACAC-3'; and sense, 5'-GGATTATGCCGGGTACTATC-3'. The oligo(dN) were generated as the phosphorothioate derivatives to maximize their stability. Gel electrophoresis of the phosphorothioates confirmed that the oligo(dN) were pure and were full-length products. After incubation overnight with the oligo(dN), media were harvested as described in Cell culture and preparation, with storage of the cellular fractions for assay of GnRH as described in Column chromatography and RIA of GnRH. Results of secretion before and after transfection with oligo(dN) were compared by Student's t-test and reported as significant if P < 0.05.

In addition, Western blotting of CFTR from GT1-7 cells treated with oligo(dN) was performed as described in Western blotting of CFTR protein in GT1-7 hypothalamic neuronal cells by using 50 µg of whole cell lysate. Densitometric readings of the Western blot corresponding to GT1-7 cells treated with control, antisense, sense, and missense oligo(dN) were recorded from each lane. Fluorographic images were digitized with an AlphaImager 2000 digital analysis system (AlphaInnotech, San Leandro, CA). Densitometric analysis of these images was performed with AlphaImager image analysis software, version 4.0 (AlphaInnotech), with two-dimensional integration of the selected band. For comparisons within an experiment, the density of the control lane (in pixels) was arbitrarily set to 1.0. A t-test was used to determine the statistical significance of changes in the densities of Western blot bands (SPSS Software, version 7.0).

5'-Terminal labeling of oligo(dN) with fluorescein to assess adequacy of cell transfection. To assess the efficacy of transfection, 5'-labeling of the oligo(dN) with fluorescein was performed at the Department of Medical Genetics, University of Pennsylvania School of Medicine. Incubation with 5'-end-labeled oligo(dN) was performed as described above under the same conditions. After overnight incubation, GT1-7 hypothalamic neurons were fixed in 4% paraformaldehyde; slides were rinsed quickly in distilled water, and placed in coverslips with Vectashield (Vector Laboratories, Burlingame, CA). All photomicrographs were obtained with a Leitz DMR research microscope (Leica, Rockleigh, NJ) equipped with a fluorescein emission filter.

Column chromatography and RIA of GnRH. For the assay of GnRH from media, the media were collected and an equal volume of 1% trifluoracetic acid (TFA) diluted in distilled water was added. Media were centrifuged at 15,000 g for 20 min at 4°C. A SEP-COLUMN containing 200 mg of C18 (Peninsula Laboratories, Belmont, CA) was equilibrated by the addition of 1 ml of 60% acetonitrile in 1% TFA in distilled water, followed by three rinses with 3 ml of 1% TFA in water. The supernatant was then added to the column and eluted by two washes with 3 ml of 1% TFA. The peptide was eluted with 3 ml of 60% acetonitrile in 1% TFA and lyophilized. For assay of GnRH from the cell fraction, the monolayers were washed with 0.1 M HCl and homogenized with Dounce until viscosity was not present.

Competitive EIA of GnRH. Lyophilized samples and standard peptide were rehydrated in enzyme immunoassay (EIA) buffer and incubated in 0.5 volumes of primary antisera and biotinylated GnRH peptide for 2 h at room temperature in a 96-well Immunoplate specifically designed to bind primary antibody (Peninsula Laboratories). The wells were washed five times with EIA buffer, streptavidin-conjugated horseradish peroxidase was added, and the wells were incubated for 60 min at room temperature. The Immunoplate was washed five times with EIA buffer, 100 µl of 3,3',5,5'-tetramethylbenzidine dihydrochloride were added, and the Immunoplate was incubated for 1/2 h. The reaction was stopped by the addition of 2 N HCl. The colorimetric reaction product was read at 450 nm in a Spectramax plate reader. A standard curve was then constructed from which quantitation of the unknown samples was made (Peninsula Laboratories).


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Identification of CFTR gene and mRNA expression by RT-PCR and nonisotopic in situ hybridization. We detected the expression of CFTR mRNA by RT-PCR. Amplification of CFTR from GT1-7 cells yielded a fragment of the same size, 257 bp, as that of the fragment produced by RT-PCR of the full-length human CFTR cDNA clone, pMT-CFTR. This clone contains the full-length CFTR transcript and has been described in previous work (29). The appropriate water control yielded no amplification; the kanamycin-positive RNA control yielded the expected 1.2-kb product (Fig. 1). The products of CFTR from GT1-7 hypothalamic neurons, amplified by PCR with the diverse primer pairs from additional exons, were identical on the basis of the CFTR gene sequence and homologies to the murine gene (Table 1).


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Fig. 1.   Detection of cystic fibrosis transmembrane conductance regulator (CFTR) and beta -tubulin mRNA from GT1-7 hypothalamic neurons by RT-PCR. Total RNA (2 µg) from GT1-7 hypothalamic neuronal cells was reverse transcribed with CFTR and beta -tubulin primers and PCR amplified with CFTR or beta -tubulin primers. Amplified products were directly stained with ethidium bromide on agarose gel. CFTR RT-PCR gave a 257-bp product, and beta -tubulin RT-PCR gave a 110-bp product, both as expected. PCRs with other primer pairs revealed amplification products of expected sizes (see Table 1). Lanes: 1, 123-bp DNA ladder (GIBCO BRL); 2, nuclease-free water; 3, kanamycin-positive control; 4, pMT-CFTR, exons 3 and 4; 5, pMT-CFTR, exons 6 and 7; 6, pMT-CFTR, exons 10 and 11; 7, pMT-CFTR, exons 14 and 15; 8, pMT-CFTR, exons 16 and 17; 9, GT1-7, beta -tubulin; 10, GT1-7, exons 3 and 4; 11, GT1-7, exons 6 and 7; 12, GT1-7, exons 10 and 11; 13, GT1-7, exons 14 and 15; 14, GT1-7, exons 16 and 17; 15, 123-bp DNA ladder.

The CFTR mRNA was detected in all nonisotopic in situ hybridization experiments conducted as part of this study. Clusters of GT1-7 neuronal cells grown on coverslips highly express CFTR mRNA, as seen in Fig. 2, A-D. There is no detectable background staining visible in Fig. 2A; intense cytoplasmic punctate and diffuse staining is reflected in clusters of the GT1-7 neuronal cells (Fig. 2B). Figure 2, C and D, represents the low-power and high-power photomicrographs of the sense hybridization conditions. Arrows represent the dendritic processes extending from the GT1-7 cells, which develop syncytia as originally described by Mellon and colleagues (28).


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Fig. 2.   Nonisotopic in situ hybridization of CFTR mRNA in GT1-7 hypothalamic neurons. Coverslips containing GT1-7 hypothalamic neurons were hybridized to 1.5 µg/ml digoxigenin-labeled sense (A) or antisense (B) cRNA probes and were developed against cloned rat brain CFTR in pGEM-T expression vector as previously described (2). C and D: low- and high-power photomicrographs of sense and antisense hybridization conditions, respectively. Coverslips of neuronal cells were developed by an alkaline phosphatase-dependent colorimetric reaction. Antisense probes produced strong signal in cell bodies, whereas sense riboprobe produced no specific signal. Arrows, dendritic processes extending from GT1-7 cells, consistent with syncytial formation as described previously (28).

cAMP-dependent 36Cl efflux in GT1-7 neuronal cells. Basal and stimulated cAMP-dependent 36Cl effluxes are reported as pseudo-first-order rate constants (kapp; units of min-1; mean ± SE) (Figs. 3, A and B, and 4). A representative time course of the basal 36Cl efflux experiments is shown in Fig. 3, A and B. In the presence of no inhibitor, 36Cl efflux increased twofold in the presence of forskolin-cAMP (Fig. 3B; P < 0.02). These results are typical for CFTR-expressing cells.


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Fig. 3.   A: representative time course of 36Cl efflux in GT1-7 hypothalamic neurons. Interpretation of exponential decay is described in METHODS. B: basal and cAMP-stimulated cAMP-sensitive Cl- efflux measured from GT1-7 hypothalamic neurons. Significant difference between basal and stimulated effluxes is depicted and further described in text. kapp, apparent pseudo-first-order rate constant.



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Fig. 4.   Effect of oligo(dN) on basal cAMP-stimulated 36Cl efflux. There is inhibition of cAMP-stimulated Cl- efflux by antisense oligo(dN) (P < 0.04) and no significant effect of either sense or missense oligo(dN) on basal or cAMP-stimulated efflux. Data are means ± SE; n = 5 experiments.

The effects of various oligo(dN) show further confirmation of the specificity of the effect of CFTR mediating these fluxes on basal and stimulated cAMP-dependent 36Cl efflux (Fig. 4, A-C). Significant inhibition of 36Cl efflux was observed for antisense oligo(dN) (P < 0.04), but not for sense or missense oligo(dN). This is consistent with a direct effect on CFTR and was confirmed as described in Electrophysiological characterization of CFTR in neuronal cells and the next section.

Western blot of CFTR protein in GT1-7 hypothalamic neurons. Western blotting of CFTR protein is shown in Fig. 5, A and B. As depicted in Fig. 4A, 3T3 CFTR and the GT1-7 hypothalamic neuronal cell lines, but not the 3T3 Mock cell line, expressed the fully glycosylated form of CFTR with an approximate molecular mass of 150-165 kDa for band C. This is consistent with previous reports (30). After incubation of pAb3145 with the CFTR tridecapeptide used for immunizing the rabbits for generating the antibody, no CFTR protein from whole cell lysates derived either from the 3T3 CFTR or GT1-7 cell lines was detected (Fig. 5B).


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Fig. 5.   Western blot of CFTR protein in immortalized GT1-7 hypothalamic neuronal cells. Peptide-inhibited conditions are shown in B; see text for details. Lane 3T3 CR, 3T3 CFTR-transfected cells; lane 3T3 MK, 3T3 Mock-transfected cells; lane GT1-7, GT1-7 hypothalamic neurons. Molecular weight markers are at left. Molecular mass of fully glycosylated band is ~150-165 kDa.

Electrophysiological characterization of CFTR in neuronal cells. CFTR functional Cl- channel activity in patch-clamp experiments is demonstrated in Fig. 6. The linear current-voltage relationship of CFTR is demonstrated in Fig. 6A, which resulted from an experiment performed with 12/20 patches, confirming that a linear-conductance, 6.9-pS Cl- channel was identified in the GT1-7 hypothalamic neuronal cells. The channel activity was activated by 100 µM IBMX and 10 µM forskolin; when the patch was excised in ATP-free solution, four of four channels ran down. The CFTR Cl- channel activity is sensitive to the same cocktail of stimulators described for CFTR in heterologous systems (36). Reactivation of the Cl- channel activity resulting from the addition of 1 mM ATP to the bath is shown in Fig. 6B; activity was also inhibited by the addition of glibenclamide (n = 2).


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Fig. 6.   A: current-voltage relationship of Cl- channels from patch-clamp experiments. For experimental details of patch-clamping of the GT1-7 hypothalamic neuronal cells, see text. Average single channel conductance was 6.9 pS (see text). B: CFTR Cl- channel activity in GT1-7 neuronal cells. Shown are representative traces of channel activity after treatment of GT1-7 cells with a cAMP stimulation cocktail consisting of 10 µM forskolin and 100 µM IBMX. Traces 1 and 2 (numbering from top) show cell-attached channel activity. There are two active channels in trace 1 and a single active channel in trace 2. Trace 3 shows activity of a single channel 30 s after patch excision; trace 4 represents rundown of channel 60 s after excision. CFTR channel activity is expected to run down in the absence of ATP. There is reactivation of CFTR channel activity with restitution of 1 mM ATP in bath as shown in trace 5. Arrows, baseline (all channels closed); dotted lines, channel levels.

Effect of antisense oligo(dN) on GnRH release and CFTR protein levels. Transfection of GT1-7 cells with oligo(dN) generated against CFTR exon 10 gave reproducible and consistent inhibitory effects on GnRH secretion (n = 6 experiments). As shown in Fig. 7, there was inhibition of GnRH release compared with control levels. Mean GnRH releases into media in the uninhibited and antisense oligo(dN)-treated conditions were 912 ± 74 and 312 ± 266 pg GnRH/4 × 106 cells, respectively (P < 0.029; Student's t-test; n = 4). There was no inhibitory effect of either missense or sense oligo(dN) after the same period of incubation, compared with either antisense oligo(dN)-treated or uninhibited conditions: mean GnRH releases were 963 ± 307 and 850 ± 150 pg GnRH/4 × 106 cells for missense and sense oligo(dN) treatment, respectively, neither of which was significantly different from the value for uninhibited conditions. However, intracellular levels of GnRH showed no changes after treatment with either sense or antisense oligo(dN). These data support the hypothesis of CFTR-dependent effects on GnRH secretion into media rather than an effect on GnRH synthesis (antisense and sense treatment produced levels of 1,400 ± 371 and 1,395 ± 384 pg GnRH/1 × 106 cells, respectively; n = 6 experiments; no significant difference).


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Fig. 7.   GnRH secretion in GT1-7 hypothalamic neuronal cells. Cells were treated with missense, sense, or antisense oligo(dN) to the CFTR mRNA as described in METHODS. Data show reduction in secretion of GnRH between control and antisense conditions (P < 0.029) but no significant differences between sense, missense, and control conditions (see text).

Furthermore, after antisense oligo(dN) incubation with GT1-7 cells, a 68% decrease in protein level compared with levels after incubation with sense and missense oligo(dN) was shown, confirming a specific effect of the oligo(dN) (Fig. 8, A and B). Figure 8B represents a graphical distribution of the densitometric recording from the lanes in Fig. 8A. Although apparently contrary to hypothesis, no greater effect on the decrease in CFTR protein after longer incubations with the oligo(dN) was confirmed (data not shown). As a control to assess the adequacy of GT1-7 cell transfection, 5'-terminal labeling of all oligo(dN) with fluorescein was performed. As shown in Fig. 9A, there is a bright fluorescence pattern that is observed within the cytoplasm and nuclei of transfected hypothalamic neurons. Cells transfected with unlabeled oligo(dN) did not demonstrate visible fluorescence, consistent with absence of transfection (data not shown). A phase-contrast micrograph of this experiment is shown in Fig. 9B. The observed cellular fluorescence supports the view that the ablation of CFTR mRNA by antisense oligo(dN) resulted in a corresponding effect on GnRH secretion.


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Fig. 8.   A: Western blot of CFTR protein from GT1-7 whole cell lysates after incubation with oligo(dN). Type of oligo(dN) pretreatment is indicated above lanes. "C" band of CFTR is appropriately depicted. B: densitometric analyses of immunoblots (3 independent experiments), performed as described in METHODS. Shown is mean pixel density ± SE, relative to control. Graph is quantitative depiction of decrease in CFTR protein level after incubation with various oligo(dN). Significance was determined in comparison to normal GT1-7 cell incubation. Normal incubation vs. incubation after sense and missense oligo(dN) pretreatment: no significant difference; normal incubation vs. incubation after antisense oligo(dN) pretreatment: P < 0.03.



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Fig. 9.   5'-Terminal labeling of phosphorothioate oligo(dN) to assess adequacy of GT1-7 hypothalamic neuronal cell transfection. Oligo(dN) labeled at 5' end were enzymatically modified to contain a fluorescein terminal label. Experiments using these labeled oligo(dN) were performed as described in METHODS. A: GT1-7 hypothalamic neurons that were fixed in 4% paraformaldehyde after overnight incubation with 1.25 µM 5'-fluorescein-tagged oligo(dN). Dense labeling is visible within multiple neuronal cells. B: phase-contrast micrograph of above image.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Sexual differentiation is regulated by mechanisms of synthesis, packaging, and secretion of neurotransmitters in the hypothalamic-pituitary axis (7, 9, 23). Secretion of hypothalamic hormones through the hypophyseal-portal system regulates hormones secreted by the anterior pituitary gland. The developmental sequence of fetal pituitary gonadotropin synthesis and secretion is integrated with morphogenesis of the hypothalamus and hypophyseal-portal system in fetal brain (24). The principal regulator of sexual differentiation involves the role of GnRH. The temporal relationship between fetal serum testosterone and luteinizing hormone suggests a role for GnRH in initiating and maintaining testosterone secretion. Testosterone levels are augmented by pituitary luteinizing hormone and human chorionic gonadotropin, leading to growth of the penis and scrotum and descent of the testes.

Effects on intracellular packaging and secretion of vesicles containing neuropeptides, e.g., GnRH, may regulate the developmental expression of luteinizing hormone and the pathogenesis of CBAVD (23). Previous data have demonstrated the expression of CFTR mRNA and protein in rat brain; in addition, one of us (A. E. Mulberg) has demonstrated the presence of the CFTR mRNA and gene in the human anterior hypothalamus, a region highly expressing GnRH (29-31, 42). In this study we demonstrated the expression of CFTR mRNA and protein and cAMP-dependent 36Cl efflux and confirmed CFTR expression with electrophysiological evidence of CFTR as a linear-conductance, approx 7-pS Cl- channel. These data were supplemented by data showing the inhibition of CFTR function, including Cl- conductance, in GT1-7 hypothalamic neuronal cells by using diphenylamine-2-carboxylate (DPC), glibenclamide, and antisense oligo(dN) against the CFTR mRNA. In addition CFTR expression detected by Western blotting and GnRH release from GT1-7 hypothalamic neurons were inhibited specifically by antisense oligo(dN).

Endocytosis and exocytosis of secretory granules involve both CCV and non-clathrin-dependent pathways. CFTR may regulate these processes. Functional CFTR has been identified in endosomes of stably transfected Chinese hamster ovary cells, human sweat duct cells, and T84 cells (1, 3, 4). CFTR protein in CCV derived from bovine brain has been documented (30); CFTR mRNA and protein are expressed within dendrites and neuronal cell bodies from specific areas of the rat diencephalon including the thalamus and hypothalamus (29, 30). These regions of the diencephalon regulate sexual differentiation, a physiological function that is abnormal in patients with CF.

The linkage of CFTR to regulation of neurosecretion may be mediated through second messengers, including cAMP. An elevation in intracellular cAMP causes secretion of luteinizing hormone-releasing hormone (LHRH) from neurons and ACTH from pituitary cells and production and secretion of somatostatin from hypothalamic neurons (13, 15-17, 19, 23). Synthetic cAMP analogs, 8-bromo-cAMP (8-BrcAMP), and the phosphodiesterase inhibitor IBMX yield similar effects (13, 15-17, 19, 23). Pituitary secretion of ACTH occurs in a regulated and constitutive fashion. 8-BrcAMP stimulates release of mature ACTH four- to fivefold but has no effect on release of the ACTH precursor. In mouse pituitary (AtT-20) cells incubated in the presence of 5 µM forskolin and 500 µM DPC, an inhibitor of CFTR Cl- conductance, ACTH secretion decreased by 48% (17). Neuronal secretion of GnRH in hypothalamus may be similarly regulated through CFTR trafficking within intracellular compartments.

CCV are associated with secretory granules in the GnRH-secreting cell line GT1-7, an immortalized hypothalamic neuronal cell line developed by genetically targeted tumorigenesis by SV40 large T antigen coupled to the LHRH promoter. A constitutive and regulated GnRH and pre-pro-GnRH secretory pathway has been demonstrated (27, 28, 40). GT1-7 hypothalamic neuronal cells maintain the pulsatile pattern of GnRH secretion that is necessary for reproduction. Activation of the protein kinase A and C pathways by 10 µM forskolin and 100 nM 12-O-tetradecanoylphorbol 13-acetate, respectively, exerts stimulatory effects on GnRH processing and secretion (40). Stimulation of GnRH secretion by forskolin and activators of the protein kinase C pathway suggests that secretion occurs through a constitutive, unregulated pathway releasing pro-GnRH precursor and a regulated secretory pathway leading to GnRH secretion (11, 40, 41).

To define the regulation of secretion of GnRH by CFTR, we extend our previous observation that multiple areas of the brain, including brain-derived CCV, express CFTR protein and mRNA. Whether CFTR regulates neuropeptide secretion, neurotransmission, and abnormal homeostatic functions in humans remains unknown but is suggested by previous work. We propose that other common nonpulmonary manifestations of CF, e.g., absence of the vas deferens, may reflect altered neuronal functions through disruption of normal neuropeptide vesicle trafficking by mutant CFTR (29, 30).

The regulation of GnRH may be involved in fetal differentiation of the vas deferens and the male reproductive tract. The exact pathway leading to the evolution of an abnormal vas deferens in the male with CF remains unclear; hypotheses include developmental defects or luminal obstruction of the vas deferens leading to atrophy and secondary infertility (10). These data support the view that a genotype-specific expression of mutant CFTR in brain may affect neuropeptide secretion in brain regions involved in the regulation of sexual differentiation. Experiments utilizing human brain must correlate with these in vitro results.


    ACKNOWLEDGEMENTS

A. E. Mulberg was supported by the Howard Heinz and BJNB Foundations and Development Funds of Children's Hospital of Philadelphia. A. E. Mulberg and S. M. Altschuler were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-02077 and R01-DK-44487, respectively. W. W. Reenstra was sponsored through BJNB, K. A. Yurko-Mauro was sponsored through Nemours, and R. C. Rubenstein was sponsored through the Cystic Fibrosis Foundations.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. E. Mulberg, Children's Hospital of Philadelphia, Division of Gastroenterology and Nutrition, 34th St. and Civic Center Blvd., Abramson 416B, Philadelphia, PA 19104 (E-mail: Mulberg{at}emailchop.edu).

Received 11 December 1998; accepted in final form 20 May 1999.


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