Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06510; and Mount Desert Island Biological Laboratory, Salisbury Cove, Maine 04672
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ABSTRACT |
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In the shark, C-type natriuretic peptide (CNP) is the only
cardiac natriuretic hormone identified and is a potent activator of
Cl secretion in the rectal
gland, an epithelial organ of this species that contains cystic
fibrosis transmembrane conductance regulator (CFTR) Cl
channels. We have cloned an ancestral CNP receptor (NPR-B) from the
shark rectal gland that has an overall amino acid identity to the human
homologue of 67%. The shark sequence maintains six extracellular Cys
present in other NPR-B but lacks a glycosylation site and a Glu residue
previously considered important for CNP binding. When shark NPR-B and
human CFTR were coexpressed in Xenopus oocytes, CNP increased the cGMP content of oocytes
(EC50 12 nM) and activated CFTR
Cl
channels
(EC50 8 nM). Oocyte cGMP increased
36-fold (from 0.11 ± 0.03 to 4.03 ± 0.45 pmol/oocyte) and
Cl
current increased
37-fold (from
34 ± 14 to
1,226 ± 151 nA) in the
presence of 50 nM CNP. These findings identify the specific natriuretic
peptide receptor responsible for
Cl
secretion in the shark
rectal gland and provide the first evidence for activation of CFTR
Cl
channels by a cloned
NPR-B receptor.
Squalus acanthias; natriuretic peptide receptor guanylyl cyclase; molecular cloning; Xenopus oocyte; guanylyl cyclase
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INTRODUCTION |
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NATRIURETIC PEPTIDES HAVE been studied extensively for their important roles in maintaining body fluid homeostasis (for reviews see Refs. 33, 37, 62). In mammals, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are cardiac peptides that exert renal hemodynamic and tubular actions resulting in diuresis and natriuresis. In contrast, the most recently discovered natriuretic peptide, C-type natriuretic peptide (CNP), was first isolated from mammalian brain and was originally considered to function exclusively in the central nervous system (34, 46). It is now known that CNP is synthesized in mammalian vascular endothelial cells (41), where it has potent vasorelaxant and antiproliferative activities (2, 13, 24). The peptide is also present in mammalian intestine, lung, and kidney but not in heart (40) and circulates at low picomolar concentrations in canine and human plasma (4, 49). However, because CNP is not natriuretic when infused in mammals, its role as a regulator of ion transport in epithelia has been challenged (4, 23).
In a primitive vertebrate, the elasmobranch shark, CNP is the only
natriuretic peptide hormone synthesized in the heart (44, 51) and
circulates in plasma at concentrations exceeding those in mammals (52).
Sharks have evolved an extrarenal osmoregulatory organ, the rectal
gland, which is highly specialized for hormone-regulated NaCl secretion
(16). CNP is a potent activator of
Cl secretion in the shark
rectal gland (44) and is ~100-fold more potent than ANP or BNP (48).
Cl
secretion in the rectal
gland occurs through an apical membrane Cl
channel highly
homologous to the mammalian cystic fibrosis transmembrane conductance
regulator (CFTR) (8, 35, 39).
Extensive studies in mammals and elasmobranchs have defined the
regulation of CFTR by the cAMP/protein kinase A (PKA) pathway (1, 8,
35, 53). cAMP-dependent processes are also involved in the trafficking
of CFTR to the cell membrane (35, 54, 58). In contrast, the role of
cGMP as a regulator of CFTR has received much less attention. cGMP
activation of CFTR Cl
channels was first observed by Lin et al. (36) in T84 cells, a
mammalian intestinal carcinoma cell line. Two ligands, heat-stable enterotoxin and guanylin, increase intracellular cGMP and activate CFTR
in T84 cells (3).
cGMP activation of CFTR could be initiated by one or more of the family
of transmembrane natriuretic peptide receptors (also called receptor
guanylyl cyclases; for review, see Ref. 19). Receptors for ANP
(designated NPR-A or GC-A) are present in renal tubular, intestinal,
and airway cells (27, 46, 47, 56). Receptors selective for CNP
(designated NPR-B or GC-B) have been cloned from rat (46), bovine (14),
human (12), and eel (26) and are expressed in intestine (26, 43), lung
(46), and kidney (7), tissues that also express CFTR. Receptors for
heat-stable enterotoxin and guanylin (designated STa-R or GC-C),
originally cloned by Schulz et al. (45), are expressed in intestine (5) and kidney (61). The Cl
secretory effect of STa acting through cGMP is virtually abolished in
CFTR (
/
) mice (38). However, regulation of CFTR
Cl
channels by a cloned
receptor guanylyl cyclase has not yet been demonstrated.
Because CNP is the only identified cardiac natriuretic peptide in the
shark and is a potent regulator of
Cl secretion in the intact
rectal gland, we sought to clone the CNP receptor from this tissue and
test the hypothesis that the receptor regulates CFTR
Cl
channels. Our findings
identify an ancestral CNP receptor with unique structural elements that
activates CFTR channels when coexpressed in
Xenopus oocytes. These observations
identify the specific natriuretic peptide receptor activating
Cl
secretion in the shark
rectal gland and have implications for the regulation of CFTR in
mammalian tissues.
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MATERIALS AND METHODS |
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In vitro perfusion of shark rectal glands.
Rectal glands were obtained from male dogfish sharks,
Squalus
acanthias, weighing 2-4 kg, which
were caught by gill nets in Frenchman's Bay, ME, and kept in marine
live cars until use, usually within 3 days of capture. Sharks were
killed by pithing the spinal cord. Rectal glands were excised, and
cannulas were placed in the artery, vein, and duct as previously
described (28, 35). For perfusion studies, rectal glands were placed in
a glass perfusion chamber, maintained at 15°C with running
seawater, and perfused with elasmobranch Ringer solution containing (in
mM) 270 NaCl, 4 KCl, 3 MgCl2, 2.5 CaCl2, 1 KH2PO4,
8 NaHCO3, 350 urea, 5 glucose, and
0.5 Na2SO4
(all from Sigma) and equilibrated to pH 7.5 by bubbling with 99%
O2 and 1%
CO2. Results are expressed as
microequivalents of Cl
secreted per hour per gram wet weight (means ± SE).
Preparation of total RNA from rectal gland tissue, degenerate primer design, PCR, and cloning of PCR fragment. Total RNA was prepared from 1 g of flash frozen shark rectal gland tissue by the method of Chomczynski and Sacchi as modified by Puissant and Houdebine (42). First-strand cDNA was synthesized using avian myeloblastosis virus RT and oligo(dT) primers (cDNA cycle kit, Invitrogen). A computer program (written by S. Aller) was designed to identify amino acid regions of high homology and low codon degeneracy in eel and human NPR-B and two sea urchin guanylyl cyclases (Arbacia punctulata and Strongylocentrotus purpuratus). The 72-fold degenerate forward oligonucleotide primer [5'-TT(C/T)AT(A/C/T)GGIGCITG(C/T)AT(A/C/T)GA(C/T)] codes for the amino acid sequence Phe-Ile-Gly-Ala-Cys-Ile-Asp that was completely conserved in all four species. A 128-fold degenerate antisense primer [5'-(A/G)TT(A/G)TTIG(C/T)(A/G)TAI(C/T)(G/T)(C/T)TCCAT] codes for highly conserved 3' sequence in these species. PCR was performed with these primers on rectal gland cDNA using Amplitaq (Perkin-Elmer) with denaturing for 1 min at 95°C, followed by 30 cycles of 95°C for 1 min, 45°C for 1 min, and 72°C for 2 min in a Techne Gene-E thermocycler. The resulting 690-bp PCR product was purified from a 1% low-melt agarose gel and ligated into the pCR2.1 TA cloning vector (Invitrogen). The insert was bidirectionally sequenced at the University of Maine automated DNA sequencing facility using an MJ Research PTC-100 thermocycler and a 373 DNA sequencer (Applied Biosystems).
cDNA library screening with shark NPR-B fragment.
One gram of frozen rectal gland tissue was homogenized in guanidinium
thiocyanate buffer (4 M guanidinium thiocyanate, 0.1 M
Tris · HCl, pH 7.5, 1% 2-mercaptoethanol, and 0.5%
sodium lauryl sarcosinate), layered onto 5.7 M CsCl-0.1 M EDTA, and
ultracentrifuged in an SW41 swinging bucket rotor at 32,000 rpm for 24 h. The RNA pellet was washed with 70% ethanol, redissolved in
Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 7.6)-0.1% SDS,
precipitated with 3 M sodium acetate (pH 5.2) and ethanol for 1 h at
20°C, and centrifuged at 12,000 g for 30 min at 4°C. The RNA was
washed in ethanol, recentrifuged, briefly dried, and resuspended in
water. Poly(A) RNA was purified from total RNA using magnetic bead
separation (PolyATtract, Promega). A ZAP Express cDNA expression
library was prepared using both oligo(dT) and random primers
(Stratagene). Library phage were transferred to Hybond-N nylon
membranes (Amersham) in duplicate. The membranes were exposed to
denaturing solution (0.5 M NaOH and 1.5 M NaCl), washed in 1.5 M NaCl,
0.5 M Tris · HCl, 10 mM EDTA (pH 7.4), and soaked in 2× SSC
(1× SSC is 0.15 MNaCl and 0.015 M sodium citrate, pH 7.0) for 3 min. DNA was fixed to the membranes by baking for 2 h at
80°C. The original shark NPR-B PCR product was labeled with
[
-32P]dCTP and
random primers (GIBCO) and used to screen the membranes essentially
according to manufacturer's protocol (Stratagene). Plasmid DNA was
prepared from four positive plaques and sequenced with vector-specific
primers by automated sequencing.
Random amplification of cDNA ends-PCR to obtain 5' and 3' ends of shark NPR-B. Shark rectal gland total RNA was prepared using the CsCl method described above. Adaptor-ligated cDNA was prepared from rectal gland total RNA exactly according to the manufacturer's protocol (Marathon cDNA amplification, Clontech). Two shark NPR-B-specific oligonucleotides (sense 5'-CAACACAAGGGAAATACCAG and antisense 5'-CTGTCAAATGCCTCTGCTTG) were used in separate PCRs using adaptor-ligated shark rectal gland cDNA. Random amplification of cDNA ends (RACE)-PCR was performed with one shark-specific primer and a second primer that anneals to the adaptor sequence attached to the ends of the shark cDNA. A 50-µl reaction mixture, containing Expand high-fidelity Taq polymerase (Boehringer Mannheim), was heated to 94°C for 2 min and then subjected to 30 cycles of 94°C for 30 s, 58°C for 30 s, and 68°C for 4 min in a Techne Gene-E thermocycler. Bands were gel purified and cloned as above and confirmed to be shark NPR-B fragments by automated DNA sequencing.
DNA sequence analysis. Sequence assembly, analysis, and alignments (nearest neighbor method) were carried out using Wisconsin Package (Genetics Computer Group) software and Lasergene (DNASTAR). Database searches of GenBank were carried out using BLAST Client and Network Entrez software (National Institutes of Health). We report percent identity as the percentage of exactly matching amino acids in aligned sequences. The nucleotide sequence of shark NPR-B was submitted to the GenBank database (accession no. AF054285).
Northern blot analysis of shark tissues.
Total RNA was isolated from flash frozen shark tissues (rectal gland,
heart, brain, kidney, liver, skeletal muscle, and spleen) using the
modification of Puissant and Houdebine (42). Total RNA (30 µg) from
each tissue was fractionated on a 1.2% agarose-formaldehyde gel,
transferred to GeneScreen nylon membranes, and fixed by baking at
80°C for 2 h; 50 ng of the original 690-bp PCR product were radiolabeled as described above. The labeled probe was added to the
hybridization solution (1% SDS, 1 M NaCl, 10% dextran sulfate, and
50% formamide), with salmon sperm DNA, and hybridization was carried
out at 42°C for 18 h. The membrane was washed in 2× SSC at
room temperature for 5 min, followed by 2× SSC-0.5% SDS at 60°C for 30 min, followed by two washes in 0.1× SSC at room
temperature for 30 min, and then the membrane was exposed to X-ray film
for 2 days at 70°C.
Generation of expression construct for oocyte injection. The forward primer 5'-ACTTGTTTTCTCCCTTCCTGATGC and reverse primer 5'-CTACAGCTTGAACAACCACTGCTCTG were used to generate the expression construct with high-fidelity PCR. Shark rectal gland cDNA was prepared as mentioned above (Invitrogen) and used as the PCR template; 25 cycles were performed at 95°C for 30 s, 63°C for 30 s, and 68°C for 4 min. The resulting 3,338-bp PCR product was cloned into pCR3.1 and bidirectionally sequenced by automated sequencing. The plasmid was linearized using Xba I endonuclease, and capped messenger RNA was synthesized using T7 in vitro transcription (mMessage mMachine, Ambion). Human CFTR plasmid (generously provided by Dr. Carol Semrad) was linearized with Kpn I and similarly transcribed into cRNA.
Oocyte preparation, coexpression of NPR-B and CFTR, and measurement
of cyclic nucleotide content.
Xenopus oocytes were manually
defolliculated and injected with cRNA as described (35). Oocytes were
injected with 15 ng shark NPR-B separately or with 10 ng of either
human or shark CFTR. Two-electrode voltage clamping was used to measure
Cl current from the
oocytes. Oocytes were clamped at
60 mV, and current was measured
as a function of time. Occasional voltage ramps (
120 mV to +60
mV) were performed to determine the current-voltage relationship. Data
were corrected for any capacitance change during the voltage ramps.
After incubation experiments, oocytes were transferred into 2 ml of
ice-cold 6% TCA and homogenized for 30 s using a Tekmar motor-driven
homogenizer. Samples were centrifuged at 5,000 g for 10 min at 4°C, and the
aqueous solution was removed from the pellet of precipitated protein.
Samples were lyophilized and resuspended in 1 ml assay buffer. RIA was
performed with cAMP and cGMP assay kits (DuPont) according to the
manufacturer's instructions. Shark CNP (44) was synthesized at the
Yale University peptide synthesis facility, and rat ANP (rANF-28) was
obtained from Peninsula Laboratories.
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RESULTS |
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Effects of CNP on Cl secretion and
cyclic nucleotide content in the in vitro perfused shark rectal gland.
When perfused with Ringer (without CNP),
Cl
secretion remained low,
~200 µeq/h/g, throughout the 40-min experiment (Fig.
1A). After 30 min of basal perfusion, the addition of increasing
concentrations of CNP resulted in a dose-dependent increase in
Cl
secretion. At maximal
concentration (10 nM), Cl
secretion increased from basal values of 182 ± 49 to 2,093 ± 142 µeq/h/g (P < 0.0001, n = 6) 8 min after the addition of
CNP. The EC50 for stimulation of
Cl
secretion by CNP in the
rectal gland was ~0.8 nM. CNP-stimulated Cl
secretion in the gland
was accompanied by marked increases in tissue cGMP content (30-, 47-, and 103-fold at 1, 3, and 10 nM CNP, respectively) without substantial
changes in cAMP content (Fig. 1B).
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Cloning of shark NPR-B.
Using degenerate primers, a 690-bp PCR product designated P-SA7 (Fig.
2A)
was amplified from shark (Squalus
acanthias) rectal gland cDNA. The
deduced amino acid sequence of P-SA7 was 78% identical to human NPR-B.
Screening of a shark rectal gland cDNA library using this PCR product
as a probe revealed three positive clones (L-SA1, L-SA4, and L-SA5)
that were homologous to NPR-B receptors on sequencing. The consensus
sequence of these library clones provided 2853 bp of shark NPR-B
sequence. To obtain the remaining 5' and 3' sequence,
RACE-PCR was performed on adaptor-ligated rectal gland cDNA with shark
NPR-B-specific primers designed from the library clones. A 2968-bp
fragment comprising the 5' sequence of shark NPR-B (Fig.
2B,
lane
1, upper band) and a 1966-bp product yielding 3' sequence (Fig. 2B,
lane
2) were amplified. Cloning and
sequencing of these products provided an additional 1055 bp of 5'
and an additional 35 bp of 3' shark NPR-B sequence. The RACE-PCR
fragments and library clones made up a total of 3943 bp of nucleotide
sequence for shark NPR-B with an open reading frame encoding 1,056 amino acids. To establish that this sequence was the product of a
single RNA transcript, PCR primers flanking the start and stop codons
were used to perform high-fidelity PCR on rectal gland cDNA. A single
product of 3322 bp was obtained (Fig.
2C) and cloned into the pCR3.1
vector (Invitrogen) for expression studies. Bidirectional sequencing of
this expression clone confirmed this construct to be native shark
NPR-B.
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Sequence analysis of cloned shark NPR-B.
The deduced amino acid sequence of the shark NPR-B receptor, shown in
Fig. 3, was 67% identical to human and
69% identical to eel NPR-B. Greatest diversity was present in the
extracellular domain, with a homology of shark to human of only 48%.
Higher homology was found in the transmembrane, kinase homology, and guanylyl cyclase domains, with identities of 55, 81, and 87%, respectively. Figure 3 shows the amino acid alignment of shark, eel,
and human CNP receptors including several proposed domains. In the
extracellular domain, shark NPR-B maintains the following elements:
1) six extracellular Cys common to
all previously cloned NPR-B receptors,
2) the NPR motif, including His-Trp
residues essential for ligand binding,
3) Val-358 present in NPR-B but not
NPR-A, and 4) four conserved
potential glycosylation sites. In the extracellular domain, shark NPR-B
differs from previously cloned NPR-B receptors in the following
elements: 1) three unique potential
glycosylation sites (Asn-137, Asn-143, and Asn-328), 2) absence of an amino-terminal
potential glycosylation site, and 3)
presence of Ala instead of Glu at position 363 in shark. The
amino-terminal glycosylation site and the Glu residue have been
proposed to be important for CNP binding (10, 15).
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Detection of NPR-B mRNA in shark tissues.
Northern blot analysis of total RNA revealed abundant expression
of NPR-B message in the shark rectal gland with a faint signal also
detected in shark kidney (Fig. 4). No
signal was detected in heart, brain, liver, skeletal muscle, or spleen.
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Functional coexpression of NPR-B and CFTR in Xenopus oocytes:
electrophysiology and cyclic nucleotide content.
To examine the hypothesis that NPR-B activates CFTR channels, we
expressed human CFTR and shark NPR-B cRNA first separately and then
together in coexpression studies in
Xenopus oocytes. In experiments in
oocytes injected with CFTR cRNA only, the addition of forskolin (5 µM), an activator of PKA, resulted in a marked increase in a negative
current (56 ± 9 to
756 ± 62 nA) and a shift in
the reversal potential from basal values (
43 ± 7 mV) to
29 ± 6 mV (n = 12 oocytes).
This negative current, with a reversal potential similar to that of
Cl
(approx.
30 mV)
was inhibited by glibenclamide and constitutes the electrophysiological
signature of CFTR Cl
channels (6, 8, 9).
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DISCUSSION |
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Sharks maintain body fluid homeostasis in the hypertonic sea by the
secretion of NaCl in tubules of the rectal gland, an accessory osmoregulatory organ that is an appendage of the gastrointestinal tract. In contrast to mammals, in which ANP and BNP are cardiac peptides, the shark employs CNP as the sole cardiac natriuretic peptide
(44, 51). CNP, stored as pro-CNP in the shark heart (44), circulates in
plasma at concentrations higher than those of ANP or BNP in mammals
(52). CNP stimulates NaCl secretion in the intact perfused gland (44,
48), monolayers of tubular cells in primary culture (60), and single
isolated perfused tubules (20). In the saline-loaded shark, Gunning et
al. (22) demonstrated that rectal gland
Cl secretion was blocked by
the natriuretic peptide receptor antagonist HS-142-1. Based on binding
studies in rectal gland membranes, both a guanylyl cyclase-coupled and
a non-guanylyl cyclase-coupled receptor have been proposed (21).
The present study identifies, by cloning and expression studies, the
specific receptor guanylyl cyclase (NPR-B) that couples to
Cl secretion in the rectal
gland. This system contrasts with mammals, in which gene knockout of
NPR-A receptors by Kishimoto et al. (32) entirely prevented the
saline-induced natriuresis observed in wild-type mice. Thus in sharks
the heart communicates with the rectal gland via a CNP/NPR-B axis,
whereas in mammals the heart communicates with the kidney through an
ANP/NPR-A system (32).
Shark NPR-B maintains the position of six conserved extracellular Cys unique to all previously cloned NPR-B. The receptor also preserves the proposed NPR motif with a His-Trp residue pair essential for ligand binding (25, 55). Shark NPR-B has seven potential glycosylation sites; four are conserved exactly or in close proximity to those of other NPR-B receptors (Asn-167, Asn-203, Asn-288, and Asn-358) and three are unique (Asn-137, Asn-143, and Asn-328). Asn-24 is present in all previously cloned NPR-B receptors. Fenrick et al. (15) showed that when this residue was mutated 90% of cyclase activity and competitive ligand binding was lost but the affinity for CNP remained unchanged. Because the expression of this mutant was comparable to wild type as estimated by Western blotting, loss of binding was attributed to improper folding of the protein. Shark NPR-B lacks Asn-24 and has no potential glycosylation site within 140 residues. Because the cloned rectal gland NPR-B receptor is highly sensitive to CNP when expressed in oocytes, it is likely that another glycosylation site in shark NPR-B functions in place of the Asn-24 of other receptors. We did observe a difference in EC50 between the transfected receptor (8-12 nM) and the native receptor in the intact gland (0.8 nM). The reason for this is uncertain but could be due to differences in the transduction pathway between these two systems.
The shark receptor also differs from all other NPR-B receptors in having an uncharged Ala at position 363 as opposed to the analogous negatively charged Glu at position 332 in human NPR-B. Duda et al. (11) found Glu-332 to be necessary for maximal activity of human NPR-B, as CNP binding and enzyme activity were reduced by 40 and 90%, respectively, when Glu-332 was mutated to Ala. Because the cloned rectal gland NPR-B receptor is highly functional, we propose that the uncharged Ala and/or other unique shark residues function to support comparable receptor activity.
Identification of specific cellular effectors of recombinant
natriuretic peptide receptors has been elusive (18). A major finding of
the present work is that the NPR-B receptor functions to activate the
CFTR Cl channel when both
proteins are coexpressed in Xenopus
oocytes. Activation of CFTR is accompanied by a marked increase in
oocyte cGMP content without change in the cAMP content. The expressed NPR-B is nearly unresponsive to maximal concentrations of ANP in
activating cGMP and CFTR, consistent with the selectivity of this
receptor for CNP. Thus an NPR-B receptor, cloned from a tissue where
CNP is the physiological activator of
Cl
secretion, has CFTR as a
specific membrane effector.
Our experiments do not define the mechanism(s) by which increases in cGMP activate CFTR in the rectal gland tubule or oocyte. In mammalian systems, several mechanisms have been proposed, including 1) cGMP activation of a membrane-bound cGMP-dependent protein kinase, type II (cGKII) (59), 2) cGMP inhibition of a cAMP phosphodiesterase (30), 3) cGMP cross-activation of a cAMP-dependent protein kinase (57), and 4) direct interaction of cGMP with the CFTR channel (50). Cross-activation of PKA and inhibition of a cAMP phosphodiesterase isoenzyme have been proposed in immortalized cell lines (T84, Caco-2, Calu-3, and 16HBE). Vaandrager et al. (59) recently proposed that cGKII may be a key mediator of cGMP-provoked activation of CFTR in intestinal cells where both proteins are colocalized (17).
Because the rectal gland is an intestinal appendage, we are currently searching for cGKII in this tissue. Because CNP does not substantially increase cAMP content in perfused rectal glands and oocytes, we speculate that inhibition of cAMP phosphodiesterase is unlikely in these systems. However, our data do not exclude involvement of PKA, protein kinase G, or other messengers that might link NPR-B and CFTR.
Regardless of the mechanism of CFTR activation, the present study
provides evidence that NPR-B receptors activate CFTR. Kelley et al.
(29, 31) recently demonstrated that CNP increases short-circuit current
and activates CFTR Cl
channels in Calu-3 airway cells and hyperpolarizes nasal epithelium in
both wild-type and cystic fibrosis (
F508) mice. The shark rectal
gland is the first intact epithelial model that supports the concept
that NPR-B receptors activate CFTR channels in tissues where both
proteins are expressed.
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ACKNOWLEDGEMENTS |
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We thank Albert George, Gabriele Hemminger, Peter Burrage, Josh Forrest, Gerhard Weber, Kristina Lübbe, Chris Smith, Lynn Matthews, and Kyle Suggs for excellent technical assistance.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants DK-34208 and P30-ES-3828 (Center for Membrane Toxicology Studies) and American Heart Association Grant 9607741S to J. N. Forrest, Jr.
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: J. N. Forrest, Jr., Dept. of Internal Medicine, Yale University School of Medicine, New Haven, CT 06510.
Received 23 September 1998; accepted in final form 26 October 1998.
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