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Am J Physiol Regul Integr Comp Physiol 291: R1157-R1164, 2006. First published May 25, 2006; doi:10.1152/ajpregu.00078.2006
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COMPARATIVE AND EVOLUTIONARY PHYSIOLOGY

Shark rectal gland vasoactive intestinal peptide receptor: cloning, functional expression, and regulation of CFTR chloride channels

Marie S. Bewley, John T. G. Pena, Florian N. Plesch, Sarah E. Decker, Gerhard J. Weber, and John N. Forrest, Jr.

Department of Medicine, Yale University School of Medicine, New Haven, Connecticut; and the Mount Desert Island Biological Laboratory, Salisbury Cove, Maine

Submitted 29 January 2006 ; accepted in final form 8 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Vasoactive intestinal peptide (VIP) is a secretagogue that mediates chloride secretion in intestinal epithelia. We determined the relative potency of VIP and related peptides in the rectal gland of the elasmobranch dogfish shark and cloned and expressed the VIP receptor (sVIP-R) from this species. In the perfused rectal gland, VIP (5 nM) stimulated chloride secretion from 250 ± 66 to 2,604 ± 286 µeq·h–1·g–1; the relative potency of peptide agonists was VIP > PHI = GHRH > PACAP > secretin, where PHI is peptide histidine isoleucine amide, GHRH is growth hormone-releasing hormone, and PACAP is pituitary adenylate cylase activating peptide. The cloned sVIP-R from shark rectal gland (SRG) is only 61% identical to the human VIP-R1. It maintains a long, extracellular NH2 terminus with seven cysteine residues, and has three N-glycosylation sites and eight other residues implicated in VIP binding. Two amino acids considered important for peptide binding in mammals are not present in the shark orthologue. When sVIP-R and the CFTR chloride channel were coexpressed in Xenopus oocytes, VIP increased chloride conductance from 11.3 ± 2 to 127 ± 34 µS. The agonist affinity for activating chloride conductance by the cloned receptor was VIP > GHRH = PHI > PACAP > secretin, a profile mirroring that in the perfused gland. The receptor differs from previously cloned VIP-Rs in having a low affinity for PACAP. Expression of both sVIP-R and CFTR mRNA was detected by quantitative PCR in shark rectal gland, intestine, and brain. These studies characterize a unique G protein-coupled receptor from the shark rectal gland that is the oldest cloned VIP-R.

Squalus acanthias; molecular cloning; Xenopus oocytes

VASOACTIVE INTESTINAL PEPTIDE (VIP) is a neurotransmitter and neuroendocrine hormone first isolated from porcine intestine (29). In numerous mammalian species, this peptide causes smooth muscle relaxation, vasodilation, exocrine secretion in the pancreas, and electrolyte and water secretion by intestinal epithelia (10, 18, 28). VIP is a member of a large family of structurally related peptides that bind to Class II G protein-coupled receptors, including secretin, pituitary adenylate cyclase activating polypeptide (PACAP), peptide histidine isoleucine (PHI), and growth hormone-releasing hormone (GHRH) (19). In mammals, G protein-coupled receptors have been identified that bind these peptides with varying affinities, including the VIP receptors VIP-R1 and VIP-R2 (also called VPAC1 and VPAC2) and the PACAP receptor (20). All previously cloned VIP-R1 and VIP-R2 have high, equal affinity for the agonists VIP and PACAP (4, 20, 21). VIP-R1 and VIP-R2 are differentiated by their affinity for secretin, VIP-R2 having no affinity for this agonist. The PACAP receptor has a high affinity for PACAP, but not for VIP (20).

The dogfish shark (Squalus acanthias) is an ancient elasmobranch species (estimated 420 million years old) and is one of the oldest jawed vertebrates (39). More than two decades ago, Stoff et al. (34) identified VIP as a secretagogue that stimulates sodium chloride secretion in the rectal gland of the dogfish shark. VIP immunoreactivity was then identified in neural elements of the rectal gland (3). This highly specialized organ, composed of tubular epithelia of a single cell type, maintains salt homeostasis in this species and is a useful model of secondary active chloride secretion (11, 31). VIP is a potent chloride secretagogue in the intact perfused gland (34), in single isolated perfused tubules (12), and in primary culture monolayers of rectal gland cells (37). VIP activates three messenger pathways in this tissue: 1) stimulation of adenylyl cyclase, increasing cellular cAMP (33) and the insertion of CFTR into apical membrane domains (22), 2) increases in intracellular Ca2+ (17), and 3) release of inositol phosphates (9). Binding studies in rectal gland membranes revealed a single high affinity binding site for VIP (32).

Only one G protein-coupled receptor, the A0-adenosine receptor, has previously been cloned and expressed from elasmobranchs (11). In this report, we describe the cloning and functional expression of the shark rectal gland sVIP-R, the first VIP-R characterized in a marine species, and the oldest VIP-R characterized to date. We compare the physiological responses to the agonists VIP, PACAP, PHI, GHRH, and secretin in the perfused shark rectal gland and the cloned shark receptor. We describe the molecular structure, functional coupling to CFTR chloride channels, and tissue distribution of this ancestral VIP-R.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 Bay, ME, and kept in tanks with flowing seawater until use, usually within 3 days of capture. Sharks were killed by pithing the spinal cord using a protocol approved by the Mount Desert Island Biological Laboratory (MDIBL) Animal Care And Use Committee. Rectal glands were excised and cannulas were placed in the artery, vein, and duct as previously described (16, 22, 27). For perfusion studies, rectal glands were placed in glass perfusion chambers, maintained at 15°C with running sea water and perfused with elasmobranch Ringer solution (27) containing 8 mM NaHCO3equilibrated to pH 7.5 by bubbling with 99% O2 and 1% CO2. Results are expressed as microequivalents of chloride secreted per hour per gram wet weight ± SE.

Preparation of primary cell culture monolayers of shark rectal gland tubules, and measurements of their transepithelial chloride transport as short circuit current. Shark rectal gland monolayers were prepared as previously described (37). Confluent cultures were mounted in a conventional Plexiglas Ussing chamber having an aperture of 0.28 cm2. Both hemichambers were independently perfused by gravity flow with room temperature (20–22°C) shark Ringer solution containing 24 mM NaHCO3gassed with 95% O2-5% CO2. Transepithelial voltage was measured with an Iowa dual voltage clamp (model 710C) and agar bridges connected to calomel electrodes using methods described previously (37). The bridges were filled with shark Ringer solution and were positioned ~1 mm from the apical and basolateral surfaces of culture. Transepithelial resistance was measured by applying bipolar current pulses every 30–60 s. Current was passed by the voltage clamp via agar bridges linked to external Ag-AgCl electrodes. The transepithelial voltage drop due to the series fluid and collagen gel resistances was compensated for by placing a collagen-coated nylon mesh lacking cells between the hemichambers. Short circuit current (Isc) was measured by determining the current necessary to clamp the spontaneous transepithelial voltage to 0 mV. Results are expressed as microampere per centimeter squared ± SE.

Preparation of total RNA from shark tissue, degenerate primer design, and cloning of PCR fragment. A cesium chloride method (Molecular Cloning, San Francisco, CA) was used to isolate total RNA from fresh shark rectal gland tissue. A DNase 1 digest was performed on the RNA, and cDNA first strand synthesis was carried out using avian myeloblastosis virus (AMV) reverse transcriptase and oligo(dT) primers (Clonetech Advantage, Palo Alto, CA). An amino acid alignment (Clustal W, generated by DNAStar Megalign software) of human, rat, guinea pig, chicken, frog, and trout VIP-R sequences (GenBank; http://www.ncbi.nlm.nih.gov) was used to design degenerate primers. Degenerate primers to transmembrane regions 2 (5'-TGCAYTGYACNMGNAAYTAYATYCA-3'), 6 (5'-AGSGGGATSAGSRKNAGNGTGGAYTT-3') and 7 (5'-TGSACCTCNCCRTTNASRAARCARTA-3') were synthesized. PCR was performed (Techne Thermocyler) with these primers on shark rectal gland cDNA by using hot start on a 30-µl reaction (1.5 min at 95°C, 2 min at 48°C, and 45 s at 72°C, and a final extension step of 2 min at 72°C). Products were directly cloned by TOPO TA-Cloning (Invitrogen, Carlsbad, CA), and clones were screened for insert size by PCR and sequenced using automated techniques (Marine DNA Sequencing Facility, MDIBL, Salisbury Cove, ME).

SMART RACE PCR to obtain full-length sequence of sVIP-R. Total RNA was isolated from fresh dogfish shark rectal gland tissue using cesium chloride method (Molecular Cloning). Reverse transcription was carried out on DNAse 1-digested RNA with oligo(dT) primers (Clonetech Advantage). Primers were designed from the partial VIP-R sequence. The sense primer: 5'- CTTCFTCCTFAFFFCCATCFCTFTCTT-3' and antisense primer: 5'-GGGGCCCGAATGACTTCGTCCTGAGGGCCATCGCTGTCTT-3' were used to amplify 3' RACE and 5' RACE fragments of sVIP-R using SMART RACE-PCR (SMART RACE cDNA Amplification Kit; Clonetech). PCR was performed for 35 cycles at 95°C for 30 s and 65°C for 3 min 30 s, using Expand Long DNA polymerase (Roche, Indianapolis, IN). PCR products were agarose purified and subcloned by TOPA-TA cloning (Invitrogen) and sequenced at the MDIBL Marine DNA Sequencing Facility.

Preparation of sVIP-R1 expression construct and cRNA for oocyte injection. The full-length expression construct was generated using a specific sense primer beginning at the start codon and antisense primer ending at the stop codon of the full-length VIP-R1. Shark rectal gland cDNA was prepared as above and used as a template for PCR using Expand Long DNA polymerase enzyme (Roche), and the following cycle conditions: 35 x (95°C 3.5 min, 95°C 30 s, 65°C 45 s, 72°C 3 min). The resulting 1,344 bp PCR product was linearized and subcloned into the pCR3.1 expression vector (Invitrogen). The purified plasmid was verified by DNA sequencing. Capped messenger RNA was synthesized from the template using T7 in vitro transcription (mMessage mMachine; Ambion, Austin, TX). Human CFTR plasmid was linearized with KpnI, and similarly transcribed into cRNA as previously described (2).

Oocyte preparation and expression of sVIP-R and CFTR. Mature female Xenopus laevis were anesthetized in a 0.15% cold solution of tricaine for 20 min, and several ovarian lobules were removed under sterile conditions through an abdominal incision per a protocol approved by the Yale University Animal Care and Use Committee. Oocytes were defolliculated in a 2.5 mg/ml solution of type I collagenase for 1 h. Mature stage V and VI oocytes were selected and stored. After 12–24 h, the oocytes were injected with either 5 ng of sVIP-R cRNA/50 nl, 5–10 ng of CFTR/50 nl, 5 ng of sVIP-R, and 5–10 ng CFTR/50 nl, or an equivalent volume of water and then stored for 36 h in modified Barth solution holding (MBSH) medium at 18°C. Oocytes were stored in MBSH medium containing (in mM) 88 NaCl, 1 KCl, 2.4 NaHCO3, 0.82 MgSO4, 0.33 Ca(NO3)2·4H2O, 10 HEPES (5 Na salt, 5 acid), buffered to pH 7.4, and 150 mg/l gentamicin sulfate.

Two electrode voltage clamping. Oocytes were used for electrophysiological study 2–3 days after injection. Glass pipettes (1.2 mm) were pulled using a microelectrode puller (Sutter Instruments, Novato, CA) to give an input resistance of 0.6–0.9 M{Omega}. Electrodes were filled with 3 M KCl. A two-electrode voltage clamp (TEV-200 Dagan Instruments, Foster City) was used to record membrane potential changes by clamping the voltage over a fixed ramp from –120 to +60 mV at a rate of 100 mV/s. The voltage clamp software (pCLAMP; Axon Instruments, Union City, CA) corrected for capacity currents and calculated reversal potentials. Conductance was calculated over a range of corrected reversal potentials ±10 mV as described previously (38). Oocytes were perifused with frog Ringer solution ND96 for 10–30 min to achieve a basal steady state. Subsequently, either VIP, PACAP, PHI, secretin, or GHRH were added at a specific concentration until a steady activated state was achieved in each (10–15 min). Current voltage ramps were taken at 1 min 30 s intervals during basal and activated conditions. Relative activation of CFTR was calculated as the ratio of conductance measured immediately before the addition of the peptide agonists to the conductance at maximal stimulation of chloride secretion after peptide addition. Data were analyzed by pCLAMP software and conductances are expressed as mean micro-Siemens ± SE. PACAP was purchased from Calbiochem (San Diego, CA), and other peptides and reagents were from Sigma-Aldrich (St. Louis, MO).

Quantitative PCR measurement of mRNA expression levels in Squalus acanthias tissues. We used the Stratagene Brilliant SYBR Green QPCR System and the Stratagene MX4000 Real-Time PCR instrument at MDIBL. Total RNA was extracted from tissues using Trizol reagent (Invitrogen). Before cDNA synthesis, RNA samples were DNase digested to avoid contamination with genomic DNA (Ambion). RNA quality was analyzed using a Agilent 2100 Bioanalyzer. A total of 3 µg RNA was reverse transcribed using a SuperScript First-Strand cDNA Synthesis system (Invitrogen). cDNA (1 µl) was amplified with primers designed to produce an amplification of ~300 bp. (CFTR sense primer: 5'-TCTCTGCCTTGGACGAATAATAGC-3', CFTR antisense primer: 5'-CACTGCCACGCCCTCATCA-3', VIP-R sense primer: 5'-GTCCTGAGGGCCATCGCTGTCTT-3', VIP-R antisense primer: 5'-GGGCCCGAATGATCCACCAATAC-3', beta-actin sense primer: 5'-CTGGCATTGTGCTAGATTCTGGTG-3', beta-actin antisense primer: 5'-AAGAGCTAGCCGTCTGCATCTCAG-3'). Primer specificity was tested by conventional PCR and all primer pairs yielded a single band. Samples were prepared in triplicate and relative expression levels were calculated using the comparative threshold cycle (Ct) method. By subtracting the average beta-actin Ct value from the average target gene Ct value, the expression levels of the target gene were normalized to the expression level of the reference gene (beta-actin). The expression levels were calculated using the following equation: 2– (target gene [sample] Ct – Actin [sample] Ct) – (target gene [standard])


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Response to increasing concentrations of VIP and related peptides on chloride secretion in the in vitro perfused shark rectal gland (Fig. 1). Rectal glands were perfused with shark Ringer for 30 min to achieve basal levels of secretion. Peptides were then added to the perfusate for the remainder of the experiment. The response to increasing concentrations of VIP is shown in Fig. 1A. Whereas 0.5 nM VIP did not stimulate chloride secretion, 1 nM VIP increased secretion to nearly half maximal values (1,242 ± 236 µeq·h–1·g–1 compared with basal values of 143 ± 10 µeq·h–1·g–1, P < 0.001). Maximal stimulation was seen at 5 nM VIP (2,604 ± 286 µeq·h–1·g–1, P < 0.001 compared with basal values). Higher concentrations did not increase chloride secretion above that observed at 5 nM. To compare the VIP response to other agonists, separate perfusions were carried out with PACAP-38, PHI, secretin, and GHRH at a concentration of 10 nM (Fig. 1B). The agonist potency profile in the perfused gland was VIP > PHI = GHRH > PACAP > secretin. Glands were also perfused with PACAP-27 and the results were identical to PACAP-38 (data not shown).


Figure 1
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  		Fig. 1. Vasoactive intestinal peptide (VIP) and related peptides stimulate chloride secretion in the in vitro-perfused shark rectal gland. All glands were perfused with elasmobranch Ringer for 30 min to reach basal rates of secretion. A: in separate experiments, VIP was added to the perfusate at 30 min at 0.5, 1, 5, 10, and 20 nM concentrations and perfusion was continued to 40 min. Cl– secretion was measured every 10 min during basal perfusion, and at 1 min intervals after the addition of VIP. VIP stimulated Cl– secretion in a dose dependent manner. Values are means ± SE, n = 5 for 10 nM, n = 4 for other concentrations. B: stimulation of chloride secretion by 10 nM concentrations of VIP, peptide histidine isoleucine anide (PHI), growth hormone releasing hormone (GHRH), pituitary adenylate cylase activating peptide (PACAP), and secretin. Values are mean chloride secretion (µeq·h–1·g–1 ± SE) 7 min after addition of agonist (*P < 0.05 compared with secretin, **P < 0.01 compared with secretin). The relative potency was VIP > PHI = GHRH > PACAP > secretin. VIP stimulated chloride secretion from 194 ± 28 to 2,351 ± 194 µeq·h–1·g–1(n = 5). The response to VIP was significantly greater than all other agonists (P < 0.01). PHI stimulated chloride secretion from 110 ± 13 to 146 ± 91 µeq·h–1·g–1 (n = 5). PHI was significantly greater than PACAP and secretin (P < 0.01) but was not significantly different from GHRH. GHRH stimulated chloride secretion from 183 ± 86 to 866 ± 225 µeq·h–1·g–1 (n = 4). GHRH-stimulated secretion was significantly greater than secretin (P < 0.05). PACAP stimulated chloride secretion from 335 ± 67 to 567 ± 188 µeq·h–1·g–1 (n = 4). PACAP was significantly greater than secretin (P < 0.01). Secretin had minimal effects on chloride secretion, only stimulating from 107 ± 30 to 245 ± 134 µeq·h–1·g–1(n = 3).

 
Experiments were also carried out by measuring chloride secretion as Isc in cultured monolayers of shark rectal gland tubular cells in which Isc is equal to net chloride flux (37). This preparation lacks the neural elements present in the perfused gland (37). In these experiments, we examined the response to three agonists: VIP, PHI, and secretin. VIP was again the most potent agonist, increasing Isc from basal values of 20 ± 6 µA/cm2 to 417 ± 175 µA/cm2 at 50 nM (n = 5 experiments) and the order of potency was VIP > PHI > secretin.

Cloning of sVIP-R. Figure 2A illustrates the PCR products obtained from seminested PCR with shark rectal gland cDNA. The 537-bp product from lane 5 was cloned, and sequencing showed 78% identity to human VIP-R1. Fig. 2B illustrates the strategy for obtaining full-length sequence. To obtain the remaining 5' and 3' ends, RACE PCR was performed on an adaptor-ligated rectal gland cDNA library with sVIP-R-specific primers designed from the degenerate 537-bp PCR product. This yielded a 5' product of ~600 bp, and a 3' product of ~900 bp. (Fig. 2B). High-fidelity PCR using start-to-stop primers amplified a single product of 1,344 bp, which was cloned into the pCR3.1 vector for expression studies (Fig. 2C). Bidirectional sequencing confirmed this clone to be sVIP-R.


Figure 2
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  		Fig. 2. Cloning of shark VIP receptor (VIP-R). A: seminested PCR of shark rectal gland cDNA yielded a 537-bp product using degenerate primers made from conserved regions of human, rat, mouse, fish, and frog VIP-Rs (lane 5). Shown are PCR with primers from TM2 and TM7 (lane 1), PCR with primers from TM2 and TM6 (lane 2), positive control using A0 adenosine receptor primers (lane 3), negative control for 1st of 2 nested PCRs (lane 4), negative control for 2nd of 2 nested PCRs (lane 6), and 1 kb plus DNA ladder (lane 7). B: strategy for obtaining full-length sequence of shark VIP-R. Sequence from 537-bp degenerate PCR product was used to design random amplification of cDNA ends (RACE) primers yielding 5' and 3' RACE-PCR fragments. *Position of the start codon and stop sequence of shark VIP-R. C: high fidelity PCR on rectal gland cDNA using primers flanking start codon and stop sequence of shark VIP-R yielded a 1,344-bp band (lane 2). Shown are 1 kb-plus DNA ladder (lane 1) and negative control with no cDNA template (lane 3).

 
Sequence analysis of cloned sVIP-R. The 447 amino acid sequence of the sVIP-R has highest identity to VIP-R1 receptors (61% identity to human and rat VIP-R1 receptors, 62% identity to mouse and goldfish VIP-R1 receptors and 71% identity to the turkey VIP-R receptor) (Fig. 3). The predicted seven transmembrane regions are highly conserved compared with human VIP-R1 (overall = 64%, TM1 = 44%, TM2 = 70%, TM3 = 81%, TM4 = 43%, TM5 = 71%, TM6 = 70%, TM7 = 75%, Fig. 3). sVIP-R has a long 137 aa NH2-terminal region and greatest diversity is seen in the extracellular domains (Fig. 3). sVIP-R contains several elements common to vertebrate VIP-Rs (see Fig. 3). These include: 1) seven Cys residues considered important for VIP binding (indicated by yellow ovals); 2) eight other residues, including a Cys-Trp motif in the second extracellular loop, considered essential for VIP binding and cAMP production (indicated by blue rectangles); 3) three potential N-glycosylation sites (purple rectangles); and 4) an RLAK motif between TM5 and TM6 essential to Gs{alpha} protein coupling (green rectangle). Phylogenetic analysis indicates that the cloned shark receptor is a VIP-R1 receptor (Fig. 4).


Figure 3
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  		Fig. 3. Amino acid alignment of shark, human, rat, turkey, and goldfish VIP-R. Seven conserved cysteine residues in the NH2-terminal extracellular domain are enclosed in yellow boxes. Conserved amino acids implicated as crucial for binding in mutagenesis studies are enclosed in blue boxes. Three conserved NH2-glycosylation sites are enclosed in purple boxes. Transmembrane domains are indicated with red boxes. A conserved Cys-Trp motif is underlined, and a GS{alpha} binding domain is highlighted in green (numbers refer to shark sequence).

 

Figure 4
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  		Fig. 4. Phylogenetic tree showing the relationship of vertebrate VIP receptors. Shark VIP-R has highest identity with the VIP-R1 receptor subtype. The tree was generated by Clustal W alignment with DNAStar MegAlign software.

 
Stimulation of chloride conductance by VIP in Xenopus oocytes coexpressing sVIP-R and CFTR. Application of VIP to oocytes coexpressing sVIP-R and CFTR resulted in an abrupt increase in chloride conductance (Fig. 5A, time course of representative experiment). To ensure that the conductance through CFTR was chloride dependent, all chloride was removed from the perifusate resulting in complete inhibition of chloride conductance (Fig. 5A). Following washout with frog Ringer, perifusion with VIP promptly restored this conductance. Addition of 300 µM glibenclamide, a known inhibitor of CFTR (30), to the perifusate inhibited VIP stimulated conductance completely and irreversibly (Fig. 5A). Control experiments were carried out with three groups of oocytes injected with: water only, VIP-R only, or CFTR only. These oocytes did not respond to VIP, but CFTR-injected oocytes responded to 10 µM forskolin and 1 mM IBMX (data not shown). A current-voltage plot of a representative experiment is shown in Fig. 5B.


Figure 5
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  		Fig. 5. Functional expression of shark VIP-R and cystic fibrosis transmembrane conductance regulator (CFTR) in Xenopus oocytes. Two-electrode voltage clamping was performed on oocytes at a holding potential of –60 mV. Oocytes were equilibrated to basal current values with frog Ringer (FR) before stimulation. A: representative experiment showing conductance of an oocyte expressing shark VIP-R and CFTR. Exposure to VIP 50 nM increased chloride conductance abruptly. The removal of Cl– from the perifusate (chloride replaced with glutamate) during maximal stimulation by VIP resulted in a sharp decline in Cl– conductance to near-basal levels. After washout with frog Ringer, the application of VIP 50 nM restimulated Cl– conductance. This conductance was immediately inhibited to basal levels by the addition of glibenclamide 300 µM to the perifusate (representative of 3 experiments). B: current voltage (I-V) plot during a 2-s voltage ramp from –120 mV to +60 mV on a representative oocyte expressing shark VIP-R and CFTR, before and after exposure to 10 nM VIP. C: VIP increases Cl– conductance (Gm) in Xenopus oocytes expressing shark VIP-R and CFTR in a dose dependent manner with an EC50 of 8 nM. Maximal stimulation was observed at 80–100 nM. Values are means ± SE (n = 3–5 oocytes each point).

 
Response to VIP in Xenopus oocytes expressing sVIP-R and CFTR. A concentration response to VIP was determined using VIP concentrations from 0.1 to 300 nM (Fig. 5C). VIP elicited a concentration-dependent, sigmoidally shaped activation of CFTR chloride conductance, with a calculated EC50of 8 nM VIP.

Comparison of VIP and related agonists in Xenopus oocytes coexpressing sVIP-R and CFTR. Experiments were next carried out to determine the relative potencies of five secretin family peptides, VIP, PHI, GHRH, PACAP, and secretin. VIP had greatest affinity for the receptor, increasing the chloride conductance of oocytes from 11 ± 3 µS to 127 ± 26 µS. GHRH increased chloride conductance to 85 ± 11.5 µS, PHI to 63 ± 8 µS, PACAP to 34 ± 12 µS, and secretin to 14 ± 2 µS. We conclude that the order of potency of agonists on the expressed functional sVIP-R, as determined by chloride conductance through CFTR chloride channels, is VIP > GHRH = PHI > PACAP > secretin, an order similar to that observed in the perfused gland (Fig. 6).


Figure 6
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  		Fig. 6. Relative potencies of VIP and related peptides in Xenopus oocytes expressing shark VIP-R and CFTR. All oocytes were perifused with frog Ringer to achieve steady-state conductance. Peptide agonists were then added at a concentration of 50 nM, and the subsequent change in Cl– conductance was measured. Relative potency of secretin, GHRH, PACAP, PHI, and VIP was established as VIP > PHI = GHRH > PACAP > secretin. Secretin increased Cl– conductance from 7 ± 2 to 14 ± 2 µS; PACAP increased Cl– conductance from 9 ± 2 to 34 ± 12 µS; GHRH increased Cl– conductance from 14 ± 3 to 85 ± 11 µS; PHI increased Cl– conductance from 8 ± 2 to 63 ± 8 µS; and VIP increased Cl– conductance from 11 ± 2 to 127 ± 26 µS. Values are means ± SE with n = 4 for secretin, PHI, and GHRH; n = 6 for PACAP; and n = 12 for VIP. *P < 0.02 compared with secretin, **P < 0.01 compared with secretin. Other significant values are: VIP stimulation was significantly greater than PHI (P < 0.03) and PACAP (P < 0.01) and GHRH stimulation was significantly greater than PACAP (P < 0.04).

 
Quantitative real-time PCR determination of the distribution of VIP-R and CFTR mRNA in tissues of Squalus acanthias. Quantitative real-time PCR (Q-PCR) analysis of VIP-R and CFTR mRNA among 11 shark tissues showed the highest levels of expression for both the VIP-R and CFTR protein mRNAs in the rectal gland. VIP-R was also highly expressed in shark intestine, brain, and stomach (Fig. 7A). CFTR was also highly expressed in shark intestine, brain, and gonad. (Fig. 7B).


Figure 7
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  		Fig. 7. Quantitative real-time PCR analysis of VIP-R and CFTR mRNA expression levels in 11 tissues of Squalus acanthias. A: relative expression of VIP-R in shark tissues. Values are means ± SE (n = 6). B: relative expression of CFTR in shark tissues. Values are means ± SE (n = 6). In both A and B, the value of one muscle sample was set equal to 1. SRG, shark rectal gland.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
VIP is a 28 amino acid polypeptide that has a wide spectrum of biological effects in vertebrates, including the digestive tract, heart, brain, endocrine glands, and the immune system (10). VIP is a member of a superfamily of related peptides, which includes PACAP, PHI, and GHRH (19). Several G protein-coupled receptors bind these peptides with varying affinities, including the VIP-R1 and VIP-R2 receptors. A characteristic of previously cloned VIP-R1 and VIP-R2 receptors is their equal affinity for the agonists VIP and PACAP (4, 20, 21).

By cloning and functional expression, the present studies identify an ancestral VIP-R that mediates chloride secretion in the rectal gland of the elasmobranch Squalus acanthias. This is the first VIP-R from a marine species to be characterized at the molecular level and is the oldest VIP-R cloned to date. The sVIP-R, when coexpressed with the CFTR chloride channel in Xenopus oocytes, has an affinity for peptide agonists (VIP > PHI = GHRH > PACAP > secretin) that is nearly identical to the agonist profile in the intact in vitro perfused shark rectal gland (VIP > GHRH = PHI > PACAP > secretin), suggesting that this receptor is responsible for the physiological action of VIP and related peptides in the gland.

We employed three systems to examine the effects of VIP and related peptides on sVIP-R: 1) perfusion of the intact organ, 2) measurements of transepithelial chloride transport in polarized primary epithelial cell monolayers lacking neural and connective tissue elements, and 3) heterologous expression and TEVC in Xenopus oocytes. These systems allowed the examination of VIP effects at three levels: whole tissue, epithelial cell, and expressed protein. Each demonstrates a similar potency order of agonists.

The sVIP-R has the following characteristics: 1) structural similarity to previously cloned vertebrate VIP-R1, not VIP-R2 receptors; 2) remarkably low affinity for the peptide PACAP compared with all other VIP-R receptors (4, 20, 21); 3) an affinity for GHRH that is greater than that reported for human, rat, or goldfish VIP-R (4); 4) absence of two residues, Glu36and Pro115, described as essential for VIP binding in human VIP-R1 (20, 24); 5) three N-glycosylation sites, including two (Asn58 and Asn69) considered necessary for delivery of the protein to the plasma membrane (5) are maintained in sVIP-R, whereas a fourth putative N-glycosylation site (Asn290) in human VIP-R1 is not maintained in sVIP-R.

The order of affinity for rat VIP-R1 receptors is VIP > PACAP > PHI > secretin. Rat VIP-R2 receptors behave similarly but have almost no affinity for secretin, a characteristic that differentiates the two receptors (36). Studies in recombinant cell lines with human VIP-Rs showed little difference in affinity toward VIP and PACAP (15, 23). The human VIP-R1 receptor in transfected transformed African green monkey kidney cells (COS-7) and Chinese hamster ovary (CHO) cell lines has an order of affinity of VIP > PACAP > secretin (7, 13). Human VIP-R2 receptors transfected in CHO cells show small preferential affinity for PACAP over VIP, while the order of affinity in COS-7 cells demonstrates affinity of VIP over PACAP (1, 35). Human and rat VIP-R1 receptors also have a low affinity for GHRH (4, 7, 36). Thus the sVIP-R, in both expression studies of the cloned receptor and in the intact perfused gland, is unique in having a very low affinity for PACAP relative to VIP and a higher affinity for GHRH compared with other VIP-Rs.

sVIP-R maintains the long NH2 terminus considered essential in binding VIP (20). Seven cysteine residues located in this NH2-terminal extracellular domain, Cys50, Cys63, Cys72, Cys86, Cys105, Cys122, and Cys285, necessary for the receptor to bind peptide agonists (14), are all conserved in the sVIP-R. Also conserved in the shark protein are other residues crucial to the human VIP-R1 receptor's ability to bind VIP and related peptides. These include residues corresponding to human Asp68, Pro87, Gly109, Trp110, Trp73 (6), Trp67 (25), and Lys143 (20). Another residue, Asp196, found to be essential for VIP binding as well as VIP-stimulated cAMP production (8) is also conserved in the sVIP-R. Additionally, a motif containing two hydrophobic residues flanked by two basic residues (RLAK), which is important for Gs{alpha} protein coupling is conserved in the third intracellular loop of sVIP-R (26).

Two residues previously reported as essential for VIP binding in human VIP-R1, Glu36 (24) and Pro115 (20), are not present in the shark receptor. This finding could account for the differing agonist affinities of the two species or could indicate that neither residue is important for the binding of ligand to the shark protein. Three N-glycosylation sites, two of which (Asn58 and Asn69) are considered necessary for delivery of the receptor to the plasma membrane, are also maintained in sVIP-R (5). A fourth putative N-glycosylation site in human VIP-R1 (Asn290) is not maintained in sVIP-R (5).

When coexpressed with the CFTR chloride channel in Xenopus oocytes, VIP and related peptides increase chloride conductance as measured by TEVC. This change in conductance indicates that the cloned sVIP-R1 is a functional protein that activates chloride secretion via the cAMP-protein kinase A- CFTR channel pathway. The ability to inhibit sVIP-R stimulated chloride conductance by removal of chloride from the perifusate, and inhibition by glybenclamide, a known inhibitor of CFTR channels (30) (Fig. 6A), are consistent with this effector pathway. The relative distribution of VIP-R and CFTR mRNA among tissues of S. acanthias, particularly rectal gland, intestine, and brain, supports a model in which the two proteins are functionally coupled (Fig. 7).

In summary, the present studies characterize a novel G protein-coupled VIP-R from the shark rectal gland that mediates VIP-stimulated chloride secretion in this highly specialized epithelial organ. This receptor has unique structural and functional characteristics compared with previously cloned mammalian VIP-Rs.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants DK-34208 and P30-ES-3828 (Center for Membrane Toxicology Studies).


   ACKNOWLEDGMENTS
 
We thank Stephen Aller, Cinda Scott, Christine Smith, Martha Ratner, Will Motley, and Suzanne Forrest for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. N. Forrest, Jr., Dept. of Internal Medicine, Yale Univ. School of Medicine, New Haven, Connecticut 06510 (e-mail: john.forrest{at}yale.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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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