Rat and guinea pig pancreatic acini possess both VIP1 and VIP2 receptors, which mediate enzyme secretion

T. Ito1, W. Hou1, T. Katsuno1, H. Igarashi1, T. K. Pradhan1, S. A. Mantey1, D. H. Coy2, and R. T. Jensen1

1 Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892; and 2 Peptide Research Laboratories, Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana 70112


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Pancreatic acini from most species possess vasoactive intestinal peptide (VIP) receptors. Recently, two subtypes of VIP receptors, VIP1-R and VIP2-R, were cloned. Which subtype exists on pancreatic acini or mediates secretion is unclear. To address this, we examined pancreatic acini from both rat and guinea pig. VIP1-R and VIP2-R mRNA were identified in dispersed acini from both species by Northern blot analysis and in rat by Southern blot analysis. With the use of the VIP2-R-selective ligand Ro-25-1553 in both species, inhibition of binding of 125I-labeled VIP to acini showed a biphasic pattern with a high-affinity component (10%) and a second representing 90%. The VIP1-R-selective ligand, [Lys15,Arg16,Leu27]VIP-(1---7)-GRF-(8---27), gave a monophasic pattern. Binding of Ro-25-1553 was better fit by a two-site model. In both rat and guinea pig acini, the dose-response curve of Ro-25-1553 for stimulation of enzyme secretion was biphasic, with a high-affinity component of 10-15% of the maximal secretion and a low-affinity component accounting for 85-90%. At low concentrations (10 nM) of Ro-25-1553 and [Lys15,Arg16,Leu27]VIP-(1---7)-GRF(8---27), which only occupy VIP receptors, a 4-fold and a 56-fold increase in cAMP occurred, respectively. These results show that both VIP1-R and VIP2-R subtypes exist on pancreatic acini of rat and guinea pig, their activation stimulates enzyme secretion by a cAMP-mediated mechanism, and the effects of VIP are mediated 90% by activation of VIP1-R and 10% by VIP2-R. Because VIP has a high affinity for both VIP-R subtypes, its effect on pancreatic acini is mediated by two receptor subtypes, which will need to be considered in future studies of the action of VIP in the pancreas.

vasoactive intestinal peptides; adenosine 3',5'-cyclic monophosphate; pancreatic secretion; vasoactive intestinal peptide receptors


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

VASOACTIVE INTESTINAL PEPTIDE (VIP) is a naturally occurring 28-amino acid peptide originally isolated from porcine duodenum that has widespread effects on many tissues, functioning primarily as a neurotransmitter (7). Pancreatic acinar cells from most species possess VIP receptors, and activation of high-affinity VIP receptors is coupled to activation of adenylate cyclase, increased cellular cAMP, and stimulation of enzyme secretion (2, 8, 10). Numerous studies have explained the relationship between VIP receptor occupation and changes in biological activity in acinar cells in different species (2, 7, 8, 10). These studies demonstrate that the exact relationship remains unclear due to the occurrence of multiple binding sites, lack of highly selective antagonists, the fact that VIP interacts with VIP and secretin receptors, both of which are present and coupled to adenylate cyclase, and that there is varying selectivity of different agonists in different species for VIP receptors over related receptors (2, 6, 18). In guinea pig, studies of binding of 125I-labeled VIP to pancreatic acinar cells indicate that these cells have two functionally distinct classes of receptors, each of which can interact with VIP and secretin and that high- and low-affinity states of the VIP receptors exist (18, 37, 38). In rat pancreatic acini, a different pattern occurs, in which four classes of receptors are necessary to account for the receptor interaction and the changes in biological activity caused by VIP and secretin (2, 6).

Recently, two subtypes of VIP receptors, a VIP subtype 1 receptor (VIP1-R) and a VIP subtype 2 receptor (VIP2-R), were cloned (5, 17, 23, 32). However, at present, it is not clear whether either or both of these VIP receptor subtypes exist on pancreatic acini. Therefore, the presence of these two subtypes on pancreatic acini could be an important factor in the relationship of the ability of VIP to occupy pancreatic acinar cell receptors and alter biological activity and could partially explain some of the complexity of binding sites previously described. Moreover, there are no functional studies or ligand-binding studies to suggest whether either or both VIP receptor subtypes are responsible for mediating the action of VIP on pancreatic acini in any species. Therefore, the purpose of the present study was to determine whether either or both VIP receptor subtypes exist in the pancreatic acini in the rat and guinea pig and to determine whether activation of the VIP1-R or VIP2-R alters cell function. Our results demonstrated for the first time that both VIP receptor subtypes exist on pancreatic acini in rat and guinea pig and that both are responsible for mediating the action of VIP on enzyme secretion in both species and are coupled to adenylate cyclase in acinar cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

Male guinea pigs (100-150 g) were obtained from Charles River (Wilmington, DE). Male Sprague-Dawley rats (80-100 g) were obtained from Taconic Farm (Germantown, NY). Chinese hamster ovary (CHO) cells and Sup-T1 cells were obtained from American Type Culture Collection (Manassas, VA). CHOP cells were a gift from Dr. James W. Dennis, Samuel Lunenfeld Research Institute, Toronto, Canada (15). Porcine vasoactive intestinal peptide (VIP) and porcine secretin were from Bachem Bioscience (King of Prussia, PA); purified collagenase (type CLSPA) was from Worthington Biochemicals (Freehold, NJ); basal medium Eagle (BME) amino mixture, BME vitamin solution, and lipofectamine transfection reagent were from Life Technologies (Gaithersburg, MD); Geneticin (G418 sulfate) was from Mediatech; pcDNA 3.1(+) and pcDNA 3.1(-) were from Invitrogen (Carlsbad, CA); cAMP, bacitracin, soybean trypsin inhibitor, 1,3-dimethylxanthine (theophylline), IBMX, triethylamine, and acetic anhydride were from Sigma (St. Louis, MO); BSA fraction V was from Miles Laboratories (Elkhart, IN); normal rabbit serum was from Calbiochem (La Jolla, CA); 125I-VIP (2,200 Ci/mmol), 125I-labeled cAMP (2,200 Ci/mmol), [alpha -32P]dCTP (3,000 Ci/mmol), and cAMP anti-serum complex were from NEN Life Science Products (Boston, MA); Phadebas amylase test reagent was from Pharmacia Diagnostics (Piscataway, NJ); silicon oil (Nyosil-50) was from W. F. Nye (New Bedford, MA); HEPES was obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN); and DMEM and Ham's F-12K medium were from Biofluid (Rockville, MD).

The standard incubation solution contained (in mM) 24.5 HEPES (pH 7.45), 98 NaCl, 6 KCl, 2 KH2PO4, 5 sodium pyruvate, 5 sodium fumarate, 5 sodium glutamate, 2 glutamine, 11.5 glucose, 0.5 CaCl2, and 1 MgCl2 and 1% (wt/vol) BSA, 0.2% (wt/vol) soybean trypsin inhibitor, 1% (vol/vol) amino acid mixture, and 1% (vol/vol) essential vitamin mixture.

Methods

Preparation of peptides. Ro-25-1553 {[Ac-Glu8,OCH-Tyr10,Lys12,Nle17,Ala19Asp25,Leu26,Lys27,28]VIP-(cyclo-21---25), a cyclic VIP analog selective for VIP2-R} (13, 14, 25, 26) and [Lys15,Arg16,Leu27]VIP(1---7)-GRF-(8---27) (a VIP analog selective for VIP1-R) (12, 14) were synthesized using standard solid phase methods as generally described previously (30). The crude hydrogen fluoride-cleaved peptides were purified on a column (2.5 × 90 cm) of Sephadex G-50, which was eluted with 1 M acetic acid. Further purification was performed by the preparative medium pressure chromatography on a column (1.5 × 45 cm) of Vydac C18 silica (10-15 mm), which was eluted with linear gradients of acetonitrile in 0.1% (vol/vol) trifluoroacetic acid (flow rate of 6 ml/min). Peptides were purified further by rechromatography on the same column with slight modifications of gradient conditions. Homogeneity of the peptides was assessed by TLC and analytical reverse-phase HPLC, and purity was at least 97% for each peptide. Amino acid analysis gave the expected amino acid ratios. Each analog gave good recovery of the molecular ion corresponding to the calculated molecular mass.

Preparation of dispersed pancreatic acini. Dispersed acini from guinea pig and rat were prepared using the modification (20) of the method described previously (27). Unless specified otherwise, dispersed acini from the pancreas of one animal were suspended in 100 ml of standard incubation solution. All incubations were at 37°C. The incubation solution was equilibrated with 100% O2, and incubations for measurement of amylase release and cAMP release were performed with 100% O2 as the gas phase.

Northern blot analysis. Total RNA was extracted from rat (r) VIP1-R and rVIP2-R stably transfected CHO cells, rat jejunum, rat whole pancreas, rat pancreatic acini, and guinea pig pancreas and guinea pig pancreatic acini that had been snap frozen in liquid nitrogen followed by pulverization in liquid nitrogen using the RNeasy Midi kit (Qiagen, Chatsworth, CA). Approximately 15 µg of total RNA samples from each tissue were subjected to denaturing gel electrophoresis in formaldehyde agarose [0.22 M and 1% (wt/vol), respectively] and then transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH) according to the methods of Thomas (33). The probe for the rVIP1-R (a 316-bp fragment) was generated from the rat jejunum. The gene-specific PCR primers used were as follows: 5'-CTGGCAGGAGCCCTTGCCTG-3' (sense) and 5'-GTTGCTGCTCATCCAGACTCG-3' (antisense). The probe for rVIP2-R (a 326 bp fragment) was generated from the rat antrum. The gene-specific PCR primers used were as follows: 5'-TCCACCCAGAATGCCGGTTTC-3' (sense) and 5'-GTGATCTTACTCTCATCCTCGG-3' (antisense). DNA sequence of the PCR products was verified by automatic sequencing on both strands. The fragments were cloned in the expression vector pCR2.1 using TA cloning kit (Invitrogen, San Diego, CA). The PCR reaction was carried out with GeneAmp PCR system (Perkin-Elmer, Foster City, CA) using the following conditions: an initial step of 3 min at 95°C; 35 cycles of 30-s denaturation at 95°C, 30-s annealing at 45°C, and 1-min extension at 75°C with a 5-min final extension at 75°C. Hybridization was carried out at 37°C using probes labeled with the random primer DNA labeling system (Life Technologies) in a buffer containing 40% (vol/vol) formamide (Fluka Chemical), 4× SCC (300 mM NaCl and 30 mM sodium citrate; Research Genetics, Huntsville, AL), 20 mM Tris, pH 7.5 (Quality Biological, Gaithersburg, MD), 10% (vol/vol) dextran sulfate (Oncor, Gaithersburg, MD), 1× Denhardt's solution (Digene Diagnostics, Beltsville, MD), and 20 µg/ml sonicated herring sperm DNA (Digene Diagnostics, Beltsville, MD). Nitrocellulose filters of Northern blot transfers were hybridized overnight with the rVIP1-R and rVIP2-R cDNA, respectively, after radiolabeling to a specific activity of about 1 × 109 cpm/µg DNA. Filters were washed sequentially, with increasing stringency, ending with a final wash in 0.1× SSC-0.1% SDS (vol/vol) at 55°C, air-dried, and exposed to X-ray films (XAR, Eastman Kodak).

Southern blot analysis. Total RNA from rat whole pancreas, dispersed rat pancreatic acini, and rat jejunum was used to synthesize first-strand cDNA. The first-strand cDNA was synthesized using 1.0 µg of total RNA with a first-strand cDNA synthesis kit using random hexamer primers (Life Technologies). For amplification from the first-strand cDNAs, the same gene-specific primers for rVIP1-R and rVIP2-R performed for Northern blot were used as described above. The PCR reaction was performed under the following conditions: an initial step of 3 min at 95°C, 35 cycles of 30-s denaturation at 95°C, 30-s annealing at 45°C, and 1-min extension at 75°C with a 5-min final extension at 75°C. The PCR products were electrophoretically separated in 1.2% (wt/vol) SeaKem GTG agarose gels (FMC BioProducts, Rockland, ME) and transferred to nitrocellulose. Nitrocellulose filters of Southern blot transfers were hybridized at room temperature with the 32P-end-labeled rVIP1-R probe (316-bp fragment) and rVIP2-R probe (326-bp fragment) as described under Northern blot analysis. The filters were then washed at room temperature, air-dried, and exposed to X-ray film for several hours.

Cell culture. The rVIP1-R/CHOP and rVIP2-R/CHOP (i.e., rat VIP1-R or rat VIP2-R transiently transfected into CHOP cells) were grown in DMEM supplemented with 10% (vol/vol) fetal bovine serum, 1% (vol/vol) antibiotics, and 200 µg/ml G418. The rVIP1-R/CHO, rVIP2-R/CHO, hVIP1-R/CHO, and hVIP2-R/CHO (i.e., rat or human VIP1-R and rat or human VIP2-R stably expressed in CHO cells) were grown in Ham's medium supplemented with 10% (vol/vol) fetal bovine serum, 1% (vol/vol) antibiotics, and 300 µg/ml G418. Cultures were maintained in incubators at 37°C in an atmosphere of 5% CO2 and 95% air.

Construction of rVIP1-R cDNA, rVIP2-R cDNA, hVIP1-R cDNA, and hVIP2-R cDNA. Total RNA was extracted from rat jejunum (contains VIP1-R) and rat antrum (contains VIP2-R), and after cDNA synthesis rVIP1-R and rVIP2-R cDNA were obtained using receptor-specific primers and RT-PCR. The full-length correct sequence was confirmed by automated sequencing on both strands using a A377 DNA sequencer (Perkin-Elmer). Both rVIP1-R and rVIP2-R cDNA were ligated into the Xba I/Hind III sites of pcDNA3.1(-) vector. hVIP1-R cDNA (hVIP1-R) was a gift from Dr. Charles D. Ulrich II, Division of Digestive Diseases, University of Cincinnati Medical Center. hVIP1-R cDNA was ligated into the Hind III/Xba I sites of pcDNA3.1(+) vector. hVIP2-R cDNA was cloned from human lymphoblast Sup-T1 cells as described previously (32) and ligated into the Hind III/Xba I sites of pcDNA3.1(+) vector. The full-length correct sequence of hVIP1-R and hVIP2-R were confirmed by automated sequencing on both strands using a A377 DNA sequencer.

Transient transfection of CHOP cells with rVIP1-R and rVIP2-R. CHOP cells were maintained in DMEM supplemented with 10% (vol/vol) fetal bovine serum, 1% (vol/vol) antibiotics (100 IU/ml penicillin and 100 µg/ml streptomycin), and 200 µg/ml G418 sulfate. Subconfluent CHOP cells were grown in 100-mm-diameter culture plates and transfected with 5 µg plasmid DNA rVIP1-R and rVIP2-R, respectively, in the expression vector pcDNA3.1(-) using the lipofectamine transfection reagent. Transfected cells were grown for 2 days and used for binding studies.

Transfection of CHO cells with rVIP1-R, rVIP2-R, hVIP1-R, and hVIP2-R and selection of stable transfectants. CHO-K1 cells were maintained in Ham's medium supplemented with 10% (vol/vol) fetal bovine serum and 1% (vol/vol) antibiotics (100 IU/ml penicillin and 100 µg/ml streptomycin). Subconfluent CHO cells were grown in 100-mm-diameter culture plates and transfected with 5 µg of plasmid DNA rVIP1-R and rVIP2-R, respectively, in the expression vector pcDNA3.1(-) and plasmid DNA hVIP1-R and hVIP2-R, respectively, in the expression vector pcDNA3.1(+), using the lipofectamine transfection reagent according to the manufacturer's instructions. Transfected cells were grown for 2 wk in medium containing 800 µg/ml G418 sulfate. Individual colonies were isolated and expanded, and cloned cells were screened for each VIP-R subtype expression by receptor binding of 125I-labeled VIP.

Binding studies. Binding of 125I-VIP to pancreatic acini was performed as described previously (8, 21, 38). Briefly, dispersed acini from the pancreas of two guinea pigs or two rats were suspended in 15-20 ml of standard incubation solution containing 0.05% (wt/vol) bacitracin. 125I-VIP was added at 50 pM without (total binding) or with 1 µM VIP (nonsaturable binding). Acini were incubated for 45 min at 37°C. Samples (100 µl) of cell suspension were centrifuged through silicon oil (d = 1.05) in Microfuge tubes to separate bound from unbound ligand. The tubes were washed twice with 4% (wt/vol) BSA in standard incubation solution, and radioactivity was determined by a Packard Auto-Gamma counter (Meriden, CT). Nonsaturable binding for 125I-VIP was <3% of total binding.

Binding of 125I-VIP to rVIP1-R/CHOP, rVIP2-R/CHOP, rVIP1-R/CHO, rVIP2-R/CHO, hVIP1-R/CHO, and hVIP2-R/CHO cells was performed by incubation in standard incubation solution containing 0.05% (wt/vol) bacitracin for 60 min at room temperature. The separation of bound from free radioactivity was obtained by centrifugation of cells through 2% (wt/vol) BSA in standard incubation solution. The tubes were washed twice with 2% (wt/vol) BSA in standard incubation solution, and radioactivity was counted. Nonsaturable binding for 125I-VIP was <5% of total binding.

For all peptides, the IC50 was calculated, which was the concentration that gave half-maximal inhibition of that seen with a maximally effective concentration of VIP (1 µM). The IC50 was calculated using the curve-fitting program Kaleidograph, and the affinity constant and binding capacity were determined using the least-squares, curve-fitting program LIGAND (24).

Assessment of amylase release from pancreatic acini. Amylase release was measured using the procedure published previously (20, 27). Briefly, dispersed acini from one rat pancreas were suspended in standard incubation solution, whereas dispersed acini from one guinea pig pancreas were suspended in standard incubation solution containing 5 mM theophylline. Incubations contained 0.5 ml of cell suspension and were at 37°C for 30 min. Amylase activity was determined using the Phadebas reagent and was expressed as the percentages of the cellular amylase released into the extracellular medium during the incubation.

For all peptides, the EC50 was calculated, which was the concentration of the peptide that gave half-maximal stimulation of a maximally effective concentration of VIP (10 nM). The EC50 was calculated using the curve-fitting program Kaleidograph.

cAMP release from guinea pig pancreatic acini. Cellular cAMP was determined by RIA as described previously (2, 37, 38). Guinea pig pancreatic acinar cells were suspended in 50-2,000 ml of standard incubation solution containing 1 mM IBMX. Incubations contained 0.5 ml of cell suspension and were at 37°C for 30 min. The concentration of pancreatic acinar cells was adjusted to maintain cAMP on the linear portion of the standard curve. Results were expressed as the value obtained with the indicated peptide (experiment) over the value obtained with no peptide added (control).

Statistical Analysis

The results are means ± SE of three or more experiments. The statistical comparisons were performed using ANOVA with the Dunnett's test, and a P value of <0.05 was considered significant. EC50 were calculated using the curve-fitting program Kaleidograph. Binding curves were fitted using the least-squares curve fitting program LIGAND (24) to calculate dissociation constant (Kd) values.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Expression of Subtypes of VIP Receptors in the Pancreas in the Rat and Guinea Pig

To determine whether both subtypes of VIP receptors existed in the pancreas in the rat and guinea pig, Northern blot analysis was used initially (Fig. 1). CHO-K1 cells stably transfected with rVIP1-R and rVIP2-R were included as a positive control as well as rat jejunum, because jejunum is reported to express moderately both VIP1-R and VIP2-R (34). The VIP1-R and VIP2-R probes specifically interacted only with the VIP1-R mRNA and VIP2-R mRNA, respectively, under the conditions used (Fig. 1A). VIP1-R mRNA and VIP2-R mRNA were identified in jejunum as well as in both whole pancreas and dispersed pancreatic acinar cells from both species (Fig. 1). Similarly, with the use of Southern blot analysis (Fig. 2), both VIP1-R and VIP2-R were detectable in both whole pancreas and dispersed pancreatic acinar cells from rat. These results indicated that pancreatic acini of both rat and guinea pig express both VIP1-R and VIP2-R subtypes.


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Fig. 1.   Northern blot analysis to detect expression of rat vasoactive intestinal peptide receptors (rVIP1-R and rVIP2-R) or guinea pig (gp) tissue mRNA in transfected Chinese hamster ovary (CHO) cells, jejunum, pancreas, and dispersed pancreatic acini. Fifteen micrograms of total cellular RNA isolated from each tissue and from pancreatic acinar cells from rat and guinea pig were analyzed by Northern blot, and subsequent gene-specific hybridization was performed as described under Methods. A: autoradiograms of mRNA from rVIP1-R- and rVIP2-R-transfected CHO-K1 cells. B: autoradiograms of mRNA from rat tissues. C: autoradiograms of mRNA from guinea pig tissues analyzed using a specific rVIP1-R or rVIP2-R probe. Rat jejunum contains both VIP1-R and VIP2-R and was used as a control. Bottom panels show positions of the 28S and 18S ribosomal RNAs.



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Fig. 2.   Autoradiograph of Southern blot after RT-PCR using gene-specific primers for rVIP1-R and rVIP2-R. Reverse transcription was performed using total cellular RNA. PCR was performed with gene-specific primers for rVIP1-R and rVIP2-R as described under Methods. Hybridization was performed using 32P-radiolabeled gene-specific probes as described under Methods. A: results with a rVIP1-R-specific probe. B: results with a rVIP2-R-specific probe.

Determination of the Presence and Function of the Two VIP Receptor Subtypes

To determine whether both VIP receptor subtypes were present and the effect of their cellular activation on cell function in acini, selective ligands for the VIP1-R and VIP2-R receptors were needed. [Lys15,Arg16,Leu27] VIP-(1---7)-GRF-(8---27) is reported to be a selective VIP1-R ligand functioning as an agonist of rVIP1-R and hVIP1-R (12). Ro-25-1553 is reported to have selectivity for the VIP2-R, functioning as an agonist of rVIP2-R (35) and hVIP2-R (13). To determine the selectivity of these two ligands, their affinities for rVIP1-R or rVIP2-R transiently expressed in CHOP cells and CHO cells stably expressing rVIP1-R, rVIP2-R, hVIP1-R, or hVIP2-R were determined (Table 1, Fig 3). As described previously (1, 11, 12, 22, 31, 34), VIP was found to interact with similar high affinity (IC50 of 0.53 ± 0.01) with CHO cells stably expressing rVIP1-R (Fig. 3A) or rVIP2-R (Fig. 3B). With both rVIP receptors stably expressed in CHO-K1 cells, the VIP dose-inhibition curve was broad, spanning at least four log units (Fig. 3). Computer analysis (LIGAND) of the VIP dose-inhibition curve (n = 3) for 125I-VIP binding demonstrated with the rVIP1-R (P = 0.006) and the rVIP2 (n = 3) (P < 0.001) CHO-K1 cells that both were fitted significantly better by a two-binding site than a single-binding site model. For the rVIP1-R stably transfected into CHO-K1 cells, the high- and low-affinity binding sites had affinities of 0.080 ± 0.27 nM and 39.3 ± 24.1 nM, respectively, with receptor densities of 135 ± 23 fmol/106 cells and 3,570 ± 1,810 fmol/106 cells. For the rVIP2-R stably transfected into CHO-K1 cells, the high- and low-affinity sites had affinities of 0.098 ± 0.065 nM and 4.6 ± 2.8 nM, respectively, with densities of 26.2 ± 12.7 fmol/106 cells and 190 ± 46 fmol/106 cells. In CHOP cells transiently transfected with rVIP1-R or rVIP2-R, VIP had a 3.2-fold higher affinity for rVIP2-R than rVIP1-R, whereas [Lys15,Arg16,Leu27]VIP-(1---7)-GRF-(8---27) had a 53-fold higher affinity for rVIP1-R and Ro-25-1553 had a >4,412-fold higher affinity for rVIP2-R (Table 1). In CHO cells stably transfected with either rVIP1-R or rVIP2-R, each of the three peptides inhibited binding of 125I-VIP; however, they differed in affinity for the two receptors (Fig. 3, Table 1). In CHO cells stably transfected with rVIP1-R or rVIP2-R, whereas VIP had similar affinity for both rVIP receptors, [Lys15,Arg16,Leu27] VIP(1---7)-GRF(8---27) had a >169-fold higher affinity for rVIP1-R and Ro-25-1553 had a 241-fold higher affinity for rVIP2-R (Fig. 3, Table 1). Similarly, in CHO cells stably transfected with hVIP1-R or hVIP2-R, VIP had a 6.4-fold higher affinity for hVIP1-R, whereas [Lys15,Arg16,Leu27] VIP-(1---7)-GRF(8---27) had a >1,765-fold higher affinity for hVIP1-R and the Ro-25-1553 had a 280-fold higher affinity for hVIP2-R (Fig. 3, Table 1).

                              
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Table 1.   Abilities of VIP, [Lys15, Arg16, Leu27]VIP-(1---7)-GRF-(8---27), and Ro-25-1553 to interact with VIP receptors in CHOP cells transiently transfected with rVIP1-R and rVIP2-R, respectively, CHO cells stably transfected with rVIP1-R and rVIP2-R, respectively, and CHO cells stably transfected with hVIP1-R and hVIP2-R, respectively



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Fig. 3.   Abilities of VIP, [Lys15,Arg16,Leu27]VIP-(1---7)-GRF-(8---27), and Ro-25-1553 to interact with VIP receptors in CHO cells stably transfected with rVIP1-R (A) or rVIP2-R (B). rVIP1-R-transfected CHO-K1 cells (0.2 × 106 cells/ml) or rVIP1-R-transfected CHO-K1 cells (1.5 × 106 cells/ml) were incubated with 50 pM 125I-labeled VIP plus the indicated concentrations of unlabeled peptide for 60 min at 23°C. Results are expressed as the percentage of the saturable binding of the 125I-VIP observed in absence of competing peptide. In each experiment, each value was determined in duplicate and results are given as means ± SE from at least 3 separate experiments.

In guinea pig pancreatic acini, inhibition of binding of 125I-VIP by VIP showed a broad dose-inhibition curve with detectable inhibition at 0.03 nM, half-maximal inhibition at 1.8 nM, and complete inhibition at 1 µM (Fig. 4A). The VIP1-R-selective ligand [Lys15,Arg16,Leu27] VIP-(1---7)-GRF-(8---27) showed a similar dose-inhibition curve to VIP but was 2.3-fold less potent, causing a half-maximal inhibition at 4.1 nM, which was significantly less (P < 0.01) than the potency of VIP (Table 2) . In contrast, the VIP2-R-selective ligand Ro-25-1553 showed a biphasic pattern of inhibition with an initial plateau representing 10% of the total binding from 1 to 10 nM (Fig. 4A). A second component of inhibition occurred at concentrations of Ro-25-1553 >10 nM, representing 90% of the total binding (Fig. 4A). Ro-25-1553 caused half-maximal inhibition of the high-affinity component at 0.43 ± 0.001 nM and half-maximal inhibition of the low-affinity component at 1.7 ± 0.1 µM (Fig. 4A; Table 2). A similar result was seen in rat pancreatic acini (Fig. 4B). Specifically, inhibition of binding of 125I-VIP by VIP in rat acini also showed a broad dose-inhibition curve with detectable inhibition at 0.03 nM, half-maximal inhibition at 1.7 nM, and complete inhibition at 1 µM (Fig. 4B). The VIP1-R-selective ligand [Lys15,Arg16,Leu27]VIP-(1---7)-GRF-(8---27) showed a similar dose-inhibition curve to VIP but was 15.8-fold less potent, causing a half-maximal inhibition at 26.9 nM. Similar to guinea pig acini, the dose-inhibition curve of Ro-25-1553 in rat acini was biphasic, with 10% of the total binding to a receptor with high affinity for Ro-25-1553 and 90% to a site with low affinity for Ro-25-1553 (Fig. 4B). Ro-25-1553 caused half-maximal inhibition of the high-affinity component at 0.21 ± 0.001 nM and half-maximal inhibition of low-affinity component at 457 ± 13 nM (Fig. 4B, Table 2). Therefore, although VIP and Ro-25-1553 had similar affinities for VIP receptors on rat and guinea pig pancreatic acini, [Lys15,Arg16,Leu27] VIP-(8---27) had a fivefold lower affinity for rat than guinea pig pancreatic acini (Table 2). To confirm further the presence of two distinct binding sites, the 125I-VIP dose-inhibition curves of VIP and Ro-25-1553 were analyzed using the least-squares curve-fitting program LIGAND (24). In guinea pig pancreatic acini, as reported previously (18, 38), computer analysis of the VIP dose-inhibition curve demonstrated that the data were significantly better fit by a two binding site model (p = 0.001). One binding site (site 1) had a high affinity for VIP (Kd of 0.21 ± 0.05 nM), and the second (site 2) had a low affinity for VIP (Kd of 82.4 ± 6.5 nM). Site 1 had a high affinity for Ro-25-1553 (Kd of 0.36 ± 0.35 nM), and each cell possessed 0.136 ± 0.025 pmol of high-affinity receptor/mg protein. Site 2 had low affinity for Ro-25-1553 (Kd of 2.94 ± 0.32 µM), and each cell possessed 2.7 ± 1.5 pmol of low-affinity binding sites/mg protein.


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Fig. 4.   Abilities of VIP, [Lys15,Arg16,Leu27]VIP-(1---7)-GRF-(8---27), and Ro-25-1553 to inhibit binding of 125I-VIP to dispersed acini from guinea pig pancreas (A) or rat pancreas (B). Acini were incubated for 45 min at 37°C with 50 pM 125I-VIP plus the indicated concentrations of the peptides. Results are expressed as the percentage of the saturable binding of the 125I-VIP observed in absence of competing peptide. In each experiment, each value was determined in duplicate, and results given are means ± SE from at least 3 separate experiments.


                              
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Table 2.   Abilities of VIP, [Lys15,Arg16,Leu27]VIP-(1---7)-GRF-(8---27), and Ro-25-1553 to interact with VIP receptors and stimulate amylase secretion from pancreatic acini in guinea pig and rat

To determine whether either or both VIP receptor subtypes mediate enzyme secretion, the ability of each selective agonist to cause amylase release from both species was examined. In guinea pig pancreatic acini, amylase release by VIP showed a broad dose-response curve with a detectable increase at 1 pM, half-maximal stimulation of increase at 0.05 nM, and maximal stimulation at 10 nM (Fig. 5A, Table 2). The VIP1-R-selective ligand [Lys15,Arg16,Leu27]VIP-(1---7)-GRF-(8---27) showed a similar dose-response curve for increasing amylase to VIP but was 34-fold less potent, causing a half-maximal stimulation at 1.7 ± 0.04 nM (Fig. 5A, Table 2). In contrast, Ro-25-1553 had a biphasic dose-response curve with a high-affinity component accounting for 15% of the maximal secretion seen between 0.01 and 1 nM (EC50 of 0.0025 ± 0.0009 nM) and a second low-affinity component accounting for 85% of the maximal secretion seen at concentrations of 1 µM (EC50 of 20.4 ± 0.9 nM) (Fig. 5A, Table 2). Similarly, in rat pancreatic acini, amylase release by VIP showed a broad dose-response curve with a detectable increase at 3 pM, half-maximal stimulation of increase at 0.09 nM, and maximal stimulation at 10 nM (Fig. 5B, Table 2). The VIP1-R-selective ligand [Lys15,Arg16, Leu27]VIP-(1---7)-GRF-(8---27) showed a similar dose response to VIP but was 96.7-fold less potent, causing a half-maximal stimulation at 8.7 ± 0.34 nM (Fig. 5B, Table 2). Similar to guinea pig acini, Ro-25-1553 had a biphasic dose-response curve with a high-affinity component accounting for 10% of the maximal secretion seen between 0.01 and 1 nM (EC50 of 0.0045 ± 0.0003 nM) and a second component accounting for 90% of the maximal secretion seen at concentrations of 1 µM (EC50 of 52.4 ± 4.9 nM) (Fig. 5, Table 2). These results indicated that activation of both subtypes can stimulate enzyme secretion; however, activation of the VIP1-R is principally responsible for enzyme secretion because its activation caused 85% and 90% of the maximal secretion, whereas VIP2-R only caused 10% and 15% of maximal secretion in the guinea pig and the rat, respectively.


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Fig. 5.   Abilities of VIP, [Lys15,Arg16,Leu27]VIP-(1---7)-GRF-(8---27), and Ro-25-1553 to stimulate amylase release from dispersed acini from guinea pig pancreas (A) or rat pancreas (B). Acini were suspended in standard incubation solution with (guinea pig acini) or without (rat acini) 5 mM theophylline for 30 min at 37°C. Results are expressed as the percentage of the stimulated response caused by a maximally effective concentration of VIP (i.e., 10 nM). In each experiment, each value was determined in duplicate, and the results shown are means ± SE from at least 3 separate experiments. Control and maximal stimulated values were 4.1 ± 0.2% and 19.2 ± 0.9% of the total cellular amylase released for guinea pig pancreatic acini and 5.2 ± 0.4% and 15.0 ± 0.54% of total cellular amylase released into the extracellular medium during the incubation for rat pancreatic acini (n = 3).

Previous studies have demonstrated that VIP causes increases in cAMP in rat and guinea pig pancreatic acini, and this increase stimulates enzyme secretion (2, 3, 18, 28, 37, 38). To investigate whether activation of both VIP1-R and VIP2-R can cause activation of adenylate cyclase and increased cAMP, we examined the ability of each selective agonist to alter cAMP levels in guinea pig pancreatic acini. Rat and guinea pig pancreatic acini possess both VIP and secretin receptors, which are both coupled to adenylate cyclase, and can both be activated by high concentrations of VIP (2, 3, 18, 29, 37, 38); therefore, it is important to test low concentrations of VIP agonist, which only activate VIP receptors. VIP at the dose of 1 nM, which only activates VIP receptors, caused a 54.2 ± 5.7-fold increase in cAMP (Table 3). The VIP1-R-selective agonist [Lys15,Arg16,Leu27]VIP-(1---7)-GRF-(8---27) at the dose of 10 nM, which only occupies VIP1-R, increased cAMP release by 56.3 ± 3.9-fold (Table 3), demonstrating that VIP and [Lys15,Arg16,Leu27]VIP-(1---7)-GRF-(8---27) have equal efficacy at low concentrations. The VIP2-R-selective agonist Ro-25-1553 at the dose of 10 nM, which only occupies VIP2-R, caused a 3.7 ± 0.3-fold increase in cAMP, which was 15.2-fold lower than that seen with VIP1-R activation by the VIP1-R-selective ligand [Lys15,Arg16,Leu27]VIP-(1---7)-GRF-(8---27) (Table 3). At high concentrations (i.e., >= 1 µM) in guinea pig acini, VIP-related peptides can stimulate cAMP through both VIP receptors and secretin receptors, which are both coupled to adenylate cyclase (10, 18, 19, 28, 37, 38). VIP, secretin, and the VIP-related peptides, [Lys15,Arg16,Leu27]- VIP(1---7)-GRF-(8---27) and Ro-25-1553, all stimulated >85-fold increase in cAMP at 1 µM concentrations, demonstrating activation of both the VIP and secretin receptor (Table 3).

                              
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Table 3.   Ability of VIP, Ro-25-1553, and [Lys15, Arg16, Leu27]VIP-(1---7)-GRF-(8--- 27) to alter cAMP in guinea pig pancreatic acini


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

High-affinity receptors for VIP have been shown by binding studies or functional assays measuring changes in cellular function in pancreatic acinar cells from a number of species, including mouse, rat, and guinea pig (18). In each of these species, activation of high-affinity VIP receptors either stimulated enzyme secretion itself or potentiated stimulated enzyme secretion by other secretagogues such as CCK or muscarinic cholinergic agents (4, 9, 18). Similar to secretin receptors, which also occur in pancreatic acinar cells of each of these species (2, 6, 18, 19), activation of high-affinity VIP receptors is coupled to activation of adenylate cyclase and increases in cellular cAMP. The exact relationships between receptor occupation, adenylate cyclase activation, and enzyme secretion in different species are complicated by the occurrence of multiple binding sites, lack of antagonists with high selectivity, and varying selectivity for different agonists in different species (2, 6, 18). For example, in rat pancreatic acini, it has been proposed that VIP and secretin interact with four classes of receptors (2, 6), of which VIP interacts with two classes in one study (2) and in another study with one class (6). In guinea pig pancreatic acini, VIP at a high concentration interacts with both the VIP and secretin receptors; moreover, the VIP receptor can exist in both low- and high-affinity states (18, 37, 38). All of these studies were performed before it was discovered that two distinct subtypes of VIP receptors exist: a 459- amino acid VIP1-R in the rat (rVIP1-R) (17) and a 437- amino acid VIP2-R in the rat (rVIP2-R) (23), which have 50% amino acid homology. VIP1-R and VIP2-R have been cloned from rat (17, 23) and human (5, 32) tissues. Both receptor subtypes are coupled to Gsalpha proteins, and their occupancy by agonists stimulates adenylate cyclase activity. VIP has a high affinity for both rat and human VIP1-R and VIP2-R, whereas secretin has a low affinity for both rat and human VIP1-R and VIP2-R (11, 12). Whether either or both of these VIP receptor subtypes exist on pancreatic acini was unclear, until the present study.

In previous studies using Northern blot analysis, VIP1-R mRNA was not detected in the rat pancreas (17) and VIP2-R mRNA was detected only in the rat pancreatic islets (16). In another study, Usdin et al. (34) did not detect either VIP1-R or VIP2-R in the rat pancreas by Northern blot analysis, whereas they detected both VIP1-R and VIP2-R in the rat pancreas by Southern blot analysis after RT-PCR. However, with the use of in situ hybridization, they showed that VIP1-R mRNA is only clearly observed in blood vessels within the pancreas and VIP2-R in islets throughout the pancreas (34). With respect to normal rat pancreas, their data suggest that rVIP1-R is expressed exclusively in the walls of blood vessels, whereas rVIP2-R is only in pancreatic islets (16, 17, 34). In normal human pancreas, VIP2-R mRNA was detected by Northern blot analysis (1) and by RNase blot analysis (36), whereas VIP1-R mRNA was not detected by Northern blot analysis (31). However, by Southern blot hybridization after RT-PCR, both VIP1-R and VIP2-R were reported to be identified in the human pancreas (22, 36). Before the present study, there were no functional studies or ligand binding studies to suggest whether either or both VIP receptor subtypes exist on pancreatic acini in any species.

A number of the results from the Northern and Southern blot analysis performed in the present study support the conclusion, combined with the functional studies of amylase release, that in both rat and guinea pig pancreatic acinar cells both VIP1-R and VIP2-R are present. First, by Northern blot analysis, both VIP1-R and VIP2-R mRNA were found in rat and guinea pig pancreas. Second, in rat pancreas with PCR with Southern blot analysis, both rVIP1-R and rVIP2-R were detected. Third, to further ascertain whether the receptors were on pancreatic acinar cells, a similar analysis was performed on dispersed acini from rat and guinea pig pancreas. In dispersed pancreatic acini from both species, both VIP1-R and VIP2-R were present. These results were not due to cross-hybridization because both probes were shown to be specific for either the VIP1-R or the VIP2-R in both the Northern and Southern blot analysis in a control tissue. Because isolated pancreatic acinar preparations can be contaminated by small numbers of pancreatic islets or centroacinar cells, the Northern and Southern blot results alone do not establish that the VIP receptors are in acinar cells. However, the ability of selective agonists for these two VIP-R receptor subtypes to stimulate enzyme secretion, discussed below, establish that these receptors are on pancreatic acinar cells.

To demonstrate the presence of both receptor subtypes directly by binding studies and to perform studies of the ability of either VIP receptor subtype to alter pancreatic acinar cell function, we needed to identify selective ligands for each VIP receptor subtype. [Lys15,Arg16,Leu27]VIP-(1---7)-GRF-(8---27) is reported to be a selective agonist for VIP1-R (12) and Ro-25-1553 is reported to have selectivity to VIP2-R (13, 35). We confirmed the selectivity of these two ligands by examining their affinities for rat and human VIP1-R and VIP2-R expressed in CHOP cells by transient expression and CHO cells stably expressing rVIP1-R, rVIP2-R, hVIP1-R, or hVIP2-R. [Lys15,Arg16,Leu27]VIP-(1---7)-GRF-(8---27) showed a 53-fold and over a 169-fold selectivity for the rVIP1-R in CHOP cells and CHO cells, respectively, and showed over a 1,765-fold selectivity for the hVIP1-R in CHO cells. Ro-25-1553 showed over a 4,412-fold and a 241-fold selectivity for the rVIP2-R in CHOP cells and CHO cells, respectively, and showed a 280-fold selectivity for the hVIP2-R in CHO cells. These results suggested that these two agonists could be useful tools for distinguishing between VIP1-R and VIP2-R in binding studies and studies of biological activity.

A number of results from the binding studies support the conclusion that both rat and guinea pig pancreatic acini possess both the VIP1-R and the VIP2-R, with the VIP1-R the predominant subtype. First, the dose-inhibition curve of the VIP2-R-selective ligand Ro-25-1553 showed a biphasic pattern of inhibition, with a high-affinity component representing 10% of the total saturable 125I-VIP binding and a second component representing 90% of the total saturable 125I-VIP binding in both species. These results demonstrated that 10% of the radioligand 125I-VIP is bound to receptors with high affinity for the VIP2-R-selective ligand and that 90% of the saturable binding of 125I-VIP is to receptors with low affinity for the VIP2-R-selective ligand. Second, the VIP1-R-selective ligand [Lys15,Arg16,Leu27] VIP-(1---7)-GRF-(8---27) gave a monophasic dose-inhibition curve of 125I-VIP binding, which was parallel to the VIP dose-inhibition curve compatible with the conclusion that 125I-VIP was primarily bound to receptor sites with high affinity for the VIP1-R ligand. Third, we verified that 125I-VIP was both binding to a site with high affinity for the VIP1-R and the VIP2-R by analyzing the 125I-VIP dose-inhibition curve of the VIP2-R-selective agonist Ro-25-1553 using a least-squares, curve-fitting program (24). The analysis in the guinea pig pancreatic acini demonstrated that the dose-inhibition curve of Ro-25-1553 was significantly better fit by a two binding site model than a one binding site model. Furthermore, one site had a high affinity (Kd = 0.36 nM) for Ro-25-1553 and the other site had a low affinity (Kd = 2.94 µM) for Ro-25-1553. The binding sites with low affinity for Ro-25-1553 (i.e., the VIP1-R) were present in 19.9-fold higher numbers than those with high affinity for Ro-25-1553 (i.e., VIP2-R).

It is known that agonist activation of VIP receptors in rat and guinea pig pancreas results in enzyme secretion (2, 10, 18). To determine whether activation of VIP1-R, VIP2-R, or both are coupled to enzyme secretion, we analyzed the ability of the selective VIP1-R and VIP2-R agonist ligands to stimulate enzyme secretion. In the present study, a number of results support the conclusion that activation of both VIP1-R and the VIP2-R stimulates enzyme secretion in both rat and guinea pig pancreatic acini, with the VIP1-R responsible for the majority of the enzyme secretion mediated by VIP receptor activation. First, the VIP2-R-selective ligand Ro-25-1553 showed a biphasic dose-response curve with a high-affinity component accounting for 10-15% of the maximal secretion caused by VIP and a low-affinity component accounting for 85-90% of the maximal secretion caused by VIP in both species. The VIP2-R-selective ligand Ro-25-1553 caused 10-15% maximal stimulation of that caused by VIP over the same concentration rate causing a 10-15% decrease in binding of 125I-VIP, supporting the conclusion that the stimulation was due to VIP2 receptor activation. Furthermore, this stimulation occurred over a concentration range (i.e., <10 nM) at which Ro-25-1553 only occupies VIP2-R. These results demonstrated that 10-15% of the maximal amylase secretion caused by VIP is through the activation of receptors with high affinity for the VIP2-R-selective ligand and that 85-90% of the maximal amylase secretion is through receptors with low affinity for the VIP2-R-selective ligand. Second, the VIP1-R-selective ligand [Lys15,Arg16,Leu27]VIP-(1---7)-GRF-(8---27) gave a monophasic dose-stimulation curve of amylase secretion that was parallel to the VIP dose-response curve, which is compatible with the conclusion that amylase secretion was primarily mediated through receptor sites with high affinity for the VIP1-R ligand. Third, at high concentrations at which the VIP1-R-selective ligand [Lys15,Arg16,Leu27]VIP-(1---7)-GRF-(8---27) and the VIP2-R-selective ligand Ro-25-1553 interact with both the VIP1-R and the VIP2-R, the efficacy of each selective analog was equal to that seen with VIP, supporting the conclusion that VIP was causing enzyme secretion by activation of both the VIP1-R and the VIP2-R.

In other cell systems, activation of either VIP1-R (17, 31) or the VIP2-R (1, 16, 23, 32) results in stimulation of adenylate cyclase and increases in cellular cAMP. In previous studies, activation of the VIP receptors in both guinea pig (10, 18, 28, 37, 38) and rat pancreatic acini (2, 6, 18) has been shown to increase cAMP, which results in enzyme secretion. A number of our results support the conclusion that activation of both VIP1-R and VIP2-R in the guinea pig pancreatic acini is coupled to increases in cAMP. First, in older studies (10, 18, 38) it was demonstrated that in guinea pig pancreas the VIP-stimulated cAMP dose-response curve is biphasic. One component is seen with VIP at low concentrations, due to VIP receptor occupation (equal10 nM), and a second component occurs at higher concentrations, corresponding to concentrations that result in occupation of secretin receptor. In the present study, therefore, it was important to investigate increases in cAMP at low concentrations (equal10 nM) of VIP agonists that only activate VIP receptors to assess only the result of VIP receptor activation or adenylate cyclase activity. At low concentrations (10 nM) of the VIP2-R-selective agonist Ro-25-1553, which would only occupy VIP2-R, a fourfold increase in cAMP was seen. These results demonstrate that occupation of the VIP2-R is coupled to adenylate cyclase and causes a fourfold increase in cAMP. Second, with a low concentration (10 nM) of the VIP1-R-selective agonist [Lys15,Arg16,Leu27]VIP-(1---7)-GRF-(8---27), which would only occupy VIP1-R, a 56-fold increase in cAMP was seen. This result demonstrates that occupation of the VIP1-R on guinea pig pancreatic acini is also coupled to adenylate cyclase and causes a 56-fold increase in cAMP. Third, VIP at 1 nM, a concentration known to activate only VIP receptors in guinea pig pancreatic acini (9, 10, 37), caused a similar increase in cAMP to that seen with the 10 nM VIP1-R agonist. These results show that <10% of the cAMP caused by 1 nM VIP is due to VIP2-R activation and the majority (>90%) is due to VIP1-R activation. Fourth, at high concentrations (i.e., >= 1 µM), VIP, secretin, and the VIP receptor subtype-selective agonists [Lys15,Arg16,Leu27] VIP-(1---7)-GRF-(8---27) and Ro-25-1553 all stimulated increases in cAMP to the same degree (i.e., >85-fold). These high-agonist concentrations were causing increases in cAMP through stimulation of both the VIP and secretin receptors (3, 9, 18, 19, 37, 38).

In conclusion, we have identified that both rat and guinea pig pancreatic acini possess both VIP1 receptors and VIP2 receptors, with the VIP1 receptor the predominant subtype. Activation of both the VIP1-R and the VIP2-R stimulates enzymes in both rat and guinea pig pancreatic acini, with the VIP1-R responsible for the majority (i.e., >85%) of the enzyme secretion mediated by VIP receptor activation. Finally, both the VIP1-R and the VIP2-R are coupled to increases in cAMP, with <10% of the cAMP caused by a low concentration of VIP (1 nM) due to VIP2-R activation and the majority (>90%) due to VIP1-R activation. These results show for the first time that both VIP receptor subtypes exist on pancreatic acini in rat and guinea pig and both are responsible for mediating the action of VIP. These results can effect future studies of activation of VIP receptors on pancreatic function in a number of ways. In future studies of the effects of any VIP/pituitary adenylate cyclase-activating polypeptide analogs on pancreatic function, it will be important to consider their abilities to interact with both the VIP1-R and the VIP2-R and activate these receptors. Differing results could be obtained from VIP itself, which is a high-affinity agonist for both VIP-R subtypes. Furthermore, in future studies of the action of VIP on pancreatic acini, it will be important to consider the participation of the different VIP receptor subtypes, a factor not included in older studies. The availability of selective agonists for each receptor subtype should make this analysis easier, and these should be valuable tools in establishing a better understanding of the physiological roles of both VIP receptor subtypes in pancreatic acini.


    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: R. T. Jensen, NIH/NIDDK/DDB, Bldg. 10, Rm. 9C-103, 10 Center Dr., MSC 1804, Bethesda, MD 20892-1804 (E-mail: robertj{at}bdg10.niddk.nih.gov).

Received 4 June 1999; accepted in final form 5 October 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastroint Liver Physiol 278(1):G64-G74