Locations and molecular forms of PACAP and sites and characteristics of PACAP receptors in canine ileum

Y. K. Mao1, Y. F. Wang1, C. Moogk2, J. E. T. Fox-Threlkeld1, Q. Xiao2, T. J. McDonald2, and E. E. Daniel1

1 Department of Biomedical Science, McMaster University, Hamilton L8N 3Z5; and 2 Department of Medicine, University of West Ontario, London, L6A 5A5 Ontario, Canada

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

In canine ileum we investigated the distribution of pituitary adenylate cyclase-activating peptide (PACAP), using immunofluorescence and radioimmunoassay and the binding of 125I-PACAP-27 to membranes. Nerve profiles immunoreactive to PACAP-27, and often to vasoactive intestinal polypeptide (VIP) as well, were found in all plexi, but PACAP was present in ~100-fold lesser amounts than VIP. High-performance liquid chromatography analysis of deep muscular plexus (DMP) synaptosomes suggested the presence of PACAP-38, PACAP-27, and a third unidentified molecular form. High- and low-affinity 125I-PACAP-27 binding sites were found in DMP synaptosomes and circular smooth muscle (CM) plasma membranes. In competition studies with DMP membranes, high (H)- and low (L)-affinity dissociation constants (Kd) and maximal binding capacities (Bmax) were as follows: Kd H = 66.9 pM, Bmax H = 101 fmol/mg; Kd L = 2.18 nM, Bmax L = 580 fmol/mg protein. The binding of 125I-PACAP-27 was fast. Dissociation was slow and incomplete in the presence of unlabeled PACAP-27 but accelerated by pretreatment with guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S). GTPgamma S or cholera toxin treatment eliminated high-affinity binding in both membranes. VIP had ~100-fold lower affinity than PACAP-27 in both membranes. Cross-linking studies identified an ~70-kDa PACAP receptor in each membrane. Thus PACAP coexists with VIP in ileal enteric nerves and acts on PACAP-preferring, possibly Gs-coupled, receptors in DMP synaptosomes and CM membranes.

pituitary adenylate cyclase-activating peptide; PACAP-27; PACAP-38; vasoactive intestinal polypeptide; synaptosome; smooth muscle; intestine

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

PITUITARY ADENYLATE CYCLASE-ACTIVATING PEPTIDE (PACAP) is a member of a family of structurally related regulatory peptides that includes secretin, glucagon, gastric inhibitory peptide, growth hormone releasing factor, and vasoactive intestinal polypeptide (VIP) (4). This neuropeptide exists in two amidated forms: PACAP-(1---38) and a shorter form, PACAP-(1---27), which possesses full biological activity (17, 18). Both PACAP and VIP have widespread distributions in the central, peripheral, and enteric nervous systems and as neurotransmitters may be involved in the regulation of gastrointestinal neuromuscular function (10, 20, 21).

Subtypes of high-affinity PACAP receptors have been identified on the basis of their relative affinities for PACAP and VIP (4, 20). One PACAP receptor has high affinity for PACAP and low affinity for VIP and may be coupled to adenylate cyclase and phospholipase C. Another PACAP receptor has equally high affinities for both PACAP and VIP. Other types may also exist. Earlier we studied the distribution of VIP receptors on canine ileum and found little evidence for their existence in circular smooth muscle (CM) membranes but specific binding associated with the deep muscular plexus (DMP) (12). PACAP sometimes relaxes and sometimes contracts CM in vitro, e.g., in the guinea pig and canine gastrointestinal tract (5-8).

The objectives of this study were 1) to compare the distributions of PACAP and VIP in enteric neurons of the canine ileum and 2) to investigate the location and nature of receptors for PACAP in the canine ileum.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Immunohistochemistry

After dissection, whole mounts of ileal tissues were incubated overnight at 4°C in phosphate-buffered saline (PBS) with rabbit anti-human PACAP-27 antibody (Peninsula, Belmont, CA) at a dilution of 1:200. The tissues were washed free of antiserum and treated with fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit immunoglobulin G (IgG) (Bio/Can, Mississauga, ON, Canada) diluted 1:50. Preparations were washed with PBS and then mounted in PBS and glycerol containing 0.1% p-phenylenediamine (PPD; pH 10) and viewed on a Leitz microscope equipped with a fluorescence epi-illuminator. Kodak T-MAX 400 film was used for black-and-white photography.

Preabsorption of the antiserum with synthetic porcine PACAP-27 (10-6 M) completely abolished staining in all cases. Nonspecific staining was minimal.

Double-label immunohistochemistry. To determine whether any of the neurons immunoreactive for PACAP also contained VIP, whole mount preparations were incubated in a complex solution containing antisera to VIP raised in a guinea pig (Peninsula) and the rabbit anti-PACAP antiserum at 4°C overnight. The tissue was washed with PBS and then incubated with lissamine rhodamine sulfonyl chloride (LRSC)-labeled donkey anti-guinea pig IgG (Bio/Can) (1:50) plus goat anti-rabbit FITC IgG (Bio/Can) (1:50) for 60-120 min at room temperature. Preparations were washed with 0.1 M phosphate buffer (PB) and then mounted in PBS and glycerol containing 0.1% PPD (pH 10). Sites of immunoreactivity of various peptides were visualized with the use of a Leitz fluorescence microscope with an I2 filter for LRSC and an N2 filter for FITC. Kodak T-MAX 400 film was used for black-and-white photography.

Synaptosomal Contents of VIP and PACAP and Molecular Forms of PACAP

Materials. PACAP-27, PACAP-38, and VIP were purchased from either Peninsula or Bachem (Torrance, CA). 3-(N-morpholino)propanesulfonic acid (MOPS), tris(hydroxymethyl)aminomethane (Tris) base, bovine serum albumin (BSA), guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S), phenylmethylsulfonyl fluoride (PMSF), and aprotinin were purchased from Sigma (St. Louis, MO). Disuccinimidyl suberate (DSS) was from Pierce Chemical (Rockford, IL).

Purified canine nerve varicosity preparations. Synaptosomes from the canine myenteric plexus (MP), the DMP, and the submucosal plexus (SMP) were prepared by tissue dissection of the canine small intestine and differential centrifugation techniques as previously described in detail (1, 2, 3, 9, 14, 15). Five preparations of canine ileal purified nerve varicosities from each of the MP, DMP, and SMP were obtained for this study. As previously described (14, 15), the purified varicosity suspensions were divided into aliquots and immediately subjected to heat inactivation (95°C for 10 min in a heating block) to denature proteolytic enzymes. After cooling, sufficient glacial acetic acid was added to each preparation to make the solution 0.5 M acetic acid, and the preparations were mixed thoroughly and centrifuged at 12,500 g for 3 min. Supernatants were stored at -70°C until performance of radioimmunoassay (RIA) or reverse-phase high-performance liquid chromatography (HPLC). The protein content of the purified varicosity suspensions was determined by the Folin phenol method of Lowry et al. (11).

RIA procedures. 125I-labeled PACAP-27 and its purified form were prepared using carrier free Na125I (New England Nuclear, Boston, MA), using the chloramine-T technique as previously described (14). After a preliminary purification by adsorption to an activated SepPak C18 cartridge (Waters Associates, Toronto, ON, Canada), the reaction mixture was applied to a C18 µBondapak column (1 × 300 mm) and subjected to HPLC using a Waters apparatus (Waters Associates). The solvent systems used were 0.12% (vol/vol) trifluoroacetic acid (TFA) (Sequenal grade; Pierce Chemical) in water (solvent A) and 0.1% TFA in acetonitrile (Fisher Scientific, Toronto, ON, Canada) (solvent B). The partially purified iodination reaction mixture was further purified with the use of an isocratic elution at 29% solvent B for 60 min. A peak of radioactivity eluting at retention time (RT) 20 min was used for binding experiments (assumed to be 2,000 Ci/mmol) and RIA.

RIA for PACAP-like immunoreactivity (PACAP-LI) was carried out with the antiserum used for the immunohistochemical procedures at a final dilution of 1:300,000. The total assay volume was 1 ml, and the buffer system used was 0.05 M PB, pH 7.4, containing 0.05 M sodium chloride, 0.1% (wt/vol) sodium azide, and 0.1% (wt/vol) BSA. The antiserum recognizes both PACAP-27 and PACAP-38 with equimolar potency; PACAP-27 was routinely used as the assay standard. The addition of 3 fmol PACAP-27 per assay tube resulted in a 10% decrease from initial binding, and a 50% decrease was produced by the addition of 20 fmol PACAP-27 per assay tube. Cross-reaction with porcine VIP (Peninsula) was 0.02%.

The RIA procedures for VIP-like immunoreactivity (VIP-LI) utilized antiserum 7913 (provided by Dr. J. Walsh, Center for Ulcer Research and Education, Los Angeles, CA). The details of the antibody specificity and sensitivities and procedures used in this laboratory have been described previously in detail (13). Radiolabeled VIP was produced as described above for production of 125I-PACAP-27, with the exception that reverse-phase HPLC purification was performed using isocratic elution at 30% solvent B for 60 min.

Reverse-phase HPLC procedures. Reverse-phase HPLC was performed using a Beckman HPLC system (System Gold) and a reverse-phase µBondapak C18 column (3.9 × 300 mm). Solvents A and B, as described in RIA procedures, were used. The solvent flow rate used was 1 ml/min. The reverse-phase chromatograms were developed using sequential linear elution gradients of 1) 26-36% solvent B over 60 min, 2) 36-65% solvent B over 30 min, 3) 65-90% solvent B over 10 min, 4) 90-100% solvent B over 10 min, and 5) a 10-min elution with 100% solvent B. The C18 columns were calibrated with synthetic PACAP-38 and PACAP-27, and the elution patterns were monitored by measuring ultraviolet absorbance patterns at 215 nm. After all calibration runs and before chromatography of experimental samples was undertaken, the system was thoroughly washed and multiple blank runs were performed to ensure no carry-over of injected peptides. Three separate aliquots of purified DMP preparations were subjected to reverse-phase HPLC under the conditions described above. One-minute eluate fractions were collected into tubes containing 100 µg BSA. All eluates were lyophilized to remove solvents, and each fraction was reconstituted with RIA buffers at an appropriate concentration.

Binding Studies

Tissue preparation. Tissue preparation and fractionation of ileal CM preparations were performed as described previously (1, 2). Briefly, following a procedure approved by the Animal Care Committee of McMaster University and the Canadian Council for Animal Care, adult mongrel dogs were euthanized by an injection of pentobarbital sodium (100 mg/kg). An incision was made in the abdomen, and after segments were removed for immunohistochemistry and suspended in PBS, the entire distal intestine was removed and suspended in ice-cold sucrose-Mg2+-MOPS buffer [25 mM MOPS, 10 mM MgCl2, and 8% (wt/vol) sucrose, pH 7.4]. Pieces ~1-in. in length were cut, cleared of the mesenteric arcade and fat, opened along the mesenteric attachment site, and pinned (mucosal surface down) on a dissecting plate on ice. The longitudinal muscle layer with attached MP was carefully peeled off, leaving a thin layer of CM (verified histologically). The CM layer was dissected off the mucosa and submucosa and placed in cold buffer solution. The tissues were blotted dry on filter paper.

For membrane preparation, 10 g of tissue was resuspended in 10 vol of buffer and homogenized with a Polytron PT20 homogenizer at 15,000 revolutions per minute (rpm) for 21 s (3 × 7 s). The homogenate was centrifuged at 1,000 g for 10 min, and the postnuclear supernatant (PNS) was collected and recentrifuged at 10,000 g for 10 min. The pellet from this centrifugation (Mit I) was saved, and the supernatant was subjected to high-speed centrifugation at 170,000 g for 60 min. The pellet thus obtained (Mic I) was resuspended in the same buffer and centrifuged at 10,000 g for 10 min. The supernatant was designated Mic II and the pellet Mit II. Protein content was measured by the Folin phenol method of Lowry et al. (11).

The Mit I (enriched in mitochondria and synaptosomes) and Mic II fractions (enriched in CM membranes) were further purified by sucrose density gradient centrifugation. Three milliliters of fraction Mit 1 were loaded on a gradient consisting of 2 ml each of 14%, 25%, 35%, 40%, and 48% sucrose and centrifuged in a Beckman SW40 rotor at 30,000 rpm for 100 min. The protein band at the interface of 8% sucrose and 14% sucrose (8%/14%) was designated fraction 1 (F1), at 14%/25% F2, at 25%/35% F3, at 35%/40% F4, and at 40%/48% F5. The pellet at the bottom of the tube was designated F6. Three milliliters of Mic II fraction were loaded on a gradient consisting of 2.5 ml each of 14%, 25%, 33%, and 48% sucrose and centrifuged at 30,000 rpm for 100 min. Four fractions were obtained: M1 at the interface of 8% and 14% sucrose (8%/14%), M2 at 14%/25%, M3 at 25%/33%, and M4 at 33%/48%.

PACAP-27 receptor binding assay. PACAP binding was performed in a total volume of 0.2 ml of 25 mM Tris · HCl buffer, pH 7.4, containing 2 mM MgCl2, 1 mM PMSF, 0.5 mg/l leupeptin, 0.7 mg/l pepstatin, 10 µl (200 KIU) aprotinin, 1% BSA (wt/vol), 150-200 pmol 125I-PACAP-27, and 10-20 µg membrane protein ( fraction Mit II or Mic II). Nonspecific binding was determined in parallel by adding excess synthetic PACAP-27 (1 µM) to the assay tube. The reaction was carried out at 25°C for 45 min. The binding reaction was terminated by quenching with 3 ml of ice-cold assay buffer. Free and bound radioligands were separated by immediate filtration through glass microfiber filters (GF/F; Whatman International, Maidstone, UK) on a 12-port Millipore filtration manifold (Millipore, Bedford, MA). The tubes and filter were washed three times with 3 ml cold buffer. Radioactivity retained on the filter was determined in a gamma counter. Nonspecific binding was 25-30% of the total binding. Nonspecific binding in these studies was determined as that measured in the presence of unlabeled excess PACAP-27. All determinations were performed in triplicate.

Saturation and competition with 125I-PACAP-27. Direct binding measurements of 125I-PACAP-27 equilibrium dissociation constant (Kd) and receptor density [maximal binding capacity (Bmax)] values were determined in triplicate for total and nonspecific binding at a series of concentrations from 0.001 to 1.5 nM 125I-PACAP-27. Specific binding was defined as the difference between total and nonspecific binding. On the basis of preliminary studies, incubation was continued for 45 min at 25°C to ensure that equilibrium was reached at all radioligand concentrations.

The ability of various unlabeled peptides to compete against 150-200 pM 125I-PACAP-27 for binding was measured by adding a series of graded concentrations of each peptide. Incubation was continued for 45 min at 25°C before free radiolabeled ligand was separated from that bound to receptor by filtration, as described above. To investigate the role of G proteins in PACAP binding, GTPgamma S (10-4 M) was added directly to the membrane preparation. Pertussis toxin (PTX) was activated by incubation with 25 mM dithiothreitol (DTT) for 20 min at 30°C. Activated PTX (4 µg/ml) was incubated with membranes in the presence of 1 mM ATP and 0.2 mM NAD for 30 min at 30°C. Cholera toxin (CTX) was activated with 50 mM DTT at 30°C for 2 h. Activated CTX (200 µg/ml) was incubated with membranes at 30°C for 30 min in the presence of 1 mM ATP, 0.2 mM NAD, and 20 mM arginine.

Kinetic binding assays. Association rate constants were measured under pseudo-first-order conditions at 25°C by incubating 150-200 pM 125I-PACAP-27 for different times in triplicate tubes containing the membrane preparation. For dissociation rate measurement, triplicate samples were prepared containing 150-200 pM 125I-PACAP-27 and the membrane preparation and incubated for 45 min at 25°C to achieve equilibrium. PACAP-27 in assay buffer was then added to the tubes at a final concentration of 1 µM at different times to prevent radioligand reassociation to receptors. All tubes were filtered simultaneously at the end of the experiment as described.

Data analysis. The binding data were analyzed by the computer program EBDA/LIGAND (16, 19) and CDATA 87 (EMF Software, Knoxville, TN) with an IBM PC/XT computer. Computer fittings of saturation and competition curves were analyzed using statistical comparisons (F test) of the residual squares of error terms after the curve fitting.

Cross-linking of 125I-PACAP-27 to its receptor. Cross-linking was performed using canine small intestinal synaptosomal membranes and CM plasma membranes (0.5 mg protein) incubated with 250 pM 125I-PACAP-27 in 1 mM MgSO4, 50 mM sodium phosphate, 1 mM PMSF, 0.5 mg/l leupeptin, 0.7 mg/l pepstatin, and 200 KIU aprotinin, pH 7.4 (phosphate-Mg2+ buffer), in a final volume of 500 µl. After 30 min incubation at 25°C, the membranes were collected by centrifugation at 12,300 g for 10 min. The pellet was washed once and resuspended in the phosphate-Mg2+ buffer. The membranes were then incubated with 5 mM DSS for 30 min at 25°C in a final volume of 250 µl. DSS stock solution (250 mM) was dissolved in dimethyl sulfoxide. The cross-linking reactions were quenched by the addition of 250 µl of a glycine solution to yield a final concentration of 20 mM glycine, followed by incubation for 10 min. After washing and centrifugation, the membranes were resuspended in 200 µl of 10% glycerol, 1% 2-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), 0.001% bromophenol blue, and 62.5 mM Tris · HCl, pH 6.8 (SDS sample buffer), and incubated for 20 min at 21°C before either immediate electrophoresis or storage at -70°C.

SDS-polyacrylamide gel electrophoresis analysis. PACAP receptors in SDS sample buffer were analyzed in 0.75-mm- thick 12% SDS-polyacrylamide gels. The gels were subjected to autoradiography (for 14 days) using Kodak X-ray film (XAR-5). Methylated molecular standards (low-molecular- weight standards from Bio-Rad, Mississauga, ON, Canada) were used to calibrate the relative molecular weight (Mr) of sample proteins.

    RESULTS
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Introduction
Methods
Results
Discussion
References

Immunohistochemistry and RIA Contents of PACAP and VIP in Enteric Plexuses

Immunohistochemistry on whole mount ileal preparations demonstrated positive staining for PACAP-LI in neuronal structures of the MP, SMP, and DMP (Fig. 1, a, c, and d). Preabsorption of the PACAP antiserum with 10-6 M PACAP-27 completely abolished all immunoreactivity (Fig. 1b), but similar preincubation with 10-6 M VIP did not affect staining for PACAP. VIP and PACAP-27 were colocalized in neurons of the MP (Fig. 1, e and f) and in neurites of the DMP (Fig. 1, g and h).


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Fig. 1.   Fluorescence micrographs showing immunohistochemical staining for pituitary adenylate cyclase-activating peptide (PACAP) in myenteric plexus (MP) (a), submucosal plexus (SMP) (c), and deep muscular plexus (DMP) (d). b: all positive PACAP staining in MP was abolished after preabsorbtion of antiserum with PACAP-27. e and f: paired micrographs of whole mount of MP from canine ileum showing colocalization of immunoreactivity for vasoactive intestinal polypeptide (VIP) (e) and PACAP (f). g and h: paired micrographs of whole mount preparations of SMP region of ileum showing immunoreactivity for VIP (g) and PACAP (h). Large arrows point to labeled nerve cell bodies, small arrows to nerve fibers. Bar in b applies to all panels and represents 50 µm.

Table 1 shows the contents of VIP and PACAP measured by RIA in synaptosomes prepared from the MP, DMP, and SMP. The PACAP-LI content of the SMP was less than that in the MP and DMP and considerably less (70-120 times) than the content of VIP-LI in all three plexuses. The ratio of VIP to PACAP in the MP and DMP differed significantly from that in the SMP.

                              
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Table 1.   VIP and PACAP RIA content in synaptosomal preparations

Figure 2 depicts two reverse-phase HPLC profiles of PACAP-LI seen in the DMP synaptosomal preparations. Peaks of immunoreactivity appeared at RT of 19-20 min and 28- to 29-min, just before the RT of human PACAP-38 and PACAP-27, respectively (Fig. 2). The relative proportions of these peaks in different preparations varied, as shown from considerably greater amounts of the 28- to 29-min RT peak to equal amounts of immunoreactivity at RT of 19-20 min and 28-29 min. In all cases, a major peak of immunoreactivity occurred at the greater RT of 32-33 min.


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Fig. 2.   Typical reverse-phase high-performance liquid chromatography profile of PACAP-like immunoreactivity (PACAP-LI) present in DMP synaptosomal preparations. Solid bars show immunoreactivity; arrows indicate elution positions of human PACAP-38 and human PACAP-27; dashed line shows elution gradient of acetonitrile.

Characterization of Membranes and Distribution of PACAP Binding Intestinal CM

Various fractions were obtained by differential centrifugation (see METHODS section) of the CM homogenate (Fig. 3). The Mit I fraction, enriched in mitochondria and synaptosomes, contained higher specific binding of [3H]saxitoxin (129 fmol/mg) compared with that in the initial PNS fraction (13 fmol/mg), a 10-fold enrichment. The F4 fraction from sucrose gradient centrifugation of the Mit I fraction contained the highest [3H]saxitoxin binding (390 fmol/mg protein), an enrichment of ~30-fold over that in the PNS. Both the Mit I and F4 fractions contained somewhat lower activities of 5'-nucleotidase, a CM membrane marker, compared with the Mic II fraction and the M2 fraction obtained from sucrose gradient centrifugation of Mic II. Mic II and M2 fractions contained the highest 5'-nucleotidase activities (Fig. 3).


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Fig. 3.   Distribution of 125I-PACAP-27 binding (fmol/mg protein), [3H]saxitoxin binding (fmol/mg protein), and specific 5'-nucleotidase activity (µmol Pi · mg-1 · h-1) in membrane fractions derived from canine intestinal circular muscle homogenates. SXT, saxitoxin; 5'NUTase, 5'-nucleotidase. See Tissue preparation for detailed description of fractions indicated on x-axes of A-C.

The distribution of PACAP binding was examined in the fractions derived from the CM homogenate (Fig. 3), using 125I-PACAP-27 concentrations of 100-200 pM. All fractions from differential centrifugation except the PNS fraction contained high levels of PACAP binding, suggesting that PACAP receptors were present in both nerve and muscle membranes. This was confirmed after sucrose gradient subfractionation by the high levels of binding found in the F4 subfraction of Mit I, which contained a high level of saxitoxin binding and a low level of 5'-nucleotidase activity (Fig. 3B). Among subfractions from sucrose gradient subfractionation of Mic II, the levels of PACAP binding and 5'-nucleotidase activity were highest and saxitoxin binding was lowest in M2 (Fig. 3C). Moreover, in M2, M3, and M4 there was a good correlation between PACAP binding and 5'-nucleotidase activity, whereas saxitoxin binding was uniformly low.

Characterization of PACAP Receptor Binding

Saturation binding studies. The amount of 125I-PACAP-27 bound in the Mit I (synaptosomal) fraction increased with increasing concentrations of the radioactive ligand (Fig. 4). Binding was saturable, and Scatchard plots revealed high (H)- and low (L)-affinity binding sites (Kd H = 66.9 ± 4.7 pM, Bmax H = 101 ± 35 fmol/mg; Kd L = 2.18 ± 1.83 nM, Bmax L = 580 ± 115 fmol/mg in Mit I; n = 3) over the range of 125I-PACAP-27 concentrations used (Fig. 4).


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Fig. 4.   125I-PACAP-27 (0.01-1.5 nM) specific binding to Mit I membranes from DMP as a function of increasing concentration of radioligand. Inset: computer-generated graph of Scatchard data analysis. High (H)- and low (L)-affinity dissociation constant (Kd) and maximal binding capacities (Bmax) are as follows: Kd H = 66.9 ± 4.70 pM, Bmax H = 101 ± 35 fmol/mg; Kd L = 2.18 ± 1.83 nM, Bmax L = 580 ± 115 fmol/mg. Data are representative of 3 such experiments done in triplicate. B/F: bound/free.

Time course of 125I-PACAP-27 binding and effect of GTPgamma S. The specific binding of 125I-PACAP-27 to the DMP membranes (Mit I) increased with time and reached equilibrium by 40 min. The time for half maximal association was 10 min, with ~90% of the total binding achieved by 30 min incubation. The specific binding at equilibrium did not change for up to 90 min of incubation (Fig. 5A). Pretreatment of the membranewith GTPgamma S did not change the association of 125I-PACAP-27 with the receptors (data not shown).


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Fig. 5.   Kinetics of association (A) and dissociation (B) of 125I-PACAP-27 binding to Mit I fraction from DMP, with specific bound ligand plotted against time. B, amount bound at a particular time; Beq, binding at equilibrium. Reaction was started by addition of membrane (10-20 µg) to incubation tubes containing 150-200 pM radioligand. Dissociation was initiated after 45 min by addition of excess (1 µM) unlabeled PACAP. Data are representative of 3 such experiments done in triplicate. For unpretreated membrane, K+11 = 0.08 ± 0.015 min-1 · nM-1, K-11 = 0.054 ± 0.049 min-1, Kd L = 0.67 ± 0.040 nM, K+12 = 0.13 ± 0.18 min-1 · nM-1, K-12 = 0.000055 ± 0.00012 min-1, Kd H = 4.1 ± 2.1 pM. For pretreated membrane with 0.1 mM guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S), K+1 = 0.069 ± 0.004 min-1 · nM-1, K-1 = 0.016 ± 0.004 min-1, Kd = 0.23 ± 0.15 nM; n = 3.

Binding of 125I-PACAP-27 to DMP membrane receptors was reversible (Fig. 5B). Excess (1 µM) unlabeled PACAP-27 was used to study the dissociation of bound PACAP from the receptor, which was incomplete after 90 min and appeared to be biphasic, i.e., the dissociation was fast during the first few minutes but later became very slow.

The calculated rate constants of dissociation were K-11 = 0.054 ± 0.049 min-1 and K-12 = 0.00006 ± 0.000012 min-1. These values lead to rate constants of association of K+11 = 0.08 ± 0.015 min-1 · nM-1 and K+12 = 0.13 ± 0.18 min-1 · nM-1. The calculated Kd values were Kd L = 0.67 ± 0.40 nM and Kd H = 4.1 ± 2.1 pM. In the presence of the GTP analog GTPgamma S the dissociation was more rapid and was eventually completed within 90 min (Fig. 5B): K-1 = 0.016 ± 0.004 min-1, K+1 = 0.069 ± 0.016 min-1 · nM-1, calculated Kd = 0.23 ± 0.15 nM; n = 3.

Competition Experiments

PACAP-27, PACAP-38, and VIP competed for 125I-PACAP-27 binding sites on synaptosomes (Mit I) (Table 2) and CM plasma membranes (Mic II) (Table 3) with differing potencies. PACAP-38 and PACAP-27 were the most effective ligands in the competition experiments; the potency order of competition was PACAP-38 = PACAP-27 >>  VIP. The inhibition constant (Ki) values of these peptides are shown in Tables 2 and 3. As is apparent from Tables 2 and 3, the slopes of displacement for PACAP-27 and PACAP-38 in the Mit I and Mic II fractions were significantly less than unity, suggesting the presence of multiple binding sites. Computer analysis of the nonlinear best fit by the CDATA 87 program consistently gave a significantly better fit to a two-site model with a small proportion of high-affinity binding sites compared with a one-site model.

                              
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Table 2.   Analysis of competition data in DMP synaptosomes (Mit I)

                              
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Table 3.   Analysis of competition data in CM plasma membrane fractions (Mic II)

Effects of GTPgamma S and CTX Treatment on VIP Binding

The binding of 125I-PACAP-27 to canine DMP synaptosomes (Mit I) and CM membranes (Mic II) pretreated with 0.1 mM GTPgamma S, 200 ng/ml CTX, or 4 ng/ml PTX are shown in Tables 2 and 3. Pretreatment of the synaptosomes or CM membrane with GTPgamma S or CTX eliminated the high-affinity binding sites. In contrast, pretreatment with PTX did not. The Ki value for the remaining low-affinity binding site was unchanged.

PACAP-27, PACAP-38, and VIP competed for 125I-VIP binding to synaptosomal (Table 4) VIP receptors with differing potencies. VIP was the most effective ligand in these competition studies; the potency order was VIP > PACAP-38 > PACAP-27. The Ki values of these peptides are shown in Table 4. As is apparent from Table 4, the slopes of displacement for VIP and PACAP-38 in the Mit I fraction were significantly less than unity, consistent with the presence of multiple binding sites. Note that PACAP-38, but not PACAP-27, had a high-affinity interaction with 125I-VIP binding sites. Computer analysis of the nonlinear best fit by the CDATA 87 program consistently gave a significantly better fit to a two-site model with a small proportion of high-affinity binding sites compared with a one-site model.

                              
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Table 4.   Competition data of 125I-VIP binding in DMP synaptosomes (Mit 1)

Covalent Cross-Linking of 125I-PACAP-27 to Membranes

When DSS was used to cross-link the radioligand to the receptor, followed by electrophoretic analysis in 12.5% polyacrylamide gels, autoradiography revealed a single radioactive band (Fig. 6) of Mr = 70,000 (n = 3) both in the Mit I and Mic II fractions. Inclusion of nonradioactive PACAP-27 reduced the extent of 125I-PACAP-27 incorporation into this band, whereas inclusion of VIP did not.


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Fig. 6.   Molecular components of 125I-PACAP-27 binding sites in canine intestinal DMP (lanes 1-3) and circular smooth muscle plasma membranes (lanes 4-6). Binding was performed in presence of 250 pM 125I-PACAP-27, either alone (-) or combined with 1 µM PACAP-27 or VIP (+). Two other experiments gave similar results.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Nerve cell bodies and nerve fibers immunoreactive for PACAP-LI were found in the MP, DMP, and SMP. This immunoreactivity was specific: antibody saturation with VIP did not affect PACAP immunostaining. PACAP-LI was found to coexist with VIP-LI in most neurons, a finding in agreement with previous work reported in airways of several species and the esophagus of humans, cats, guinea pigs, and sheep (22, 23).

RIA demonstrated the presence of PACAP-LI in the three synaptosomal preparations, with the content of PACAP-LI greater in the MP and DMP than in the SMP. In all synaptosomal preparations, the content of PACAP-LI appeared to be considerably less than that of VIP-LI. However, the exact extent of the recognition of canine PACAP molecules by the antiserum raised against human PACAP-27 cannot be determined, as the primary structures of canine PACAP-27 and PACAP-38 are unknown. Reverse-phase HPLC analysis of the PACAP contents in the DMP preparations demonstrated heterogeneity of molecular forms. Major peaks of immunoreactivity appeared at RT 19-20 min, 28-29 min, and 32-33 min. The first two peaks correspond closely to the human PACAP-38 and PACAP-27 elution positions at 21 and 30 min, respectively. This suggests that canine PACAP molecules have different primary structures from their human counterparts. The third peak of immunoreactivity, occurring at 32-33 min, remains unidentified but may represent either a truncated form of PACAP or a precursor PACAP molecule. Hence, both PACAP-27 and PACAP-38 molecular forms apparently exist in DMP synaptosomal preparations together with an as yet unidentified molecular form.

The membrane fractions used in this study have been well characterized by electron microscopic examination and by nerve membrane markers such as binding with saxitoxin and omega -conotoxin GVIA (1, 2, 3) and by smooth muscle membrane markers such as 5'-nucleotidase. 125I-PACAP-27 binding sites were found in DMP synaptosomes and CM plasma membranes. Hence, PACAP receptors appear to be located on membranes of both the DMP synaptosomes and CM plasma membranes. The presence of PACAP receptors on both nerve and muscle is consistent with functional studies carried out in vivo and ex vivo in the canine intestine, which suggested that PACAP-27 released acetylcholine as well as nitric oxide and a noncholinergic excitant in canine intestine to affect CM contractions (5, 6). After tetrodotoxin blockade of nerve function, PACAP-27 was directly inhibitory to CM.

Kinetic studies demonstrated that with addition of excess unlabeled PACAP-27, the bound 125I-PACAP-27 was incompletely dissociated. Pretreatment of membrane with 0.1 mM GTPgamma S or CTX, but not PTX, enhanced the rate of dissociation or eliminated high-affinity binding. This suggests that the PACAP receptor on both nerve and muscle membranes may be coupled to a G protein, possibly Gs. Presumably PACAP-27 activates adenylate cyclase in nerve endings and CM. If so, receptor activation would result in enhanced mediator release from nerves and decreased CM contractions (6).

Competition studies demonstrated that PACAP receptors in both DMP and in CM membranes were PACAP preferring, i.e., the VIP/PACAP type I receptor. However, VIP-preferring receptors in DMP were also previously found (12). Surprisingly, this VIP receptor had a relatively high affinity for PACAP-38. By analyzing the cross-linked 125I-PACAP-27 receptor complexes on SDS-polyacrylamide electrophoresis gels, the molecular mass of PACAP receptors in the DMP and in the CM membranes was determined to be 70 kDa (4, 10). The radioactive bands were completely abolished by PACAP-27. These findings are consistent with the competition studies. In combination with previous in vivo functional studies (5, 6), these studies establish the presence of functional PACAP-preferring receptors in the canine ileum.

Many nerve fibers and cell bodies in the MP, DMP, and SMP exhibited PACAP-LI, which often coexisted with VIP-LI. Canine ileal PACAP receptors are of the PACAP-preferring subtype, are located in both the DMP synaptosomal and CM membranes, and are distinct from those for VIP.

    ACKNOWLEDGEMENTS

The secretarial support of G. Kellett is gratefully acknowledged.

    FOOTNOTES

This study was supported by the Medical Research Council of Canada.

Address for reprint requests: E. E. Daniel, Dept. of Biomedical Science, McMaster University, Hamilton, Ontario, Canada L8N 3Z5.

Received 24 February 1997; accepted in final form 15 October 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Ahmad, S., H.-D. Allescher, H. Manaka, Y. Manaka, and E. E. Daniel. [3H]saxitoxin as a marker for canine deep muscular plexus neurons. Am. J. Physiol. 255 (Gastrointest. Liver Physiol. 18): G462-G469, 1988[Abstract/Free Full Text].

2.   Ahmad, S., H.-D. Allescher, H. Manaka, Y. Manaka, and E. E. Daniel. Biochemical studies on opioid and alpha 2-adrenergic receptors in canine submucosal neurons. Am. J. Physiol. 256 (Gastrointest. Liver Physiol. 19): G957-G965, 1989[Abstract/Free Full Text].

3.   Ahmad, S., J. Rausa, E. Jang, and E. E. Daniel. Calcium channel binding in nerves and muscle of canine small intestine. Biochem. Biophys. Res. Commun. 159: 119-125, 1989[Medline].

4.   Christophe, J. Type I receptor for PACAP (a neuropeptide even more important than VIP?). Biochim. Biophys. Acta 1154: 183-199, 1993[Medline].

5.   Daniel, E. E., J. E. T. Fox-Threlkeld, Z. Woskowska, and T. J. McDonald. Pituitary adenylate cyclase activating peptide (PACAP) enhances canine circular muscle (CM) contractions by stimulating release of acetylcholine (ACh) (Abstract). Gastroenterology 110: A654, 1996.

6.  Daniel, E. E., Z. Woskowska, and J. E. T. Fox-Threlkeld. Pituitary adenylate cyclase activating peptide (PACAP) better enhances [3H]acetylcholine release than vasoactive intestinal peptide (VIP) from canine nerve ending in ileal circular muscle (CM) (Abstract). Can. J. Physiol. Pharmacol.. 96, Suppl. A200, 1996.

7.   Katsoulis, S., A. Clemens, H. Schworer, W. Creutzfeldt, and W. E. Schmidt. PACAP is a stimulator of neurogenic contraction in guinea pig ileum. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28): G295-G302, 1993[Abstract/Free Full Text].

8.   Katsoulis, S., W. E. Schidt, R. Schwarzhoff, U. R. Folsch, J. G. Jin, J. R. Grider, and G. M. Makhlouf. Inhibitory transmission in guinea pig stomach mediated by distinct receptors for pituitary adenylate cyclase activating peptide. J. Pharmacol. Exp. Ther. 278: 199-204, 1996[Abstract].

9.   Kostka, P., S. N. Sipos, C. Y. Kwan, L. P. Niles, and E. E. Daniel. Identification and characterization of presynaptic and postsynaptic beta -adrenoreceptors in the longitudinal smooth muscle/myenteric plexus of dog ileum. J. Pharmacol. Exp. Ther. 251: 305-310, 1989[Abstract].

10.   Lam, H. C., K. Takahashi, M. A. Ghatei, S. M. Kanse, J. M. Polak, and S. R. Bloom. Binding sites of a novel neuropeptide pituitary adenylate cyclase activating polypeptide in the rat brain and lung. Eur. J. Biochem. 193: 725-729, 1990[Abstract].

11.   Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951[Free Full Text].

12.   Mao, Y. K., W. Barnett, D. H. Coy, G. Tougas, and E. E. Daniel. Distribution of vasoactive intestinal polypeptide (VIP)-binding in circular muscle and characterization of VIP-binding in canine small intestinal mucosa. J. Pharmacol. Exp. Ther. 258: 986-991, 1991[Abstract].

13.   McDonald, T. J., S. Ahmad, H.-D. Allescher, P. Kostka, E. E. Daniel, W. Barnett, and E. Brodin. Canine myenteric, deep muscular, and submucosal plexus preparations of purified nerve varicosities: content and chromatographic forms of certain neuropeptides. Peptides 11: 95-102, 1990[Medline].

14.   McDonald, T. J., F. L. Christofi, B. D. Brooks, W. Barnett, and M. A. Cook. Characterization of content and chromatographic forms of neuropeptides on purified nerve varicosities prepared from guinea pig myenteric plexus. Regul. Pept. 21: 69-83, 1988[Medline].

15.   McDonald, T. J., Y. F. Wang, Y. K. Mao, R. M. Broad, M. A. Cook, and E. E. Daniel. PYY: a neuropeptide in the canine enteric nervous system. Regul. Pept. 44: 33-48, 1993[Medline].

16.   McPherson, G. A. A practical computer based approach to the analysis of radioligand experiments. Comput. Programs Biomed. 17: 107-114, 1983[Medline].

17.   Miyata, A., A. Arimura, R. R. Dahl, N. Minamino, A. Uehara, L. Jiang, M. D. Culler, and D. H. Coy. Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells. Biochem. Biophys. Res. Commun. 164: 567-574, 1989[Medline].

18.   Miyata, A., L. Jiang, R. R. Dahl, C. Kitada, K. Kubo, M. Fujino, N. Minamino, and A. Arimura. Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38). Biochem. Biophys. Res. Commun. 170: 643-648, 1990[Medline].

19.   Munson, P. J., and D. Rodbard. LIGAND: a versatile computerized approach for characterization of ligand binding systems. Anal. Biochem. 107: 220-239, 1980[Medline].

20.   Shivers, B. D., T. J. Gorces, P. E. Gottschall, and A. Arimura. Two high affinity binding sites for pituitary adenylate cyclase activating polypeptide have different tissue distributions. Endocrinology 128: 3055-3065, 1991[Abstract].

21.   Sundler, F., E. Ekblad, A. Absood, R. Hakanson, K. Koves, and A. Arimura. Pituitary adenylate cyclase activating peptide: a novel vasoactive intestinal peptide-like neuropeptide in the gut. Neuroscience 46: 439-454, 1992[Medline].

22.   Uddman, R., A. Luts, A. Absood, A. Arimura, M. Ekelund, H. Desai, R. Hakanson, G. Hambreans, and F. Sundler. PACAP, a VIP-like peptide in neurons of esophagus. Regul. Pept. 36: 415-422, 1991[Medline].

23.   Uddman, R., A. Luts, A. Arimura, and F. Sundler. Pituitary adenylate cyclase activation peptide (PACAP), a new VIP-like peptide in the respiratory tract. Cell Tissue Res. 265: 197-201, 1991[Medline].


AJP Gastroint Liver Physiol 274(1):G217-G225
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