Immunohistochemical localization of PACAP in the ovine digestive system

Immunohistochemical localization of PACAP in the ovine digestive system

Peptides.Vol. 14, pp. 449-455, 1993 0196-9781/93 $6.00 + .00 Copyright © 1993PergamonPressLtd. Printedin the USA. Immunohistochemical Localization ...

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Peptides.Vol. 14, pp. 449-455, 1993

0196-9781/93 $6.00 + .00 Copyright © 1993PergamonPressLtd.

Printedin the USA.

Immunohistochemical Localization of PACAP in the Ovine Digestive System KATALIN KOVES,*t AKIRA ARIMURA,*t I SANDOR VIGH,*t A N I K O S O M O G Y V A R I - V I G H * t A N D JIM M I L L E R ~

*U.S.-Japan Biomedical Research Laboratories, Tulane University Hebert Center, Belle Chasse, LA 70037, ?Departments of Medicine, Anatomy, and Physiology, Tulane University School of Medicine, New Orleans, LA 70112, andS:Department of Epidemiology, Louisiana State University School of Veterinary Medicine, Baton Rouge, LA 70803 Received 8 July 1992 KOVES, K., A. AR1MURA, S. VIGH, A. SOMOGYVA.RI-VIGH AND J. MILLER. Immunohistochemical localization of PACAP in the ovine digestivesystem. PEPTIDES 14(3) 449-455, 1993.--The localization ofimmunoreactive PACAP (PACAPIR) in the entire length of the sheep gastrointestinal tract and the pancreas was studied by an immunohistochemical method. PACAP-IR-containing nerve fibers innervated the longitudinal smooth muscle layer of the mucosa in the esophagus, stomach, and small and large intestines, the muscular layer of the stomach and intestine, Brunner's gland of the duodenum, and the walls of small arteries. PACAP-IRfibersalso innervated the exocrine acini, isletsof Langerhans, and the small arteries in the connective tissue septa of the pancreas. These findings suggest a regulatory role of PACAP in the digestive system. Pituitary adenylate cyclase activating polypeptide


PITUITARY adenylate cyclase activating polypeptide with 38 amino acid residues, PACAP38, and a truncated form with 27 residues, PACAP27, were isolated from the ovine hypothalamus during the search for novel hypothalamic hypophysiotropic hormones. They were isolated by screening for adenylate cyclase stimulating activity in rat pituitary cell cultures (17,18). Both PACAP38 and PACAP27 appear to be produced from a single precursor having 176 amino acid residues (8). The N-terminal 28 residues of PACAP are homologous to the amino acid sequences of members of the glucagon/secretin family ofpeptides. They show the greatest homology (68%) with vasoactive intestinal peptide (VIP). Despite considerable homology with VIP, the precursor for PACAP is very different from that for VIP (7). Immunohistochemical studies have shown that PACAP27and PACAP38-immunoreactive (IR) neuronal processes occur in the forebrain of several species, e.g., sheep (11), nonhuman primates (26), man (26), and rats (10). A comparison of the distribution of PACAP- and VIP-IR neuronal processes revealed that these two peptides have different distributions in the septum, thalamus, hypothalamus, and posterior pituitary (10). Vasoactive intestinal peptide, which is present in the forebrain (1,13,24) and the peripheral nervous system (15,25), was first isolated from porcine small intestine (21), and was found in various portions of the digestive system using immunohistochemistry (13,20,27). It is likely that PACAP also occurs in the gut and plays a regulatory role in digestive organs. Studies using



radioreceptor assays or autoradiography (5,23) indicate that PACAP receptors are present in the central nervous system and in peripheral organs. Vasoactive intestinal peptide and PACAP share binding sites in the lung, liver, prostate gland, and seminal vesicle, but not in the brain, anterior pituitary, or in the adrenal medulla. The present study compares the distribution of PACAP and VIP immunoreactivities in various regions of the digestive system of sheep. METHOD

Animals Four castrated male and three intact female sheep (7 months old; Suffolk or Louisiana Native, Louisiana State University breed) were used. The animals were sacrificed with an overdose of T-61 Euthanasia Solution (0.3 ml/kg). Parts of the digestive system were removed and rapidly immersed in 0.1 M phosphatebuffered (pH 7.4) 4% paraformaldehyde or Bouin's solution. The tissues were fixed for 3 days at room temperature.

Antiserum The antiserum (No. 88121-3) against synthetic PACAP27 (synthesized by Dr. C. Kitada Takeda Chemical Industries, Tsukuba Laboratories, Tsukuba, Japan) was generated in rabbit and partially characterized as previously described (11). The titer

Requests for reprints should be addressed to Akira Arimura, M.D., Ph.D., U.S.-Japan Biomedical Research Laboratories, Tulane University Hebert Center, Bldg. 30, 3705 Main St., Belle Chasse, LA 70037.




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FIG. 1. ELISA titration of the antiserum against PACAP27 (No. 88121-3). Polystyrene microtiter plates were coated with 1 ug/ml of peptide. The antiserum exhibited a significant titer for PACAP27 (P27) and no cross-reactivity with the other peptides tested. Each value is the mean of two measurements. Dilution of antiserum was 1:3000 and 1:6000, respectively. Optical absorbance was read at 490 nm. See peptide abbreviations in the Method section. NSB, nonspecific binding.

and the specificity of the antiserum were tested using an enzyme linked immunosorbent assay (ELISA) (3,4,12). The following peptides present in the digestive system were examined for crossreactivity: VIP, peptide histidine isoleucin (PHI), glucagon (GLU), somatostatin (SS), neuropeptide Y (NPY), cholecystokinin (CCK), bombesin (B), motilin (M), gastrin (G), gastrinreleasing peptide (GRP), fl-endorphin (r-E), dynorphin (DYN), and Leu-enkephalin (L-E). The method used has been described elsewhere (4). Other peptides have also been tested for crossreactivity with this antiserum (11).

Immunohistochemistry Sections from various regions of the digestive system were cut at 50 tsm on a Lancer Vibratome (St. Louis, MO). Some tissues were soaked in a 15% sucrose solution in phosphate buffer, 0.12 M, pH 7.7 (PB), until they sank and then the sections were cut on a cryostat (Ames, Elkhart, IN) at 6 /~m. Endogenous peroxidase activity was blocked with 0.05% H202 and 2% normal lamb serum in PB. The sections were placed in PB containing 1% Triton X-100 and 2% normal lamb serum overnight at 4°C. Following three washes, the sections were preincubated in 10% normal lamb serum, and then incubated with the antiserum against PACAP27 ( 1:6000 dilution) or with an antiserum against VIP (1:10,000 dilution) (6) for 48 h at 4°C. To reduce nonspeciflc staining, the P A C A P antiserum was preabsorbed with liver and kidney powders (Arnel, New York, NY) for 3 h at r o o m temperature and centrifuged. After incubations with the primary antiserum, tissue sections were processed further using the Vectastain (ABC) kit (Vector, Burlingame, CA). Tissue-bound peroxidase activity was revealed using the nickel-intensified diaminobenzidine as the chromogen. The following controls were performed. 1. Sections were incubated in a n o n i m m u n e serum instead of the PACAP antiserum. 2. Sections were incubated in the primary antiserum without the second and the third antisera. 3. The PACAP antiserum was preadsorbed with 18 #g of PACAP27 using a solid-phase method or with 10 ~zg of VIP in solution.

4. The VIP antiserum was preabsorbed with 10 ug VIP or 10 /zg PACAP.

Solid-Phase Adsorption Method To verify the specificity of immunostaining, a solid-phase adsorption method (9) was used to remove the PACAP antibodies from the PACAP antiserum. This is an alternative procedure to preabsorption of the antiserum with PACAP27 in solution. The advantage of this method is its ability to test the effectiveness of removal of PACAP antibodies from the antiserum using ELISA.

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FIG. 2. Effectiveness of removing the PACAP antibodies from the PACAP antiserum with solid-phase adsorption. The graph shows the ELISA titer of the antiserum treated in either control wells or PACAP27-coated wells after 6th, 12th, or 18th transfers. After the 6th and 12th transfers a considerable titer for PACAP27 was still observed; however, it was much lower than the titer of the PACAP antiserum treated in control wells. After the 18th transfer, all antibodies against PACAP27 were removed. Polystyrene mierotiter plates were coated with 1 ~tg/ml of PACAP27 (P27). Each value is the mean of two measurements. The dilution of the antiserum was 1:6000. Optical absorbance was read at 490 nm. NSB, nonspecific binding.



FIG. 3. PACAP immunoreactivity in esophagus and stomach. (a,c) Mucosal layer of the esophagus; (b,d) muscular layers of the stomach. Arrows indicate longitudinal PACAP-IR fibers running along a papilla of connective tissue underneath the epithelium, and cross-sectionedimmunoreactive fibersamong transected smooth muscle cells in the muscular layer of the mucosa (a,c). Arrowheads indicate fibers in the inner (b) and middle zones (d) of the muscular layers of the stomach. E, epithelium; C, connective tissue layer; M, smooth muscle layer. ABC method, 50 um thick vibratome sections. Dilution of the antiserum was 1: 6000. Bar = 50 ~m.

Eighteen wells of a 24-well, flat-bottomed polystyrene tissue culture plate were coated with 1 ~tg of PACAP27 in 1 ml of carbonate-bicarbonate buffer (pH 9.6) and another 18 wells were treated with buffer only. These wells were used as controls. The plates were kept at 4°C overnight. After washing the wells, 1 ml of the PACAP antiserum was added to the first PACAP-coated well and another 1 ml was added to the first control well. The samples were shaken for 1 h at 50 rpm using an orbital shaker (American Rotator V) at room temperature. The antisera were transferred to the second well of each plate and shaken for 1 h. This procedure was repeated 16 times more and then antisera were used for immunohistochemistry. After the 6th, 12th and 18th transfers, 100 #1 ofPACAP antisera was tested for PACAP antibody titer by ELISA. RESULTS

Specificity of the PACAP Antiserum The ELISA results indicated that the PACAP antiserum (No. 88121-3) exhibited a significant titer for PACAP27 and no crossreactivity with other peptides tested (Fig. 1).

Removal of PACAP Antibodies From the PACAP Antiserum The effectiveness of removing the PACAP antibodies from the antiserum was tested with ELISA. After the 6th and 12th

transfers, the PACAP antiserum treated in PACAP-coated wells still exhibited a low, but significant, antibody titer for PACAP27 (Fig. 2). After the 18th transfer, PACAP antibodies were undetectable and there was no immunolabeling when used for immunohistochemistry. Both untreated and preabsorbed antisera were used in our immunohistochemical study. The immunostaining with the untreated PACAP antiserum that was not seen with the preadsorbed antiserum was considered specific for PACAP.

Distribution of PACAP Irnmunoreactivity Esophagus. PACAP-IR fibers were present in the muscular layer of the mucosa (Fig. 3a), which consisted of a longitudinal smooth muscle layer. In the connective tissue, especially in the submucosal layer or underneath the epithelium, PACAP-IR fibers innervated small arteries. The striated muscle layer did not contain PACAP-IR fibers. Stomach. PACAP-IR fibers were present mainly in the muscular layer of the stomach (Fig. 3b,c) including cardia, corpus, antrum, and pylorus. The pyloric sphincter was heavily innervated by PACAP-IR fibers. Scattered immunolabeled fibers were present in the mucosa, mainly in its muscular layer. PACAPimmunolabeled neuronal cell bodies were rarely found in the myenteric ganglia. However, PACAP-IR fibers surrounded unlabeled perikarya in these ganglia.



Small intestine. The muscular layers throughout the small intestine [duodenum (Fig. 4), jejunum, and ileum] were innervated by PACAP-IR fibers. Few immunolabeled fibers were present in other layers of the small intestine except for the duodenum. In the submucosa of the duodenum, Brunner's glands were heavily surrounded by PACAP-IR fibers. The myenteric ganglia contained some immunolabeled perikarya, Large intestine (including rectum). PACAP-IR fibers were present in the muscular layer (colon, Fig. 5a,c). The distribution of immunoreactivity was similar to that observed in the jejunum and ileum. Salivary glands. The submandibular and sublingual glands did not contain PACAP immunoreactivity.

Pancreas. PACAP-IR fibers were present in the connective tissue septa separating the lobes of glands. The fibers penetrated the islets of Langerhans, surrounded exocrine acini, or innervated arteries (Fig. 6). In all parts of the digestive system small arteries and arterioles were frequently innervated by PACAP-IR fibers (Figs. 4e and 6b). No immunostaining was observed with the PACAP27preadsorbed antiserum (Fig. 4t). However, the immunostaining was present with the PACAP antiserum treated in the control wells of the solid-phase adsorption method, or with the VIPpreabsorbed antiserum. There was no staining when the sections were incubated with a n o n i m m u n e serum or only with the primary antiserum.

FIG. 4. Distribution of PACAP immunoreactivity in the duodenum. (a) Mucosal layer; (b) submucosal layer; (c) muscular layer; (d) myenteric ganglion; (e) subperitoneal layer; (f) submucosal and muscular layers. Arrows indicate a few fibers in the deepest layer of the mucosa (a), and a dense fiber network in the submucosa surrounding Brunner's glands (b), in the circular and longitudinal smooth muscle layers (c), and in the subserosa innervating an artery (e). Arrowheads indicate immunolabeled neuronal perikarya in the myenteric ganglion (d). No immunostaining was observed when the section was stained with the antiserum from which PACAP antibodies were removed by a solid-phase adsorption method. The same method was used as in Fig. 3. Bar 25 um in (d), 50 um in (a-c) and (e), and 100 um in (f).




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FIG. 5. PACAP (a,c) and VIP (b,d) immunoreactivity in the colon. (a,b) Mucosal layer; (c,d) smooth muscle layer. A few PACAP-IR fibers were present in the mucosa, but a dense VIP fiber network was observed in this layer surrounding the Lieberkiihn gland. The density of fiber staining in the muscular layer was similar for PACAP and VIP. Arrows indicate single fibers, arrowheads indicate the myenteric plexus between the circular and longitudinal smooth muscle layers. The same method was used as in Fig. 3. Bar = 50 urn.

Distribution of VIP Immunoreactivity Vasoactive intestinal peptide-immunoreactive fibers were observed in all layers of the gastrointestinal tract, surrounding Lieberkfihn glands in the mucosal layer (colon, Fig. 5b). Vasoactive intestinal peptide-immunoreactive fibers were also found in the Brunner's glands in the submucosal layer of the duodenum and in the smooth muscle layers (colon, Fig. 5d); these fibers also innervated small arteries and arterioles in the gut, and they innervate all parts of the digestive system (19,20,27). Vasoactive intestinal peptide immunostaining was abolished after liquidphase absorption of the VIP antiserum with VIP but was not affected after preincubation with PACAP27. DISCUSSION Our results provide morphological evidence for innervation of the gut by PACAP-IR fibers in sheep. Vasoactive intestinal peptide (13,20,27) and PACAP immunoreactivities are present throughout the digestive system. PACAP-IR fibers innervating the circular and longitudinal muscular layer were observed in the entire length of the gastrointestinal tract. Few transected PACAP-containing fibers were present in the thin muscular layer of the stomach mucosa and in the small and large intestines. However, dense networks of immunoreactive fibers were observed in the well-developed longitudinal muscular layer of the esophageal mucosa. PACAP-IR fibers were not present in the salivary glands. In the pancreas, PACAP-IR fibers innervated

the exocrine acini and penetrated into the islets of Langerhans. In all parts of the digestive system, PACAP-IR fibers were seen to innervate small arteries and arterioles, as was previously observed in ovine and rat brains (10,11). The distributions of PACAP- and VIP-IR fibers were different in the digestive system. The VIP-IR fibers innervated all layers of the gastrointestinal tract. In contrast, no dense network of PACAP-IR fibers was demonstrated in the mucosal layer. In the duodenum, Brunner's glands in the submucosa were heavily innervated by PACAP-IR fibers. Recent physiological results indicate that PACAP may be important in the functions of the digestive system. PACAP has a dose-dependent inhibitory effect on spontaneous smooth muscle contractions (19). In contrast to VIP, PACAP has a potent inhibitory effect of acetylcholine- and carbachol-stimulated smooth muscle contractions (19). PACAP also stimulated the glucose-dependent insulin release from the islets of Langerhans, as well as lipase and amylase release from the exocrine pancreas (22). Highly selective receptors for PACAP were observed in membranes from a rat pancreatic acinar cell line, AR 4-2J (2). The presence of PACAP-IR fibers innervating the smooth muscle layer of arteries correlates with the observation that IV administration of PACAP produced a vasodepressor response in anesthetized rats (17). The depressor activity of PACAP was comparable to that of VIP (17). However, PACAP and VIP exhibit differential effects on the pulmonary and hindquarters vascular beds of the cat, suggesting that vascular







FIG. 6. PACAP immunoreactivity in the pancreas. (a) Connective tissue septum surrounded by exocrine acini and an islet of Langerhans; (b) a high power detail of (a); (c) islet of Langerhans; (d) exocrine acini. Arrows indicate fibers penetrating the islet (b,c), innervating an artery (b) or among exocrine acini (d). In the connective tissue, branches of immunoreactive peripheral nerves are indicated by arrowheads. A, artery; EX, exocrine acini; LI, islet of Langerhans; S, connective tissue septum. The same method was used as in Fig. 3. Bar = 50 urn.

responses induced by the two peptides may, in part, be mediated by different receptors (16). It is not k n o w n whether P A C A P and VIP show different effects on the intestinal small arteries and arterioles. In summary, we have localized PACAP, a new member of the glucagon-secretin family of peptides, in the digestive system with immunohistochemistry. PACAP-IR fibers mainly inner-

vated the smooth muscle layers and small arteries of the gastrointestinal tract and Brunner's glands, as well as the exocrine and endocrine parts of pancreas. Our work supports the view that PACAP plays multifunctional regulatory roles in the digestive system. PACAP may control smooth muscle relaxation, blood flow, the activity of Brunner's gland, and the secretion of the exocrine and endocrine pancreas.

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