PACAP, a VIP-like peptide, in neurons of the esophagus

Regulatory Peptides, 36 (1991) 415-422

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© 1991 Elsevier Science Publishers B.V. All rights reserved. 0167-0115/91/$03.50 REGPEP 01116

PACAP, a VIP-like peptide, in neurons of the esophagus R. Uddman l, A. Luts 2, A. Absood 3, A. Arimura 5, M. Ekelund 4, H. Desai 1, R. Hhkanson 3, G. H a m b r e a u s 4 a n d F. S u n d l e r 2 ~Department of Otorhinolaryngology, General Hospital, Malm6 (Sweden), 2Department of Medical Cell Research, and 3Pharmacology, University of Lund, Lund (Sweden), 4Department of Surgery, University Hospital, Lund (Sweden) and s U.S.-Japan Biomedical Research Laboratories, Tulane University Herbert Center, School of Medicine, New Orleans, LA (U.S.A.)

(Received 23 May 1991; revised version received 30 August 1991; accepted 3 September 1991) K e y words." PACAP; VIP; SP; Immunocytochemistry; Esophagus

Summary The lower esophagus of guinea-pig, cat, sheep and man was analyzed for pituitary adenylate cyclase activating peptide (PACAP), a novel vasoactive intestinal peptide (VIP)-like peptide, using immunocytochemistry and radioimmunoassay. PACAPimmunoreactive nerve fibers were numerous in the longitudinal and circular muscle layers of sheep and man, moderate in numbers in cat, while being few in the esophagus of guinea-pig. A few PACAP-immunoreactive nerve cell bodies and numerous nerve fibers were seen in the myenteric ganglia of the esophagus of cat, sheep and man. In the lower esophagus of cat, sheep and man all PACAP-containing nerve cell bodies and nerve fibers stored VIP. The results of radioimmunoassay of PACAP in extracts of specimens from man were in good agreement with the immunocytochemical findings. High performance liquid chromatography revealed one major peak of PACAP-like immunoreactivity in extracts of human esophagus. We suggest that neuronal PACAP may serve to modulate motor activity and secretion in the lower esophageal sphincter region.

Correspondence: Rolf Uddman, Department of Otorhinolaryngology,Maim0 General Hospital, S-21401 Malm6, Sweden.

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Introduction

The esophagus is richly supplied with peptide-containing nerve fibers. Among the first neuropeptides to be detected in the esophagus was vasoactive intestinal polypeptide (VIP) [1], a 28 amino acid peptide with a widespread distribution in the peripheral nervous system [2]. The VIP precursor contains also peptide histidine isoleucine/ methionine (PHI/PHM) [3,4], a VIP-like peptide with a bioactivity profile similar to that of VIP [5,6]. VIP/PHI belong to a family of peptides that includes also helodermin and helospectin isolated from the salivary gland venom of the Gila monster (see, e.g., Ref. 7). Recently, still another member of the VIP/PHI family was isolated from ovine hypothalami and named pituitary adenylate cyclase activating peptide (PACAP) [8,9]. Hypothalamic PACAP occurs in two forms, PACAP 27, and the C-terminally extended PACAP 38. PACAP27 has 68~°/0 sequence homology with VIP. PACAP 27 and PACAP 38 appear to coexist in many tissues, but their concentration ratio varies greatly from one tissue to another (Arimura, unpublished observations). Ligand binding studies have revealed high specific binding of PACAP in various peripheral tissues, such as lung, liver and duodenum [10]. In the present report, we describe the occurrence and distribution of PACAPimmunoreactive nerve fibers and PACAP immunoreactivity in the esophagus of guineapig, cat, sheep and man.

Materials and Methods

Immunocytochemistry Five adult cats and three guinea-pigs were killed by an overdose of pentobarbitone. In the cats, specimens were taken from the lower esophagus. In the guinea-pigs, specimens were taken from both the sub- and supradiaphragmatic part of the esophagus. Esophageal specimens from sheep were obtained at a local abbatoir. In addition, specimens from the lower esophagus were taken from nine patients with carcinoma of the stomach. The tissue specimens comprised both the longitudinal and the circular muscle layers. Two specimens included the whole esophageal wall. Conventional histology was applied in order to exclude specimens with neoplastic infiltration. The specimens were immersed for 12 h in an ice-cold solution of 2~o formaldehyde, buffered to pH 7.2 with 0.1 M phosphate buffer, and containing 0.2~o picric acid. They were then rinsed in a Tyrode solution containing 10~o sucrose for 48 h, frozen on dry ice and sectioned in a cryostat at 10 #m thickness. Cryostat sections were processed for the immunocytochemical demonstration of PACAP using indirect immunofluorescence [ 11]. The PACAP antiserum (code No. 88121-3) was raised in a rabbit against ovine PACAP 27 conjugated with bovine thyroglobulin [ 12]. For immunocytochemistry it was used in a dilution of 1 : 640. The antiserum is directed against the C-terminal region of PACAP 27 and it does not recognize PACAP 38, VIP, PHI, helodermin or helospectin I and II as tested by preincubation of each of the peptides (10 #g/ml diluted antiserum) (see also Ref. 12). Of the antisera against VIP one was raised in rabbit (code No. 7852, MILAB, Maim0, Sweden) and used in dilution 1 : 640; another was raised

417 in guinea-pig (code No. 8701, MILAB, MalmO) and used in a dilution of 1 : 640. The sections were exposed to the peptide antibodies for 20 h at 4 °C in a moist chamber. The site of the antigen-antibody reaction was revealed by application of fluorescein isothiocyanate (FITC)-labeled antibodies against immunoglobulin G (IgG) (Dakopatts, Copenhagen, Denmark) in a dilution of 1 : 320 for 1 h at room temperature. The VIP antisera used have been shown not to cross react with other structurally related peptides such as glucagon, secretin, helodermin, helospectins and gastric inhibitory peptide [ 13,14]; they were now shown not to cross react with PACAP (10 #g/ml of PACAP 27 or PACAP 38). Although the antisera used seem specific, cross reaction with other still unidentified peptides or proteins sharing amino acid sequences with the antigenic peptide cannot be excluded. Therefore, it is appropriate to refer to the immunoreactive material as PACAP-Iike or VIP-like. For brevity, however, the shorter terms PACAP and VIP will be used henceforth. For double immunostaining the VIP antiserum raised in guinea-pig was used in combination with the PACAP antiserum. Second antibodies were labelled with FITC and tetramethylrhodamine isothiocyanate (TRITC), respectively. Briefly, sections were first incubated with PACAP antiserum and with TRITC-labeled second antibodies. The sections were then incubated with VIP antiserum. The latter antibodies were demonstrated with FITC-labeled second antibodies. In order to exclude inappropriate binding of the second antibodies controls were run as follows: sections were incubated either using the rabbit primary antiserum followed by incubation with anti-guinea-pig second antibodies or the guinea-pig primary antiserum followed by antirabbit second antibodies. No inappropriate binding could be demonstrated. The sections were examined in a fluorescence microscope fitted with the appropriate filter settings for viewing TRITC and FITC fluorescence alternately.

Radioimmunoassay Smooth muscle specimens, comprising the longitudinal and circular muscle layers from the lower esophageal region of man, obtained during surgery, were analyzed for PACAP immunoreactivity. They were extracted by boiling in 0.9~o NaC1 for 10 min followed by homogenization (Polytron, 1-2 min). The homogenates were centrifuged at 3000 g for 10 min at + 4 °C. The sediment was re-extracted by boiling in 0.5 M acetic acid for 15 min, followed by homogenization and centrifugation as above. The pooled supernatants were freeze-dried and dissolved in 0.05 M phosphate buffer (pH 7.5) for radioimmunoassay (RIA), using antiserum No. 88121-3. The antiserum does not recognize PACAP 38, helodermin, helodermin fragments: (1-30) NH2, (1-31) NH2, and (1-33) NH2, helospectin (I and II), VIP, PHI, secretin, glucagon, salmon calcitonin, CGRP or growth hormone releasing factor. The detection limit for PACAP 27 is 20 pg/ml; the intraassay variation is 5~o. The PACAP-like immunoreactivity in the extracts diluted in parallel with the PACAP 27 standard curve. The concentrations of PACAP-Iike peptides were expressed as pg/g wet weight.

Chromatogrtq~hic amth'sis Extracts from the esophagus of man were analyzed by HPLC. The HPLC system consisted of a Waters model 204 liquid chromatograph with a reverse phase column of /~-Bondapak C-18. The solvent system was 0.08~o trifluoroacetic acid with a 28-58 o/ /O (v/v) linear gradient of acetonitrile over 60 min followed by 10 min of isocratic elution at 58~o (flow rate of 1.0 ml per min). Fractions of 0.5 ml were collected and assayed for P A C A P 27.

Results A rich supply of PACAP-immunoreactive fibers was seen in the lower esophagus of sheep and man where nerve fibers occurred in both the longitudinal and circular smooth muscle layers (Fig. 1). In addition, PACAP-immunoreactive nerve fibers were numerous in the submucosa of the esophagus of sheep and man where immunoreactive fibers occurred close to small blood vessels and glands. A moderate supply of PACAPimmunoreactive nerve cell bodies and a rich supply of nerve fibers were seen in the myenteric plexus (Fig. 2). In the esophagus of the cat a few PACAP-containing nerve cell bodies were seen. PACAP-immunoreactive nerve fibers were moderate in number; they were few in the esophagus of the guinea-pig and restricted to the muscle layers of the subdiaphragmatic part. Double immunostaining of sections from the esophagus of cat, sheep and man revealed that all PACAP-immunoreactive nerve cell bodies and nerve fibers were VIP-immunoreactive (Fig. 2).

Fig. 1. Homo esophagus. Numerous PACAP-immunoreactivenerve fibers are seen among bundles of smooth muscle ( × 350).

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Fig. 2. Sections from human (a,b) and sheep (c,d,e,f) esophagus. Simultaneousimmunostaining for PACAP (a,c,e) and VIP (b,d,f,). Fine varicose nerve fibers containing PACAP-immunoreactivity are seen among bundles of smooth muscle. Note a seemingly total coexistence PACAP (a) and VIP (b). PACAP-immunoreactive nerve cell bodies in the myenteric plexus also contain VIP (c and d). Numerous PACAP-immunoreactive nerve fibers are seen in the plexus. Nerve fibers surrounding a blood vessel in the circular muscle layer containing PACAP (e) also show immunoreactivity for VIP (f). (a,b x 350; c - f × 300).

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Fig. 3. HPLC elution profile of PACAP-immunoreactivematerial (drawn line) in extracts from the esophagus of man. The elution position of synthetic ovine PACAP and the elution gradient is indicated (broken line). Immunochemistry revealed moderate concentrations of PACAP in the lower esophageal wall of man (0.92 + 0.13 ng/g, n = 7). H P L C of extracts of human specimens revealed one PACAP-immunoreactive peak, co-eluting with authentic PACAP-27 (Fig. 3).

Discussion In the present study, PACAP-immunoreactive nerve fibers were found to be numerous in the non-vascular smooth muscle of the esophagus of sheep and man. In addition, PACAP-immunoreactive fibers were seen close to glands and small blood vessels. In the myenteric ganglia of the esophagus a moderate number of PACAP-immunoreactive nerve cell bodies and a large number of PACAP-immunoreactive nerve fibers was seen. In the non-vascular smooth muscle layers of the cat esophagus PACAP-immunoreactive fibers were moderate in number. The subdiaphragmatic part of the guinea-pig esophagus harboured only few PACAP-immunoreactive fibers. H P L C analysis of extracts from the lower esophageal region of man revealed one single peak of immunoreactive PACAP, co-eluting with synthetic P A C A P 27. Double immunostalning revealed that in the feline, ovine and human esophagus all PACAP-immunoreactive nerve fibers and all PACAPcontaining nerve cell bodies in the myenteric ganglia stored in addition VIP. This is in agreement with recent findings in the airways of several species [ 15] and in the human gut [ 16,17 ]. In the feline and ovine gut on the other hand PACAP-immunoreactive nerve fibers are distinct from those storing VIP [ 17]. These data indicate a difference between the esophagus and gut in these species with respect to the chemical coding of the PACAP-immunoreactive neurons.

421 P A C A P belongs to a family o f n e u r o p e p t i d e s that includes VIP, P H I , helospectins and helodermin. The N - t e r m i n a l portion o f P A C A P displays m a r k e d h o m o l o g y with VIP and P H I [8,9]. However, the C-terminal region ( 2 9 - 3 8 ) o f P A C A P 38 does not show h o m o l o g y with any other k n o w n regulatory peptide. Recent studies have shown P A C A P to be distributed in the brain (Ref. 12, Kivipelto, personal c o m m u n i c a t i o n ) and in neuronal elements in the gut [ 16,17]. Like VIP, P A C A P lowers the b l o o d pressure when injected in anesthetized rats ( A b s o o d , personal communication). However, P A C A P is much more potent than VIP in activating adenylate cyclase [8]. Despite numerous studies little is k n o w n about the basic m e c h a n i s m s that control the relaxation o f the lower esophageal sphincter (LES). M a n o m e t r i c a l and electrophysiological studies have indicated that the distal esophagus possesses a neuronal noncholinergic, nonadrenergic inhibitory system [18,19]. VIP relaxes the L E S by a direct action on the sphincter muscle [ 2 0 - 2 3 ] . F r o m the present findings it is possible that also P A C A P participates in the sphincter regulation. However, further studies are n e e d e d to identify the functional role o f P A C A P in the esophagus.

Acknowledgements This work was s u p p o r t e d by grants from the Swedish M e d i c a l Research Council (No. 04X-4499, 04X-1007 and 17X-6859) and P~ihlsson's F o u n d a t i o n .

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