Cell Tissue Res (1991) 265:19%201 Cell and Tissue Research �9 Springer-Verlag 1991

Pituitary adenylate eyelase-aetivating peptide (PACAP), a new vasoactive intestinal peptide (VIP)-like peptide in the respiratory tract Rolf Uddman 1, Anders Luts 2, Akira Arimura 3, and Frank Sundler 2

1 Department of Otorhinolaryngology, Malta6 General Hospital, S-21401 Malm6, Sweden, 2 Department of Medical Cell Research, University of Lund, S-22362 Lund, Sweden, 3 U.S.-Japan Biomedical Research Laboratories, Tulane University Herbert Center, School of Medicine, New Orleans, LA 70112, USA

Accepted February 25, 1991

Summary. Pituitary adenylate cyclase-activating peptide (PACAP) is a vasoactive intestinal peptide (VIP)-like peptide recently isolated from ovine hypothalami. Nerve fibers displaying PACAP immunoreactivity were found in the respiratory tract of rats, guinea pigs, ferrets, pigs, sheep and squirrel monkeys. A moderate supply of PA- CAP-immunoreactive fibers was seen in the nasal muco- sa of guinea pigs. Few to moderate numbers of PACAP- containing fibers occurred in the tracheo-bronchial wall of rats, guinea pigs, ferrets, pigs, sheep and squirrel mon- keys. The fibers were distributed beneath the epithelium, around blood vessels and seromucous glands, and among bundles of smooth muscle. In the lungs, the im- munoreactive fibers were observed close to small bron- chioli. A few PACAP-immunoreactive nerve cell bodies were seen in the sphenopalatine and otic ganglia of guin- ea pigs. Simultaneous double immunostaining of the re- spiratory tract of sheep and ferrets revealed that all PA- CAP-containing nerve fibers stored VIP. We suggest that neuronal PACAP may take part in the regulation of smooth muscle tone and glandular secretion.

Key words: Pituitary adenylate cyclase-activating pep- tide (PACAP) - Vasoactive intestinal peptide (VIP) Immunocytochemistry - Respiratory tract - Autonomic innervation Mammals

The respiratory tract is invested with a rich supply of nerve fibers of an adrenergic and non-adrenergic nature (cf. Uddman and Sundler 1987). The role of adrenergic and cholinergic neurons in the respiratory tract is well established. More recently, several biologically active peptides have emerged as neurotransmitter candidates. Among the first neuropeptides to be detected in the re- spiratory tract was vasoactive intestinal polypeptide (VIP), a 28-amino acid peptide with a widespread distri-

Offprint requests to: R. Uddman

bution in the peripheral nervous system (Sundler et al. 1988a). The VIP precursor also contains peptide histi- dine isoleucine/methionine (PHI /PHM) (Tatemoto and Mutt 1981), a VIP-like peptide sharing several biological activities with VIP (Obata et al. 1981 ; Itoh et al. 1983). Many of the VIP-containing neurons seem to be identi- cal with cholinergic neurons in the upper airways; there is evidence for the coexistence of VIP and acetylcholine in parasympathetic ganglia supplying the respiratory tract (Lundberg et al. 1979, 1984; Uddman et al. 1983). VIP/PHI belongs to a family of peptides that, in addi- tion, contains helodermin and helospectin (for refer- ences, see Cauvin et al. 1989). These latter peptides have been isolated from the venom of the lizard Gila monster (Hoshino et al. 1984; Parker et al. 1984). There is recent evidence that helodermin/helospectin-like peptides are present also in mammals, where they seem to be distrib- uted mainly in endocrine cells (thyroid C-cells and ad- renomedullary cells) and in the central nervous system (Robberecht et al. 1985; Sundler et al. 1988b; Bjartell et al. 1989; Grunditz et al. 1989). Recently, still another member of the family has been discovered and desig- nated pituitary adenylate cyclase-activating peptide (PA- CAP) (Miyata et al. 1989). PACAP occurs in two forms: PACAP 27 and PACAP 38 (Miyata et al. 1989, 1990). Both peptides are amidated, PACAP 27 constituting the N-terminal (VIP-like) port ion of PACAP 38.

In the present study, we report on the occurrence and distribution of PACAP-immunoreactive nerve fibers in the respiratory tract of several mammals.

Materials and methods

Adult rats, guinea pigs, ferrets, pigs, sheep and squirrel monkeys (at least 5 of each species) were used. The laboratory animals were killed by an overdose of pentobarbitone. Specimens from pigs and sheep were collected at a local abattoir. Specimens from the nasal mucosa, the larynx, trachea, bronchi and the lung were taken for immunocytochemical analysis. In addition, the sphenopalatine gan- glia and the otic ganglia from 3 guinea pigs were analyzed. These ganglia are known to contribute to the innervation of the airways.

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Fig. 1. a Guinea pig, trachea. Delicate PACAP-immunoreactive nerve fibers among bundles of smooth muscle, b Rat, larynx. Fine varicose fibers (arrowheads) penetrate the epithelium, e Guinea pig, pulmonary parenchyma. Scattered PACAP-immunoreactive nerve

fibers close to a blood vessel (arrowhead). d Sheep, pulmonary parenchyma. A cluster of PACAP-immunoreact ive cells within the epithelium of a bronchiolus, a, d x 350; b, e x 250

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Fig. 2a-d. Sections from sheep airways. Double immunostaining for PACAP (a~ e) and VIP (b, d). PACAP and VIP coexist in the same nerve fibers in the smooth muscle (a, b). In the pulmonary

parenchyma, a PACAP-immunoreactive nerve fiber runs in the wall of a small bronchus contains VIP (e, d)

The specimens were immersed in an ice-cold fixative solution composed of 2% formaldehyde, buffered to pH 7.2 with 0A M phosphate buffer, and 0.2% picric acid, for 12 h. They were then rinsed in a Tyrode solution containing 10% sucrose for 48 h, frozen on dry ice and sectioned in a cryostat at a thickness of 10 gm.

The cryostat sections were processed for the immunocytochemi- cal demonstration of PACAP using the indirect immunofluores- cence method (Coons 1958). The PACAP antiserum (code no. 88121-3) was raised in rabbit against sheep PACAP-27 and used at a dilution of 1:640. The antiserum is directed against the C- terminal region of PACAP-27 but also recognizes PACAP-38 (K6ves et al. 1990). One antiserum against VIP was raised in rabbit (code no 7852, MILAB, Malm6, Sweden) and used at a dilution of 1:640; another was raised in guinea pig (code no 8701) and used at a dilution of 1:640 (Luts et al. 1989). The sections were exposed to the peptide antiserum for 24 h at 4~ in a moist chamber. The site of the antigen-antibody reaction was revealed by application of fluorescein isothiocyanate (FITC)-labeled anti-

bodies against immunoglobulin G (IgG) (MILAB, Malm6, Swe- den) at a dilution of 1:320 for 1 h at room temperature. Control sections were exposed to antiserum that had been preabsorbed with an excess amount of the antigen (10-100 gg of synthetic pep- tide per ml diluted antiserum).

Our absorption tests showed that the PACAP 27 antiserum does not crossreact with VIP, helodermin, bombesin or SP. For further details regarding the properties of the antiserum, see K6ves et al. (1990). The VIP antisera used have been shown not to cross- react with other peptides, such as glucagon, secretin, helodermin and gastric inhibitory peptide; neither do they crossreact with PACAP (10 gg/ml PACAP 27 added). However, crossreaction with other peptides or proteins sharing amino acid sequences with the examined peptide cannot be excluded. Therefore, it is appropriate to refer to the immunoreactive material as PACAP-like 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

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pigs was used in combination with the PACAP antiserum. Second antibodies were labeled with FITC and tetramethyl rhodamine 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. Controls were run to exclude unexpected crossreactivity of the second antibodies. The sections were examined in a fluores- cence microscope fitted with the appropriate filter settings for view- ing TRITC and FITC fluorescence, alternately.

Results and discussion

Few to moderate numbers of PACAP-immunoreact ive nerve fibers could be seen in the respiratory tract of all animals studied. A moderate supply of PACAP-im- munoreact ive nerve fibers was observed around blood vessels and seromucous glands in the nose of the guinea pig. In the bronchial wall, a moderate supply of PACAP- immunoreact ive fibers occurred beneath the epithelium, in the smooth muscle layer, and around blood vessels and small seromucous glands in the lamina propria.

PACAP-containing fibers appeared close to small bronchi and bronchioli in peripheral parts of the lung (Fig. 1). A few fibers seemed to have no obvious connec- tion to bronchi or blood vessels. In the pulmonary pa- renchyma of sheep, a few neuroepithelial bodies, i.e., small clusters of endocrine cells, were immunoreact ive for PACAP (Fig. 1).

The sphenopalatine and otic ganglia contained a sparse supply of PACAP-immunoreact ive perikarya. Double immunostaining performed on sections f rom sheep trachea and ferret pulmonary parenchyma re- vealed that virtually all PACAP-immunoreact ive nerve fibers contained VIP (Fig. 2). A subpopulat ion of the VIP-containing fibers seemed to lack PACAP.

There is growing evidence that VIP-related peptides occur in addition to VIP in the mammal ian peripheral neuroendocrine system. Thus, helodermin-like immuno- reactivity has been demonstrated in thyroid C-cells, in noradrenaline-containing cells of the adrenal medulla and in endocrine cells of the airways in a number of species (Sundler et al. 1988b; Bjartell et al. 1989; Luts et al. 1990). Recently, PACAP, another VIP-related pep- tide, has been isolated f rom sheep hypothalami, se- quenced (Miyata et al. 1989, 1990) and found to occur in neuronal elements in the brain (K6ves et al. 1990). Preliminary immunochemical and immunocytochemical observations indicate the presence of PACAP immuno- reactivity in neuronal elements in several peripheral or- gans, including the gut. Furthermore, a high density of specific PACAP binding sites has been observed in lung membranes of the rat (Gottschall et al. 1990). In the present study, PACAP-containing fibers are seen in the airways of all animals examined. In the nose, the fibers are moderate in number; they are more frequent in the trachea and bronchi. Generally, the fibers occur close to small seromucous glands, around small blood vessels within the smooth muscle and beneath the epithelium. Thus, the distribution of PACAP-containing fibers differs to some extent f rom that of VIP-containing fibers. This is exemplified by the comparat ively rich sup-

ply of VIP-containing fibers in the upper respiratory tract and the more limited innervation by PACAP-con- taining fibers (Uddman et al. 1978, 1985; Christofides et al. 1984). Double immunostaining reveals that, in the sheep and ferret, the majori ty of the PACAP- immunore- active fibers also store VIP. Like VIP, PACAP may in- duce vasodepression when injected in anesthetized rats. PACAP shows a pituitary adenylate cyclase-stimulating activity 1000 times more potent than VIP in hypotha- lamic extracts (Miyata et al. 1989). In view of the abun- dance of PACAP binding sites in the airways (Gottschall et al. 1990), the present data provide a morphological basis for a functional role of PACAP in the neuronal regulation of the airways.

Acknowledgement. This work was supported by grants from the Swedish Medical Research Council (no. 04X-4499 and 17X-6859).

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