Cell Tissue Res (2001) 305:341–349 DOI 10.1007/s004410100415
REGULAR ARTICLE
N. Mirabella · C. Squillacioti · G. Germano E. Varricchio · G. Paino
Pituitary adenylate cyclase activating peptide (PACAP) immunoreactivity in the ureter of the duck Received: 21 July 2000 / Accepted: 20 April 2001 / Published online: 20 June 2001 © Springer-Verlag 2001
Abstract The presence, distribution and colocalisation of pituitary adenylate cyclase activating peptide (PACAP) immunoreactivity have been studied in the duck ureter by using Western blot analysis, radioimmunoassays (RIA) and immunohistochemistry. The presence of both PACAP-38 and PACAP-27 was demonstrated, PACAP-38 being the predominant form. PACAPimmunoreactive fibres and neurons were found in all the ureteral layers. Double immunostaining showed that PACAP was almost completely colocalised with vasoactive intestinal peptide (VIP). Moreover, PACAP was found in substance P (SP)-containing ureteral nerve fibres and in SP-containing dorsal root ganglion neurons. RIA performed on denervated ureters demonstrated that almost half of the ureteral PACAP was extrinsic in origin. These findings suggest that, in birds, PACAP has a role in diverse nerve-mediated ureteral functions. Keywords Radioimmunoassay · Urinary tract · Vasoactive intestinal peptide (VIP) · Campbell khaki ducks, Anas platyrhynchos (Aves)
Introduction Pituitary adenylate cyclase activating peptide (PACAP) is a peptide of the glucagon/vasoactive intestinal peptide (VIP)/secretin family of peptides. It was originally isolated from the ovine hypothalamus and found to exist as two forms with 27 amino acid (PACAP-27) and 38 amino acid (PACAP-38) residues (Miyata et al. 1989, 1990), both derived from a 176-amino-acid precursor (Kimura et al. 1990). The amino acid sequence of PACAP is highN. Mirabella (✉) · C. Squillacioti · G. Germano · G. Paino Department of Biological Structures, Functions and Technology, University of Naples “Federico II”, Via Veterinaria 1, 80137 Naples, Italy e-mail:
[email protected] E. Varricchio Department of Animal Production Sciences, University of Basilicata, Potenza, Italia
ly conserved throughout phylogeny (Ogi et al. 1990; Chartrel et al. 1991; Yasuhara et al. 1992; Parker et al. 1993; Matsuda et al. 1997). PACAP immunoreactivity has been observed in the brain (Arimura et al. 1991; Arimura 1992; Köves et al. 1991; Vigh et al. 1991; Yon et al. 1992; Ghatei et al. 1993; Masuo et al. 1993; Hannibal et al. 1995a, 1997) and in autonomic neurons and nerve fibres supplying several organs and tissues, including the respiratory system (Cardell et al. 1991; Uddman et al. 1991b; Hauser-Kronberger et al. 1996), the digestive system (Uddman et al. 1991a; Sundler et al. 1992; Köves et al. 1993; Ny et al. 1994, 1995; McConalogue et al. 1995; Portbury et al. 1995; Hannibal et al. 1998), genital organs (Shioda et al. 1994; Fahrenkrug and Hannibal 1996; Steenstrup et al. 1996) and ocular tissue (Wang et al. 1995). Furthermore, it has been found in capsaicin-sensitive sensory neurons of the dorsal root ganglia and trigeminal ganglion (Moller et al. 1993; Zhang et al. 1993, 1996; Mulder et al. 1994). A considerable amount of evidence suggests that PACAP has a role as a ubiquitous chemical messenger with a vast range of biological effects (Läuffer et al. 1999). It has been found in high concentration in the hypothalamus (Arimura et al. 1991; Ghatei et al. 1993; Masuo et al. 1993; Hannibal et al. 1995a) and is considered to be a hypothalamic-pituitary regulatory peptide (Köves et al. 1990; Arimura et al. 1991; Kivipelto et al. 1992; Hannibal et al. 1995a, 1995b; Nussdorfer and Malendowicz 1998). Both PACAPs exert their biological activity by binding to three different types of receptors (Harmar and Lutz 1994): the PAC1 receptor and the VPAC1 and VPAC2 receptors. The PAC1 receptor binds to PACAP with a much higher affinity than to VIP, whereas the VPAC1 and VPAC2 receptors bind to PACAP and VIP with similar affinities. In the mammalian urinary tract, PACAP has been demonstrated immunohistochemically in the nerve fibres innervating blood vessels and smooth muscle and in those supplying the subepithelial plexus. Moreover, it
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has been found to be almost completely colocalised with calcitonin gene-related peptide (CGRP) in capsaicin-sensitive nerves and, hence, is considered to be a sensory neurotransmitter (Fahrenkrug and Hannibal 1998). In birds, PACAP immunoreactivity has been observed in the digestive system, where it has been found to coexist with substance P (SP) and VIP (Sundler et al. 1992), and in the brain (Peeters et al. 1998). Moreover, PAC1 receptors have been found to be expressed in the chicken brain, gonads, pituitary and adrenal glands, intestine, pancreas, lung, heart and kidney, thus suggesting that PACAP affects a variety of functions both in the avian brain and peripheral tissues (Peeters et al. 1999). No data are presently available concerning the presence, distribution and colocalisation of PACAP in bird urinary organs. It should be remembered that the avian ureter, compared with that in mammals, comprises the entire urinary tract and plays an important role not only in the moving, but also in the physio-chemical modification and emission of urine. In addition, it exhibits a dense intrinsic and extrinsic innervation, suggesting a role of ureteric neurons in controlling ureteric motility, blood flow, the secretory mechanisms of the epithelium and the opening and/or closing of the papilla ureteralis (Sann et al. 1997; Mirabella et al. 1999, 2000). In the peripheral nervous system, PACAP may act as a neurotransmitter or neuromodulator and have a role in the regulation of smooth muscle motility and epithelial secretion. Thus, detailed information regarding the distribution, coexistence with other neurotransmitters and origin of ureteral PACAP is essential for understanding the part played by this neuropeptide in the neuronal control of ureteric function in birds. In the present study, we have therefore investigated the presence of PACAP in the duck ureter by means of Western blot analysis and radioimmunoassay (RIA). Immunohistochemistry has been performed to examine the distribution of immunoreactive PACAP and its possible colocalisation with the sensory neuropeptides CGRP and SP and with the related peptide VIP. Furthermore, the origin and possible sources of PACAP-immunoreactive nerve fibres have also been investigated.
Materials and methods Twenty-five adult Campbell khaki ducks (Anas platyrhynchos) of both sexes were used. Tissue extraction Fresh segments of ureter were collected, immediately frozen on dry ice and stored at –80°C until extraction. The tissue extraction procedure was performed according to Hannibal et al. (1995a). Briefly, frozen segments of ureter were weighed on dry ice and boiled in 10 volumes of demineralised water for 25 min followed by homogenisation in an Ultra-turrax homogeniser and centrifugation at 1000 g for 10 min. The supernatant was decanted. The sediment was homogenised with 5 volumes of 0.5 M acetic acid and mixed with the boiling water supernatant. This mixture was centrifuged and the supernatant was decanted and freeze-dried.
Electrophoresis and Western blot analysis Aliquots of reconstituted ureter extracts and synthetic peptide PACAP-38 (code 8920; Peninsula Laboratories) were subjected to electrophoresis on 18% sodium dodecyl sulphate (SDS)-polyacrylamide (Bio-Rad, Calif.) gel. After electrophoresis, gel was transferred to nitrocellulose by using a semidry apparatus (Bio-Rad) according to the manufacturer’s instructions. The membrane was incubated for 1 h at 42°C in 5% bovine serum albumin (BSA; Sigma, St Louis, Mo., USA) in TBST (150 mM NaCl, 20 mM TRIS-HCl pH 7.4, 0.3% Tween-20), washed with TBST and incubated 2 h at room temperature in rabbit antiserum specific for PACAP-38 (code IHC 8920; Peninsula Laboratories) diluted 1:2000 in TBST containing 1% BSA. The membrane was washed three times with TBST, incubated 1 h with anti-rabbit IgG peroxidase conjugate (code A 9169; Sigma) diluted 1:3000 in TBST-1% BSA and washed three times with TBST. Proteins were visualised by an enhanced chemiluminescence kit (Amersham, UK). Marker proteins (coloured protein molecular weight markers; Rainbow, Amersham) were used to estimate the molecular weight of each band. Immunohistochemistry and colocalisation studies The ducks were anaesthetised by intramuscular injection of Ketamine (25 mg/kg). They were then sacrificed by exanguination. Both the pars renalis and the pars pelvica (King 1993) of each ureter were collected. The synsacral section of the spinal cord, synsacral dorsal root and synsacral sympathetic chain ganglia were also collected. The specimens were fixed in 4% paraformaldehyde in a 0.1 M phosphate-buffered saline (PBS) solution at pH 7.5 for 2 h. Successively, they were placed in PBS containing 0.1% sodium azide and 10% sucrose and stored overnight at 4°C. The following day, the samples were transferred to a mixture of PBS-sucrose-azide and OCT compound (Tissue Tek, Elkhart, Ind., USA) in a ratio of 1:1 for 24 h before being embedded in 100% OCT. Coronal and sagittal sections were cut (10–50 µm thick) and then immunostained according to the fluorescent technique. They were preincubated in 10% normal goat serum (NGS) (i200/001, UCB) in PBS containing 0.1% Triton X-100 and 0.1% sodium azide overnight at 4°C and rinsed (3×10 min) in 0.1% Triton X-100, 0.1% sodium azide in PBS prior to the exposure to primary antiserum. Antisera against PACAP-27 (rabbit, IHC 8922), PACAP-38 (rabbit, IHC 8920), SP (guinea-pig, GHC 7451), CGRP (guinea-pig, GHC 6009) and VIP (guinea-pig, GHC 7161), all purchased from Peninsula Laboratories, were used for singleand double-labelling techniques. For single-labelling, a working dilution of 1:200 was used for each primary antiserum. After an overnight incubation at 4°C, excess serum was washed off with 3×10 min changes in 0.1% Triton X-100, 0.1% sodium azide in PBS. The sections were then incubated for 2 h at 4°C in goat antiguinea-pig (TRITC: AP 193 R; FITC: AP 193 F; Chemicon International) or in goat anti-rabbit (TRITC: AP 187 R; FITC: AP 187 F; Chemicon international) antibodies (TRITC: tetramethylrhodamine isothiocynate; FITC: fluorescein isothiocyanate), both diluted 1:100, according to the antiserum that had been used as primary antibody. After being washed (3×10 min) in 0.1% Triton X-100, 0.1% sodium azide in PBS, the sections were mounted in 1:1 PBS-glycerol, examined and photographed. The specificity of the immunoreactions was tested by omitting, alternatively, the primary or the secondary antibody and using buffer instead. No immunoreaction was detected in control tests. In addition, the specificity of the PACAP-27, PACAP-38 and VIP antisera and their cross-reactivity were tested by absorption of each primary antisera either with excess homologous (up to 100 µg/ml antiserum as the final dilution) or with excess heterologous (up to 200 µg/ml antiserum as the final dilution) antigens (VIP 7161; PACAP-27 8922 and PACAP-38 8920; Peninsula Laboratories). No cross-reaction was found between the PACAP and VIP antibodies; however, PACAP-27 and PACAP-38 antibodies were found to cross-react.
343 For double-labelling the sections were incubated in a 1:1 mixture of PACAP/VIP, PACAP/SP or PACAP/CGRP antisera. Each antiserum was diluted 1:100. After being washed (3×10 min) in 0.1% Triton X-100, 0.1% sodium azide in PBS, the sections were incubated in a 1:1 mixture of secondary antibodies that had each an initial dilution of 1:50. The secondary antibodies and the incubation times were the same of those used for single-labelling. To ensure that no cross-reactivity of secondary detection system occurred, the primary antisera were alternatively omitted in control tests. No cross-reactivity was observed. The specimens were observed by using a Leitz microscope equipped with epifluorescence and the appropriate filter sets for viewing FITC and TRITC. Some of the specimens were examined by using a confocal laser scanning microscope (CLSM 3, Zeiss): FITC was visualised in the green channel by using an Ar+ 488 nm laser and an LP 520 filter; TRITC was visualised in the red channel by using an HeNe 543 nm laser and the same LP 520 filter. In order to improve the visualisation of any colocalisation, colour confocal laser scanning micrographs were used. These micrographs were obtained by superimposing separate images that were alternatively taken in the green and the red channels from a single microscopic field. Surgical denervation The ducks were anaesthetised by an intramuscular injection of Ketamine (25 mg/kg), intubated with an orotracheal tube and administered a mixture of oxygen (1 l/min) and 2%–3% isoflurane. After the peritoneal cavity was incised, the pelvic segment of the left ureter was exposed. Denervation was performed by carefully stripping off the tunica adventitia from the underlying tunica muscularis. The right ureter of each operated duck was left intact as a control. The ducks were allowed to survive for 48 h, at which time they were sacrificed and their ureters were collected. After collection, all specimens were processed according to the described single-labelling immunohistochemical procedures for the detection of PACAP immunoreactivity. Radioimmunoassays Five normal and five denervated animals were used. The extracted samples were reconstituted and analysed by RIA with kits specific for PACAP-38 (rabbit, RIK 8920) and PACAP-27 (guinea-pig, RIK 8922G), both purchased from Peninsula Laboratories. These kits were constituted by specific antisera for PACAP-38 and PACAP-27 and labeled 125I-peptides. Synthetic PACAP-38 and PACAP-27 (Peninsula Laboratories) were used as standards. According to the data provided by the manufacturer, the PACAP-27 antiserum shows 11% of cross-reactivity with PACAP-38. All tissue extracts were assayed in duplicate in at least two different dilutions. The detection limits of the PACAP-38 and PACAP-27 assays were 2.2 pmol/l and 3.2 pmol/l, respectively. The working ranges of the PACAP-38 and PACAP-27 assays were 2.2–282 pmol/l and 3.2–407 pmol/l respectively. In the denervated specimens, only PACAP-38 was determined.
Results Western blot analysis The results of the Western blot analysis are shown in Fig. 1. The ureter tissue extracts and the synthetic peptide reacted with the anti-PACAP-38 antibody. The extracts formed a well-defined single band with an electrophoretic mobility similar to that of the synthetic PACAP-38.
Fig. 1 Detection of PACAP-38 immunoreactivity by Western blot analysis. Lane A Tissue extract of duck ureter, lane B synthetic PACAP-38. Protein markers are expressed in kDa
Immunohistochemistry PACAP immunoreactivity was widely distributed in all the ureteral layers. PACAP-positive neurons were primarily located in the muscular plexus (Fig. 2a, c). To a lesser extent, they were also located in the lamina propria, the adventitial ganglia and along the route of the adventitial nerves (Fig. 2a, d). A large number of positive varicose nerve fibres were found around adventitial (Fig. 2a) and intramural blood vessels, within the adventitial and muscular nerve fibre bundles and in the mucosal lamina propria where they formed a very dense network in the subepithelial zone (Fig. 2a, b). In the adventitial ganglia, PACAP-positive varicose nerve fibres were observed to form pericellular baskets around negative cells. PACAP immunoreactivity was also observed in neurons of the synsacral dorsal root (Fig. 2e) and synsacral sympathetic chain ganglia (Fig. 2f) and in fibres reaching the dorso-lateral side of the dorsal horn in the synsacral section of the spinal cord (see below). Double immunostaining disclosed that PACAP and VIP were almost completely colocalised in ureteral neurons and nerve fibres (Fig. 3h–n). Moreover, PACAP immunoreactivity was found in SP-positive neurons of the synsacral dorsal root ganglia (Fig. 4a, b) and in SP-positive fibres in the ureteral wall (Fig. 3a–g) and in the dorso-lateral side of the dorsal horn of the synsacral section of the spinal cord (as mentioned above; Fig. 4c, f). Compared with the controls, marked reductions of PACAP-positive nerve fibres were observed in the muscular and mucosal layers of the denervated ureters (Fig. 5a, b).
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Fig. 3 PACAP/SP (a–g) and PACAP/VIP (h–n) colocalisation studies. Light microscopy (a–d, h–k) and confocal laser scanning microscopy (e–g, l–n). PACAP (pacap) and SP (sp) were colocalised in nerve fibres located underneath the epithelium (a–d) and in nerve fibres contacting a PACAP-positive muscular neuron (e–g). PACAP and VIP (vip) were almost completely colocalised in nerve fibres located underneath the epithelium (h–k) and in neu-
rons and nerve fibres of the ureteral muscular plexus (h–i, l–n). In g, PACAP was visualised in the red channel and SP in the green channel. In n, PACAP was visualised in the green channel and VIP in the red channel. In both of the colour micrographs, the colocalised structures appear yellow (ep epithelium, ml muscular layer, arrows nerve fibres, asterisks PACAP-positive/SP-negative neuron, arrowheads PACAP/VIP-positive neurons). Bar 25 µm
Fig. 2a–f Distribution of PACAP immunoreactivity (ep epithelium, arrows PACAP-positive neurons, arrowhead PACAP-positive nerve fibres around an adventitial blood vessel). Light microscopy (a, b, d) and confocal laser scanning microscopy (c). PACAP-positive nerve fibres and nerve cells were distributed in all ureteral layers (a–d). PACAP-positive nerve fibres formed a dense plexus
underneath the epithelium (a, b). PACAP-positive neurons were located in the muscular (a, c) and adventitial (a, d) plexus, in the dorsal dorsal root ganglia along the synsacral section of the spinal cord (e) and in the synsacral sympathetic chain ganglia (f). The position of d is marked in a. Bar 25 µm
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Fig. 4a–f Synsacral section of the spinal cord. PACAP (pacap) and SP (sp) were coexpressed in dorsal root ganglion cells (a, b) and in nerve fibres (c–f) reaching the dorso-lateral side of the dorsal horn (dh dorsal horn, arrows PACAP/SP-positive nerve fibres). Bar 50 µm
Fig. 5 Denervated (a) and control (b) ureters. PACAP-immunoreactive muscular (arrows) and subepithelial (arrowheads) nerve fibres are clearly reduced after denervation (ep epithelium, ml muscular layer). Bar 50 µm
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Radioimmunoassays In the normal ureters, the concentrations (means ± SEM) of PACAP-27 and PACAP-38 were 0.25±0.01 and 2.52±0.16 pmol/g. of wet weight, respectively. In the denervated ureters, the concentration (mean ± SEM) of PACAP-38 was 1.22±0.05 pmol/g.
Discussion The results of the present study are consistent with the presence of both PACAP molecular forms in the duck ureter. Antisera raised against the mammalian sequence of PACAP were used. These antisera were raised against either the N-terminal 27-amino-acid sequence or the whole PACAP sequence; both were capable of reacting in tissue sections and with tissue extracts of the duck ureter. Among avian species, the PACAP amino acid sequence has been determined only in the chicken (Yasuhara et al. 1992). This sequence is similar to that of mammalian species; however, it differs in one amino acid close to the N-terminal part and in a series of six amino acids close to the C-terminal part. Western blot analysis has shown that the anti-mammalian PACAP-38 antiserum used in the present study recognises a PACAP-like peptide in the ureter of the duck. In birds, the presence of PACAP has been immunohistochemically detected in the chicken brain (Peeters et al. 1998) and gastrointestinal system (Sundler et al. 1992). However, no data have previously been available regarding the quantity of PACAP immunoreactivity in avian tissues. In the ureter of the duck, PACAP is expressed at a lower level than in the rat urinary tract (Fahrenkrug and Hannibal 1998). A comparison of the different segments of the urinary tract shows however that the quantity of PACAP-38 immunoreactivity in the duck ureter (2.52±0.16 pmol/g wet wt.) is similar to that reported in the rat urinary bladder (2.9±0.2 pmol/g wet wt.) by Fahrenkrug and Hannibal (1998). As reported in several mammalian and non-mammalian tissues (Arimura et al. 1991; Hannibal et al. 1998; Montero et al. 1998; Mikkelsen et al. 1995; Fahrenkrug and Hannibal 1996), PACAP-38 is also the dominant form in the duck ureter. RIA and immunohistochemistry performed in normal and denervated ureters have shown that almost a half of the ureteral PACAP is extrinsic in origin. At least some of the extrinsic PACAP-positive ureteral nerve fibres may represent primary sensory afferents. This hypothesis is supported by the presence of PACAP immunoreactivity in SP-positive ureteral fibres, in SP-positive dorsal root ganglion neurons, and in SP-positive dorsal horn fibres. PACAP immunoreactivity has also been observed in capsaicin-sensitive, SP-positive and CGRP-positive nerve fibres supplying the rat urinary tract (Fahrenkrug and Hannibal 1998) and there is a strong evidence that PACAP functions as a sensory neuropeptide in the rat peripheral nervous system (Moller et al. 1993).
The presence of PACAP-immunoreactive neurons in the synsacral sympathetic chain ganglia suggests that these ganglia may represent another source of PACAPpositive fibres extrinsic to the avian ureter. This hypothesis is supported by the finding that, in birds, the ureteral innervation is supplied by branches that originate from the pudendal (pelvic) nerve, which, in turn, receives fibres from the nearby sympathetic chain ganglia (Dubbeldam 1993). In addition, PACAP is also expressed in several rat sympathetic ganglia (Nielsen et al. 1998). However, PACAP immunoreactivity has not been detected in any of the sympathetic ganglia supplying the guinea-pig gastrointestinal tract (Portbury et al. 1995). Our colocalisation studies have disclosed that PACAP and VIP are almost completely coexpressed in ureteral neurons and nerve fibres. This is quite different from the rat urinary tract in which PACAP has been colocalised only in a small number of VIP-positive fibres (Fahrenkrug and Hannibal 1998). In a preceeding study (Mirabella et al. 2000), VIP has been demonstrated to be largely colocalised with nitric oxide synthase (NOS) in cranio-caudally projecting fibres intrinsic to the duck ureter. As a consequence, a population of PACAP/ VIP/NOS-positive neurons intrinsic to the ureter does indeed seem to exist in the duck. These neurons may serve as inhibitory motor neurons in the regulation of ureteral motility. This is supported by the finding that PACAP is a potent relaxant of vascular and non-vascular smooth muscle (Katsolius et al. 1993; McConalogue et al. 1995; Naruse et al. 1996; Ozawa et al. 1997; Yoshida et al. 2000) and, hence, it may play a complementary role to VIP and NO in mediating inhibitory non-adrenergic noncholinergic neurotransmission in the avian ureter. We have found numerous PACAP-positive varicose nerve fibres in the subepithelial zone of the lamina propria. PACAP-positive fibres have also been observed in the subepithelial zone of the rat urinary tract (Fahrenkrug and Hannibal 1998). In mammals, the rich subepithelial nerve plexus in the urinary tract is generally considered to have a sensory function (Karahan et al. 1993; Lincoln and Burnstock 1993; Wakabayashi et al. 1993; Jen et al 1995). As discussed above, some of the PACAP-positive subepithelial fibres may play a similar role in avian ureter. However, since the ureteral epithelium is highly mucosecretive and, hence, has a role in preventing excessive precipitation of uric acid in colloidal suspension (Johnson 1979), it can be hypothesised that the PACAP-positive subepithelial fibres also play a role in the regulation of mucosecretion. The observations that PACAP has important secretomotor properties (Cox 1992; Fuchs et al. 1996; Takeuchi et al. 1997) support this hypothesis. In conclusion, PACAP immunoreactivity is wide distributed in the ureter of the duck. Compared with the mammalian urinary tract, in which PACAP is considered to play a primary role in sensory neurotransmission, this peptide seems to affect further nerve-mediated activities, such as mucosecretion and smooth muscle motility, in the avian urinary tract.
348 Acknowledgements The authors thank Mr. A. Calamo and Ms. S. Alì for their careful technical assistance.
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