I. Introduction

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The secretory activity of the adenohypophysis is regulated by aminergic (mainly dopaminergic) and peptidergic hypothalamic neurons (Elde and Hökfelt, 1979; Stumpf and Jennes, 1984; Ju et al., 1991). Five neuropeptides have been isolated from ovine and porcine hypothalamic extracts, or from a human pancreatic tumor, and characterized by the groups of Roger Guillemin, Andrew Schally, and Willy Vale, based on their ability to either stimulate or inhibit the secretion of anterior pituitary hormones: thyrotropin-releasing hormone (TRH; Boler et al., 1969; Burgus et al., 1969), gonadotropin-releasing hormone (GnRH; Amoss et al., 1971; Matsuo et al., 1971), somatostatin (Brazeau et al., 1973; Esch et al., 1980; Böhlen et al., 1981), corticotropin-releasing factor (CRF; Vale et al., 1981), and growth hormone-releasing factor (GRF; Guillemin et al., 1982; Rivier et al., 1982b). All of these hypophysiotropic neurohormones are synthesized in hypothalamic neurons, whose axons project toward the median eminence, and are transported to the anterior pituitary by the capillaries of the portal system. Another common feature of these hypothalamic neurohormones is that they are generally widely distributed in the central nervous system (CNS) and in peripheral organs, and that they exert a large array of biological activities in addition to their hypophysiotropic actions. After the primary structure of GRF had been determined in 1982, it was commonly thought that all major hypophysiotropic neurohormones had been identified. However, the subsequent characterization of other neuropeptides capable of regulating the activity of anterior pituitary cells, such as pituitary adenylate cyclase-activating polypeptide (PACAP; Miyata et al., 1989) and prolactin (PRL)-releasing peptide (Hinuma et al., 1998), has shown that this view was incorrect.

PACAP has been originally isolated from an extract of ovine hypothalamus on the basis of its ability to stimulate cAMP formation in rat pituitary cells (Miyata et al., 1989). Hypothalamic neurons containing PACAP project toward the median eminence and terminate in the vicinity of the capillary loops of the hypothalamo-pituitary portal system. Like other hypophysiotropic neurohormones, PACAP is contained in extra-hypothalamic neurons as well as in numerous peripheral tissues. Consistent with its widespread distribution, PACAP has been found to exert pleiotropic effects including modulation of neurotransmitter release, vasodilation, bronchodilation, activation of intestinal motility, increase of insulin and histamine secretion, as well as stimulation of cell multiplication and/or differentiation.

	II. PACAP

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To isolate novel hypophysiotropic neuropeptides, the group of Arimura has screened fractions from an extract of 4300 ovine hypothalamus by monitoring their stimulatory effect on adenylyl cyclase activity in cultured rat anterior pituitary cells. Using this approach, they have isolated in pure form a peptide, found to markedly increase cAMP formation, that they termed pituitary adenylate cyclase-activating polypeptide. Characterization of the peptide revealed that it comprises 38 amino acid residues and is C-terminally -amidated (Miyata et al., 1989). Two years later, the primary structure of this 38-amino acid form of PACAP (PACAP38) was determined in the European green frog Rana ridibunda, a species that diverged from the line leading to mammals some 280 million years ago (Chartrel et al., 1991; Hoyle, 1998). Frog PACAP38 appears to contain only one amino acid substitution (Val³⁵  Ile), which may be accomplished by the exchange of a single nucleotide in the cDNA sequence (Chartrel et al., 1991). The sequence of PACAP38 comprises an internal cleavage-amidation site (Gly²⁸-Lys²⁹-Arg³⁰), suggesting that the PACAP precursor can generate a 27-residue -amidated polypeptide (PACAP27). Consistent with this hypothesis, Miyata et al. (1990) have isolated from the ovine hypothalamus another fraction capable of stimulating adenylyl cyclase activity in adenohypophysial cells that, on characterization, happened to correspond to the N-terminal 27-amino acid sequence of PACAP38. Thus it appears that the structure of the biologically active region of PACAP, corresponding to the PACAP27 sequence, has been totally preserved during evolution, from amphibians to mammals. The sequence of PACAP27 shows 68% identity with vasoactive intestinal polypeptide (VIP), identifying PACAP as a member of the VIP-glucagon-GRF-secretin superfamily of structurally related peptides (Fig. 1; Campbell and Scanes, 1992; Segre and Goldring, 1993).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Amino acid sequences of the different members of the PACAP-VIP-GRF-glucagon superfamily in human. -, amino acids identical with those of PACAP38. Adapted from Kieffer and Habener, 1999.

Conformational analysis of PACAP27 by two-dimensional NMR and circular dichroism spectroscopy has shown an initial disordered N-terminus sequence of eight amino acid residues followed by a region, from amino acid residues 9 to 24, that consists of four distinct domains (Inooka et al., 1992). The first domain, encompassing residues 9 to 12, forms a -turn-like conformation whereas the three others are composed of distinct helical regions that extend from residues 12 to 14, 15 to 20, and 22 to 24, respectively. The conformation of PACAP38 mirrors that of PACAP27 in its N-terminal region whereas the C-terminal segment exhibits a short helix attached by a flexible hinge to the 1-27 region (Wray et al., 1993). The biological importance of the three structural domains of PACAP38 has been investigated using truncated PACAP analogs (see Section III, D).

The three-dimensional structure of PACAP exhibits substantial similarities with those of other members of the VIP/glucagon family (Braun et al., 1983; Gronenborn et al., 1987; Wray et al., 1993). In particular, both PACAP27 and VIP possess two helices separated by a disordered region, but the position of the first -helix of PACAP27 is shifted by two residues toward the C-terminus, and the conformation of the second helix of PACAP27 is closer to an -helix than that of VIP. These minor conformational differences between PACAP27 and VIP may contribute to the selectivity of the peptides for their receptors (Inooka et al., 1992).

The cDNA encoding the PACAP precursor has been characterized in several vertebrate species (Ogi et al., 1990; Ohkubo et al., 1992; Arimura and Shioda, 1995; Okazaki et al., 1995) and in a protochordate, the ascidian Chelyosoma productum (McRory and Sherwood, 1997). In humans, the cDNA encodes a 176-amino acid prepro-protein, which comprises a 24-amino acid signal peptide (Hosoya et al., 1992). In all mammalian species studied so far, the sequence of PACAP38 is located in the C-terminal domain of the precursor (Fig. 2). The cDNA sequences of humans (Ohkubo et al., 1992), sheep (Kimura et al., 1990), rat (Ogi et al., 1990), and mouse prepro-PACAP (Okazaki et al., 1995) have revealed the existence of a 29-amino acid peptide delimited by basic residues at its N- and C-terminal extremities, located upstream of PACAP38 (Fig. 2). This peptide, which exhibits moderate structural homology with PACAP27, has been termed PACAP-related peptide (PRP) (Ogi et al., 1990; Wray et al., 1995; Hoyle, 1998). In mammals, the overall organization of the PACAP precursor exhibits strong similarities with that of the VIP precursor (Fig. 2). In particular, the VIP precursor encompasses a VIP-related peptide, called peptide histidine-methionine (PHM) amide in humans (Itoh et al., 1983; Bodner et al., 1985; Christophe et al., 1989) or peptide histidine-isoleucine (PHI) amide in sheep (Bounjoua et al., 1991), rat (Nishizawa et al., 1985), mouse (Lamperti et al., 1991), and chicken (McFarlin et al., 1995), which possesses moderate amino acid identity with VIP. The degree of similarity between PACAP27 and PRP (22%) or VIP and PHI (37%) is less than that between PACAP and VIP (68%) or PRP and PHI (44%), respectively. Thus it is assumed that intragenomic duplication of a VIP/PACAP ancestor sequence has occurred before duplication of the whole ancestor gene (Ohkubo et al., 1992). A proposed model describing the evolutionary process leading to the generation of distinct precursors for PACAP, VIP, glucagon, GRF, and secretin in mammals is presented in Fig. 3. In submammalian vertebrates and the tunicate Chelyosoma productum, the PACAP precursor comprises both GRF and PACAP (Fig. 3) (Parker et al., 1993; McRory et al., 1995, 1997; McRory and Sherwood, 1997; Alexandre et al., 2000) (See section II, I).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Schematic representation of the human PRP/PACAP and PHM/VIP precursors. The general organization of the two precursors is presented and the sequences of PRP and PHM as well as PACAP and VIP have been aligned. The conserved amino acids are indicated in black and the percentage of amino acid identity between PRP and PHM as well as PACAP27 and VIP are indicated. SP, signal peptide.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Hypothetical schemes depicting the evolutionary history of the PACAP/VIP/glucagon/GRF/secretin gene family. Adapted from Ohkubo et al., 1992.

In mammals, the primary structure of the PACAP precursor reveals the existence of seven mono- or dibasic residues that can be cleaved by various prohormone convertases (PCs) including PC1, PC2, PC4, PC5, PC7, furine, and PACE4 (Seidah et al., 1994, 1998). In the rat, cleavage at three dibasic sites, i.e., Arg⁷⁹-Arg⁸⁰, Lys¹²⁹-Arg¹³⁰, and Arg¹⁷⁰-Arg¹⁷¹, generates a large intermediate precursor of PRP (big PRP) and a glycine-extended form of PACAP38 (Fig. 4). Cleavage at the single Arg¹¹⁰, followed by hydrolysis of this C-terminal Arg residue by carboxypeptidases E, H, or M, generates PRP (Rouillé et al., 1995). The Gly¹⁶⁹ residue is used by peptidyl glycine -amidating monooxygenase (Eipper et al., 1992a,b) for the amidation of the Lys¹⁶⁸ residue at the C-terminal extremity of PACAP38. Finally, the tripeptide Gly¹⁵⁸-Lys¹⁵⁹-Arg¹⁶⁰ can be cleaved to generate the -amidated PACAP27 isoform (Fig. 4). Processing of the PACAP precursor has been studied in Chinese hamster ovary (CHO)-K1 cells transfected with the human PACAP cDNA (Okazaki et al., 1992). Characterization of the various peptides secreted in the incubation medium by HPLC combined with radioimmunoassay (RIA) detection has confirmed that processing of the PACAP precursor actually yields to the formation of PACAP38, PACAP27, and PRP (Okazaki et al., 1992).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Schematic representation of the post-translational processing of the rat PACAP precursor. The nature and allocation of each cleavage and amidation site is specified. PAM, peptidyl glycine -amidating monooxygenase. SP, signal peptide.

In the rat hypothalamus, PC1 and/or PC2 are intensively expressed in nuclei enriched with PACAP-immunoreactive neurons, supporting the hypothesis that these two endopeptidases could be involved in the processing of the PACAP precursor (Köves et al., 1994a; Zheng et al., 1994; Dong et al., 1997). Cotransfection experiments in GH4C1 cells have confirmed that both PC1 and PC2 can actually process the rat PACAP precursor to generate mature PACAP38 and PACAP27 (Li et al., 1999). In the testis, where PACAP is particularly abundant, PC4 can process the PACAP precursor to generate both PACAP38 and PACAP27 (Li et al., 1998).

The gene encoding PACAP has been cloned in humans (Hosoya et al., 1992) and mouse (Yamamoto et al., 1998). The overall architecture of the two genes is similar, with the exception of the 5'-untranslated region of the mouse gene, which encompasses two exons as a result of alternative splicing of the transcription initiation domain. The human PACAP gene is composed of five exons, the sequence of PRP being encoded by exon 4 and that of PACAP by exon 5 (Fig. 5). Northern blot analysis has revealed the presence of a 3-kb PACAP mRNA in the rat hypothalamus (Hosoya et al., 1993; Hannibal et al., 1995a). A shorter transcript with a truncated 5'-untranslated region has been characterized in the rat testis (Hurley et al., 1995). Similarly, shorter PACAP mRNA has been found in the mouse, bovine, and human testis (Hurley et al., 1995). It has also been reported that another short PACAP transcript is produced in sympathetic neurons (Harakall et al., 1998).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Organization of the human PACAP gene and PACAP mRNA. The five exons are boxed and numbered. The untranslated regions of exons 1 and 5 are denoted by dashed lines. Exon domains encoding PRP and PACAP are hatched. Arrows indicate the locations of binding sites for potential transcriptional factors. CRE, cAMP response element; Inr-like, initiator-like element; P1, promotor region 1; P2, promotor region 2; TRE, 12-O-tetradecanoylphorbol 13-acetate response element. GHF-1, growth hormone factor 1.

The promoter sequence of the PACAP gene (about 400 bp) comprises two regions, termed P1 and P2 (Fig. 5), which correspond, respectively, to an initiator-like sequence and a CT-rich domain with GC boxes (Jankowski and Dixon, 1987; Ohkubo et al., 1994). Surprisingly, the promoter region of the human PACAP gene does not contain any apparent TATA or CAAT box, which are normally required for accurate initiation of transcription (Hampsey, 1998). In contrast, the PACAP promoter possesses two cAMP-response-like elements, a 12-O-tetradecanoylphorbol 13-acetate response element and a pair of sequences homologous to the consensus sequence for pituitary-specific factor growth hormone factor 1-binding sites, which are known to play a role in the tissue-specific expression of growth hormone (GH) (Bodner et al., 1988; Dolle et al., 1990; Castrillo et al., 1991). Investigation of the promoter activity has revealed that PACAP is constitutively expressed and that transcription of the PACAP gene can be enhanced by cAMP, 12-O-tetradecanoylphorbol 13-acetate, and even by PACAP itself (Suzuki et al., 1994a; Hashimoto et al., 2000).

The structural organization of the PACAP gene is similar to that of the VIP gene (Lamperti et al., 1991) and GRF gene (Mayo et al., 1985), confirming that all three genes originate from a common ancestral sequence through gene duplication (Fig. 3). In humans, the PACAP gene has been localized by Southern blotting and in situ hybridization to the P11 region of chromosome 18. This region is associated with holoprosencephaly, the most common hereditary developmental defect of the forebrain in humans, suggesting that PACAP might be involved in the control of brain development (Hosoya et al., 1992; Chang et al., 1993; Golden, 1998).

Soon after the characterization of PACAP, the distribution of the peptide was determined in the brain of mammals (Arimura et al., 1991; Köves et al., 1991; Vigh et al., 1991; Kivipelto et al., 1992; Ghatei et al., 1993) and amphibians (Yon et al., 1992). The distribution of PACAP-immunoreactive cells and fibers in the rat brain was schematically presented in a previous review (Gonzalez et al., 1998). In rat, RIA measurements have revealed that the highest concentrations of PACAP occur in the hypothalamic area (Arimura et al., 1991; Ghatei et al., 1993). Reversed-phase HPLC analysis showed that PACAP38 is by far the predominant form, PACAP27 representing less than 10% of the total peptide content in brain tissue (Arimura et al., 1991; Ghatei et al., 1993; Masuo et al., 1993; Hannibal et al., 1995a; Piggins et al., 1996).

The mapping of PACAP-expressing neurons has been investigated by in situ hybridization and immunocytochemistry (Table 1). In the rat hypothalamus, PACAP-immunoreactive neurons are primarily located in the parvo- and magnocellular neurons of paraventricular and supraoptic nuclei (Köves et al., 1991, 1994b; Kivipelto et al., 1992; Ando et al., 1994; Kimura et al., 1994; Hannibal et al., 1995a,b; Piggins et al., 1996). PACAP mRNA is expressed in the paraventricular and arcuate nuclei (Hannibal et al., 1995b; Murase et al., 1995). A dense accumulation of PACAP-immunoreactive fibers is found in the internal zone of the median eminence and in the vicinity of the capillaries of the hypothalamo-hypophysial portal system (Köves et al., 1990, 1991; Kivipelto et al., 1992; Tamada et al., 1994; Hannibal et al., 1995a,b; Mikkelsen et al., 1995). Quantification of PACAP by RIA has shown that the concentration of the peptide in the rat portal blood is significantly higher than in the peripheral blood, indicating that PACAP released by hypothalamic nerve terminals is actually transported to the pituitary (Dow et al., 1994). Regional distribution studies revealed that significant amounts of PACAP38 are also found in extrahypothalamic regions, including the substantia nigra, nucleus accumbens, septum, globus pallidus, cerebral piriform cortex, and pons (Ghatei et al., 1993; Masuo et al., 1993). In the limbic system, PACAP-like immunoreactive fibers are detected in the amygdaloid complex and in the mediodorsal and paraventricular nuclei of the thalamus (Köves et al., 1991; Masuo et al., 1993; Takahashi et al., 1994; Palkovits et al., 1995). In the lateral septum area, a dense network of immunoreactive fibers innervates blood vessels (Köves et al., 1991). In situ hybridization has revealed the presence of scattered PACAP-expressing cell bodies in the cingulate and frontal cortex (Mikkelsen et al., 1994). PACAP and its mRNA also have been detected in the cerebellum (Ghatei et al., 1993; Mikkelsen et al., 1994; Takahashi et al., 1994; Hannibal et al., 1995a; Nielsen et al., 1998a). Specifically, PACAP-like immunoreactivity (PACAP-LI) is localized in the soma and dendrites of Purkinje cells, whose axons directly contact granule cells (Nielsen et al., 1998a). In the medulla oblongata, the majority of perikarya exhibiting PACAP-LI are found in the commissural and medial subnuclei of the solitary nucleus, the dorsal motor vagal nucleus, the nucleus ambiguous, the ventrolateral medulla, the ventral medullary surface, and the caudal raphe nuclei, supporting the hypothesis that PACAP may act as a regulator of visceral functions (Legradi et al., 1994). In the spinal cord, PACAP mRNA is expressed in a subpopulation of sensory neurons of the dorsal root ganglia (Mulder et al., 1994), and numerous PACAP-immunoreactive fibers are found in the superficial layer of the dorsal horns (Moller et al., 1993; Dun et al., 1996a).

The location of PACAP-containing neurons also has been investigated in the CNS of nonmammalian vertebrates, including birds (Peeters et al., 1998), amphibians (Yon et al., 1992, 1993b), and fishes (Matsuda et al., 1997a,b; Montéro et al., 1998). Globally, the distribution of PACAP-immunoreactive cells exhibits a high degree of similarity with that of mammals. In particular, in the brain of the frog Rana ridibunda, prominent groups of PACAP-containing neurons are located in the hypothalamus, i.e., in the anterior preoptic area, the ventral magnocellular preoptic nucleus, the suprachiasmatic nucleus, the ventral hypothalamic nucleus, and the posterior tubercle (Yon et al., 1992). Similarly, in the primitive teleost fish Anguilla anguilla, PACAP-containing neurons are primarily located in the parvo- and magnocellular subdivisions of the preoptic nucleus (Montéro et al., 1998).

The distributions of PACAP and VIP in the CNS are substantially different (Masuo et al., 1993). For instance, in the thalamus a few VIP fibers were found running up the wall of the third ventricle whereas a dense network of PACAP fibers was observed in the central thalamic nuclei (Köves et al., 1991). In the bed nucleus of stria terminalis, PACAP fibers appear to surround unstained, round-shaped neuronal cell bodies, whereas the VIP fibers are homogeneously distributed. PACAP-immunoreactive fibers are also found in the lateral septum of the hypothalamus where only a few VIP fibers are observed (Köves et al., 1991). In the magnocellular neurons, PACAP but not VIP is colocalized with oxytocin (Köves et al., 1994b). In the brainstem, VIP-LI is present in the mesencephalic periaqueductal gray and the dorsal and linear raphe nuclei whereas PACAP-LI is abundant in the paraventricular nucleus (PVN) and the dorsal vagal complex. The bed nucleus of the stria terminalis contains a very high concentration of PACAP and VIP-LI but no double-labeled cells have been detected (Kozicz et al., 1997). In contrast, both PACAP and VIP-immunoreactive fibers appear to innervate the wall of cerebral blood vessels (Jansen-Olesen et al., 1994).

In peripheral tissues, as in the brain, PACAP38 is by far the major molecular form but the proportions of PACAP27 and PACAP38 vary between the different organs (Arimura et al., 1991). For instance, in the colon, PACAP27 represents 30% of the total immunoreactivity whereas, in the testis, PACAP27 is hardly detectable (Arimura et al., 1991). The occurrence of different proportions of the two peptides in various tissues can be likely ascribed to the existence of different sets of PC enzymes.

The presence of PACAP mRNA and PACAP has been detected in most endocrine glands (Table 2). In particular, PACAP is found in the different lobes of the pituitary gland (Arimura and Shioda, 1995; Rawlings and Hezareh, 1996; Arimura, 1998). In the anterior pituitary, PACAP is observed in a subpopulation of gonadotrope cells (Mikkelsen et al., 1995; Köves et al., 1998). In the ventral part of the neural lobe, PACAP is contained in nerve fibers with large terminal boutons (Mikkelsen et al., 1995). At the ultrastructural level, PACAP-LI appears to be located in dense core granules contained in neurosecretory fibers (Kimura et al., 1994). PACAP-immunoreactive elements are also found in the gonads (Shioda et al., 1994), adrenal (Arimura et al., 1991), parathyroid (Luts and Sundler, 1994), and endocrine pancreas (Table 2; Arimura and Shioda, 1995; Love and Szebeni, 1999). In rat, the highest amounts of PACAP are found in the testis. In fact, the concentration of PACAP in the testis is higher than in the whole brain and exceeds the concentration of any other known peptides (Arimura et al., 1991). In situ hybridization studies have shown that PACAP mRNA is present in germ cells and not in Sertoli or Leydig cells (Shioda et al., 1994). Electron microscopic studies have revealed that PACAP is located in acrosoma caps and granules of primary spermatocytes but the peptide has not been observed in mature spermatids (McArdle, 1994; Shioda et al., 1994; Hannibal and Fahrenkrug, 1995). In the ovary, the concentration of PACAP is much lower than in the testis, and the peptide appears to be contained in nerve fibers (Steenstrup et al., 1995). Intense expression of PACAP mRNA has also been observed in the granulosa cells of preovulatory follicles (Ko et al., 1999). The adrenal gland contains a high concentration of PACAP (Arimura et al., 1991; Watanabe et al., 1992; Ghatei et al., 1993). In mammals, PACAP is found in the adrenal medulla (Shiotani et al., 1995), where it is contained both in chromaffin cells (Holgert et al., 1996) and in fibers (Frödin et al., 1995; Moller and Sundler, 1996). In contrast, in the frog adrenal gland, PACAP-LI is restricted to nerve fibers that contact either chromaffin cells or steroid-producing cells (Yon et al., 1993a). Similarly, in mammals, the parathyroid gland and the intrapancreatic ganglia are innervated by PACAP-containing fibers (Luts and Sundler, 1994; Filipsson et al., 1998a; Love and Szebeni, 1999).

Large amounts of PACAP-LI are found in all parts of the gastrointestinal tract (Arimura et al., 1991; Hauser-Kronberger et al., 1992; Ghatei et al., 1993; Mao et al., 1998; Vincze et al., 1999). The presence of PACAP-immunoreactive cell bodies has been observed in the myenteric ganglia throughout the gastrointestinal tract, and the existence of intrinsic neurons has been confirmed by in situ hybridization (Shen et al., 1992; Hannibal et al., 1998). Numerous PACAP-containing nerve fibers have been visualized along the circular muscle fibers and in the longitudinal smooth muscle layer of the esophagus (Uddman et al., 1991a; Köves et al., 1993; Olsson and Holmgren, 1994). PACAP-LI has also been detected in various exocrine glands of the alimentary canal, e.g., the parotid and submandibular glands, the liver, and the exocrine pancreas (Arimura et al., 1991; Fridolf et al., 1992; Moller et al., 1993; Luts and Sundler, 1994). In the urinary bladder, networks of PACAP-immunoreactive fibers are found in the vicinity of blood vessels (Moller et al., 1993; Fahrenkrug and Hannibal, 1998). In the airways, PACAP-immunoreactive fibers innervate smooth muscle bundles and blood vessels in the trachea as well as small bronchioles in the lung (Cardell et al., 1991; Uddman et al., 1991b; Hauser-Kronberger et al., 1996; Shigyo et al., 1998). In the immune system, PACAP is expressed in various lymphoid tissues including the thymus, spleen, and duodenal mucosa (Gaytan et al., 1994), and in peritoneal macrophages (Pozo et al., 1997). The occurrence of PACAP mRNA has been demonstrated in the superior cervical ganglion (Nogi et al., 1997b). Depolarization of these neurons stimulates the release of PACAP27 and PACAP38 and causes a concomitant increase of PACAP mRNA and peptide (Brandenburg et al., 1997). A few PACAP-positive perikarya are also present in the sphenopalatine and otic ganglia (Uddman et al., 1991b, 1999). In the eye, PACAP-LI is present in fibers innervating the iris sphincter and in cell bodies scattered in the ciliary ganglia (Wang et al., 1995; Elsas et al., 1997; Olianas et al., 1997; Samuelsson-Almen and Nilsson, 1999) and in fibers of the ganglion cell layer of the retina (Hannibal et al., 1997; Seki et al., 1997).

In peripheral organs, in contrast to the CNS, PACAP and VIP often appear to be coexpressed by the same cells. For instance, colocalization of PACAP and VIP has been demonstrated in nerve fibers and cell bodies in the human and sheep esophageal sphincter (Uddman et al., 1991a; Ny et al., 1995), in the human and chicken gut (Sundler et al., 1992), and in the ovine respiratory tract (Uddman et al., 1991b). Nerve fibers containing both PACAP and VIP are also found in other tissues, notably in the parathyroid glands of cat and sheep (Luts and Sundler, 1994), and in the gill arch of the goldfish Carassius auratus (De Girolamo et al., 1998).

The PACAP gene is differentially expressed in brain tumors. PACAP mRNA is present in most gliomas but is detected in only one-fifth of meningiomas (Vertongen et al., 1995a). PACAP mRNA and PACAP-LI are abundant in human neuroblastomas (Suzuki et al., 1993; Takahashi et al., 1993a; Vertongen et al., 1997a; Waschek et al., 1997). Double-staining experiments have demonstrated that PACAP and VIP are colocalized and intensely expressed in most pancreatic carcinoma, neuroblastoma, and pheochromocytoma tumors (Fahrenkrug et al., 1995). VIP has been reported to exert an autocrine stimulation of neuroblastoma cell growth and differentiation. The presence of PACAP suggests that it could also control neuroblastoma cell tumor proliferation (O'Dorisio et al., 1992; Pence and Shorter, 1992). Most pituitary tumors contain large amounts of PACAP. Because pituitary cells are programmed to proliferate in response to cAMP (Lin et al., 1992), it is conceivable that in pituitary adenomas, PACAP contributes to tumorigenesis (Spada et al., 1996). Overexpression of PACAP has also been reported in ovarian tumors (Odum and Fahrenkrug, 1998) and in pheochromocytomas (Takahashi et al., 1993b).

The evolution of the content of PACAP during development has been studied in detail in the CNS of rodents (Shuto et al., 1996; Waschek et al., 1998; Skoglösa et al., 1999b,c). In the mouse embryo, PACAP mRNA is present in the brain as early as embryonic day 9.5 (E9.5) (Shuto et al., 1996; Waschek et al., 1998), and the mRNA level increases during the prenatal period to reach a maximum at birth. In situ hybridization histochemistry revealed that the PACAP gene is widely expressed in the neural tube of the mouse at E10.5 (Shuto et al., 1996; Waschek et al., 1998). PACAP mRNA is found in differentiating neurons, suggesting that PACAP may control proliferation or differentiation of neuroblasts during neural tube development. PACAP is readily measurable by RIA in the rat brain at E14 (Masuo et al., 1994; Tatsuno et al., 1994). Immunoreactive nerve fibers are observed in the spinal cord and ganglia at E16 (Nielsen et al., 1998b). In the septum and hypothalamus, the content of PACAP increases gradually from birth to postnatal day 60 (P60). In the cortex, hippocampus, thalamus, and midbrain, PACAP levels increase more rapidly from P10 to P20 and reach a plateau at P30 (Masuo et al., 1994). In the striatum and cerebellum, the content of PACAP is very high at birth and during the first postnatal weeks and then decreases gradually from P20 to adulthood. In the developing rat cerebellum, PACAP is expressed in Purkinje cells (Nielsen et al., 1998a; Skoglösa et al., 1999b), which are known to regulate the survival of granule cells.

The ontogenesis of PACAP has also been described in the brain of the frog Rana ridibunda (M. Mathieu, L.Yon, I. Charifou, M. Trabucchi, M. Vallarino, C. Pinelli, R.K. Rastogi and H.Vaudry, submitted). PACAP-immunoreactive neurons are found soon after hatching (stages IV-VII of development; Taylor and Kollros, 1946) in the dorsal thalamus, and appear later (stages VII-IX) in the dorsal and ventral infundibular nuclei of the hypothalamus. PACAP-immunoreactive fibers are seen in the median eminence during the premetamorphic period (stages XIII-XVIII), suggesting that PACAP could be involved in the activation of the pituitary-thyroid axis, which is required for the onset of metamorphosis (Tata, 1998). Reversed-phase HPLC analysis combined with RIA detection indicates that PACAP38 is, by far, the predominant molecular form present in the frog brain at all developmental stages (M. Mathieu, L.Yon, I. Charifou, M. Trabucchi, M. Vallarino, C. Pinelli, R.K. Rastogi and H.Vaudry, submitted).

The primary structure of PACAP has been totally conserved among those mammalian species yet studied, i.e., human (Ohkubo et al., 1992), sheep (Miyata et al., 1989), rat (Ogi et al., 1990), and mouse (Okazaki et al., 1995). The sequence of PACAP has now been determined in several representative species of nonmammalian vertebrates, including the chicken Galus domesticus (McRory et al., 1997), the frog Rana ridibunda (Chartrel et al., 1991), the salmon Oncorhynchus nerka (Parker et al., 1993), the catfish Clarias macrocephalus (McRory et al., 1995), and the tunicate Chelyosoma productum (McRory and Sherwood, 1997) (Fig. 6). A partial sequence of PACAP that is identical with the first 28 amino acids of mammalian PACAP38 has also been characterized in the lizard Gila monster salivary gland (Pohl and Wank, 1998), and the presence of PACAP-LI has been documented in the brain and ovary of the crested newt, Triturus carnifex (Gobbetti et al., 1997). The primary structure of the 1-27 region of PACAP, which is responsible for the biological activity of the peptide, has been fully conserved in lizard, frog, salmon, and catfish, whereas the PACAP27 sequences of the chicken and stargazer exhibit only one amino acid substitution (Fig. 6). In contrast, the C-terminal portion of PACAP, which is not required for the biological activity of the peptide, is more variable (Fig. 6). Globally, the sequence of PACAP has been better preserved than that of VIP (Chartrel et al., 1995) and far more conserved than that of GRF across vertebrates (M. Montéro, L.Yon, D. Kikuyama, S. Dufour and H.Vaudry, submitted). The fact that evolutionary pressure has acted to strongly preserve the bioactive sequence of PACAP indicates that the peptide must exert important physiological functions. In support of this notion, a PACAP-like peptide has been identified in the insect Drosophila melanogaster (Feany and Quinn, 1995), and this peptide has been found to modulate ionic conductances at the neuromuscular junction (Zhong, 1995; Zhong and Pena, 1995).

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Comparison of the amino acid sequences of PACAP from various vertebrate species and a protochordate. Percentages indicate amino acid identity between PACAP38 from different nonmammalian vertebrates and mammalian PACAP38, and between lizard or tunicate PACAP27 and mammalian PACAP27. -, amino acids identical with those of human PACAP. The potential cleavage-amidation sites are underlined.

Two different genes for PACAP are present in the tunicate Chelyosoma productum (Fig. 6; McRory and Sherwood, 1997). Each of these genes encodes both PACAP and a GRF-like peptide (Fig. 3). Nucleotide sequence similarities suggest that the two tunicate PACAP genes arose from exon duplication followed by gene duplication. In salmon, a cDNA that encodes both PACAP and a GRF-like peptide has been characterized (Parker et al., 1993). A cDNA encoding both PACAP and GRF-like peptide has also been cloned in the catfish Clarias macrocephalus (McRory et al., 1995), frog Rana ridibunda (Alexandre et al., 2000), and chicken Gallus domesticus (McRory et al., 1997). In salmon, catfish, and chicken, alternative splicing of the primary transcript generates a shorter precursor that contains only PACAP (Parker et al., 1993; McRory et al., 1995, 1997). In contrast to all submammalian species investigated so far, in mammals, GRF and PACAP precursors are encoded by two distinct genes (Mayo et al., 1985; Hosoya et al., 1992). Based on primary sequence homologies among existing peptides of the GRF superfamily (Fig. 1), it is possible to construct a hypothetical evolution tree of these genes (Campbell and Scanes, 1992). The organization of the mammalian prepro-GRF and prepro-PACAP cDNAs suggests that the two genes arose from duplication of an ancestral gene with subsequent exon loss (Fig. 3; Parker et al., 1997). Within the PACAP-VIP-glucagon-GRF-secretin gene superfamily, the PACAP gene appears to be closely related to the VIP one (Ogi et al., 1990). Furthermore, by comparison of the peptide sequences and geological record, one can predict the changes that have occurred during the evolution of the VIP-glucagon-GRF-secretin superfamily. According to these chronological analyses, duplication of a common ancestral gene yielding to the PRP/PACAP and PHI/VIP genes may have occurred some 750 million years ago (Campbell and Scanes, 1992).

	III. The PACAP Receptors

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Two classes of PACAP binding sites have been characterized on the basis of their relative affinities for PACAP and VIP (Table 3). Type I binding sites, which have been originally characterized in the anterior pituitary and hypothalamus using ¹²⁵I-PACAP27 as a radioligand, exhibit high affinity for PACAP38 and PACAP27 (K_d  0.5 nM) and much lower affinity for VIP (K_d > 500 nM) (Cauvin et al., 1990; Gottschall et al., 1990, 1991; Lam et al., 1990; Suda et al., 1992). Type II binding sites, which are abundant in various peripheral organs including the lung, duodenum, and thymus, possess similar affinity for PACAP and VIP (K_d  1 nM) (Gottschall et al., 1990; Lam et al., 1990). Subtle differences in the ability of PACAP38 and PACAP27 to displace ¹²⁵I-PACAP27 from its recognition sites in the CNS suggest the existence of two subsets of type I binding sites (Cauvin et al., 1991; Robberecht et al., 1991b). Similarly, type II binding sites have been subdivided into two classes, depending on their affinity for secretin (Hubel, 1972) and helodermin (Christophe et al., 1986): classical VIP binding sites exhibit low affinity for secretin (Christophe et al., 1981, 1989; Robberecht et al., 1982, 1988) whereas helodermin-preferring binding sites possess higher affinity for helodermin than for VIP or PACAP, and no affinity for secretin (Robberecht et al., 1984, 1998; Gourlet et al., 1991a; Shima et al., 1996; Solano et al., 1996). Careful characterization of ¹²⁵I-PACAP27 binding on membrane preparations indicated that the expression of type I and type II binding sites is not cell-specific and that most of the tissues possess various proportions of each receptor subtype (Tatsuno et al., 1990; Nguyen et al., 1993).

Type I PACAP binding sites were first isolated from a tumoral cell line derived from the rat exocrine pancreas (Buscail et al., 1990). Cross-linking of ¹²⁵I-PACAP27 to cell membrane preparations made it possible to isolate a 65-kDa protein (Buscail et al., 1990). In the porcine brain, type I PACAP binding sites exhibit an apparent molecular mass of 60 kDa (Schäfer and Schmidt, 1993; Schäfer et al., 1994). The extent of N-glycosylation of type I PACAP binding sites appears to be rather low compared with other glycosylated receptors (Klueppelberg et al., 1989; Feldman et al., 1990), but it is similar to those of type II PACAP or glucagon receptors (Iwanij and Hur, 1985; Raymond and Rosenzweig, 1991). In the bovine brain, type I PACAP binding sites have a molecular mass of 57 kDa and are coupled to a Gs protein (Ohtaki et al., 1990, 1993). Type I PACAP binding sites purified from bovine brain membranes were used to sequence the N-terminal portion of the protein (Ohtaki et al., 1993). The amino acid sequence was subsequently used to clone the type I PACAP receptor (see Section III, C).

Type II PACAP binding sites have been isolated in pure form from bovine brain membranes (Ohtaki et al., 1990). The protein has an apparent molecular mass of 45 kDa, i.e., very similar to that previously reported for the VIP receptor (Couvineau et al., 1986a,b).

Three PACAP receptors have been cloned so far and termed PAC1, VPAC1, and VPAC2 receptors (Table 3) by the International Union of Pharmacology according to their relative affinity for PACAP and VIP (Harmar et al., 1998).

The PACAP-specific receptor (PAC1-R) cDNA sequence was first determined from a pancreatic acinar carcinoma cell line (Pisegna and Wank, 1993). This PAC1-R cDNA, which encodes a 495-amino acid protein with seven putative membrane-spanning domains, exhibits a high degree of sequence identity with the glucagon, secretin, and calcitonin receptor cDNAs. The PAC1-R has subsequently been cloned in humans (Ogi et al., 1993; Pisegna and Wank, 1996), bovine (Miyamoto et al., 1994), rat (Hashimoto et al., 1993; Hosoya et al., 1993; Morrow et al., 1993; Spengler et al., 1993; Svoboda et al., 1993), and mouse (Hashimoto et al., 1996b). The PAC1-R has also been cloned in the goldfish Carassius auratus (Wong et al., 1998), and the frogs Rana ridibunda (Alexandre et al., 1999) and Xenopus laevis (Hu et al., 2000). Five variants resulting from alternative splicing in the third intracellular loop region have been identified in rat (Spengler et al., 1993). The splice variants are characterized by the absence (short variant) or presence of either one or two cassettes of 28 (hip or hop1 variant) or 27 (hop2 variant) amino acids (Journot et al., 1994). The presence of the hip cassette impairs adenylyl cyclase stimulation and abolishes phospholipase C (PLC) activation, suggesting that the various cassettes are involved in second messenger coupling (Table 3). In the brain and pituitary, the short variant is the most abundant form, whereas the hop variant predominates in the testes and adrenal gland (Spengler et al., 1993). A very short splice variant of PAC1-R, characterized by a 21-amino acid deletion in the N-terminal extracellular domain, has also been characterized (Pantaloni et al., 1996; Dautzenberg et al., 1999). The existence of this 21-amino acid sequence influences the receptor selectivity for the PACAP38 and PACAP27 isoforms and determines the relative potencies of the two peptides in stimulating PLC. Another PACAP receptor variant termed PAC1-R transmembrane domain (TM) 4 has been cloned in the rat cerebellum (Chatterjee et al., 1996). This latter receptor differs from the short variant of the PAC1-R by discrete sequence substitutions located in TMs II and IV. Surprisingly, activation of PAC1-R TM4 has no effect on adenylyl cyclase or PLC activity, but causes calcium influx through L-type voltage-sensitive calcium channels (Table 3). The mouse PAC1-R gene spans more than 50 kb and is divided into 18 exons (Aino et al., 1995). The proximal promoter region has no apparent TATA box but contains a CCAAT box and two potential specific protein 1-binding sites that act as transcriptional activators (Dynan and Tjian, 1983; Skak and Michelsen, 1999). The rat PAC1-R gene has been localized on chromosome 4 (Cai et al., 1995) and spans 40 kb with 15 exons (Chatterjee et al., 1997). The intron/exon organization of the PAC1-R gene is very similar to that of the other members of the secretin receptor family. Alternative splicing of the PAC1-R gene also occurs in the untranslated region and could represent a regulatory mechanism involved in tissue-selective expression of the gene and/or in mRNA stability. The human PAC1-R gene is located in region p15 of chromosome 7 (Brabet et al., 1996).

The VIP/PACAP receptor, subtype 1 (VPAC1-R) was first cloned from a rat lung cDNA library by cross-hybridization with a secretin receptor cDNA. The rat VPAC1-R cDNA encodes a 459-amino acid protein (Ishihara et al., 1992) and exhibits 50% amino acid sequence identity with the rat PAC1-R (Pisegna and Wank, 1993). The human VPAC1-R cDNA was characterized from a HT29 human colonic adenocarcinoma cell line library. The human VPAC1-R comprises 457 amino acids and possesses 84% sequence identity with the rat VPAC1-R (Sreedharan et al., 1993). The VPAC1-R gene spans 22 kb and is composed of 13 exons ranging in size from 42 to 1400 base pairs (Sreedharan et al., 1995; Pei, 1997). The promoter region encompasses several potential binding sites for nuclear factors, including specific protein 1, activator protein-2, or autotumorolytic fraction. The human VPAC1-R gene is located on region p22 of chromosome 3 (Sreedharan et al., 1995). Selective substitution of amino acids His¹⁷⁸  Arg and Thr³⁴³  Lys, Pro, or Ala by directed mutagenesis results in constitutive activation of the VPAC1-R with respect to cAMP production (Gaudin et al., 1998, 1999). The VPAC1-R also has been cloned in the goldfish Carassius auratus (Chow et al., 1997) and the frog Rana ridibunda (Alexandre et al., 1999). The fact that the frog VPAC1-R exhibits pharmacological characteristics of both VPAC1 and VPAC2 receptors in mammals should help to decipher the structure-activity relationships of the VIP/PACAP receptor family.

The VIP/PACAP-receptor, subtype 2 (VPAC2-R) was cloned initially from a rat pituitary cDNA library (Lutz et al., 1993) and subsequently from a human placenta cDNA library (Adamou et al., 1995). The rat and human VPAC2-R proteins exhibit 87% amino acid identity (Gagnon et al., 1994; Svoboda et al., 1994; Adamou et al., 1995). Northern blot analysis indicates that two VPAC2-R mRNAs of 4.6 and 2.3 kb are expressed in the human skeletal muscle, heart, brain, placenta, and pancreas (Adamou et al., 1995). The VPAC2-R gene is located in region q36.3 of chromosome 7 in humans (Mackay et al., 1996), and on chromosome 4 in rats (Cai et al., 1995). The human VPAC2-R is encoded by 13 exons, and the human gene spans 117 kb (Lutz et al., 1999b).

A number of PACAP analogs have been synthesized to identify the determinants responsible for the recognition and activation of the receptors (Fig. 7). As previously reported for other members of the glucagon-GRF-secretin family, the N-terminal region of PACAP appears to play a crucial role for the biological activity of the peptide. In particular, it has been shown that the deletion of the His¹ residue causes a 50-fold decrease in the affinity of PACAP27 for rat and human PAC1-R (Gourlet et al., 1991b; Bitar and Coy, 1993). Deletion of the His¹ residue of frog PACAP38 abolishes its adenylyl cyclase-stimulating activity on adenohypophysial fragments (Yon et al., 1993b). Suppression of the His¹ and Ser² residues reduces by 3000-fold the potency of PACAP27 to stimulate adenylyl cyclase in AR4-2J rat pancreatic acinar cells (Robberecht et al., 1992a). Replacement of the Ser² residue by Ala has little effect whereas substitution of Ser² by Phe or Arg decreases by 1000-fold the ability of PACAP27 analogs to stimulate adenylyl cyclase (Hou et al., 1994). Substitution of the Asp³ residue by Asn markedly reduces the stimulatory effect of PACAP27 on adenylyl cyclase (Hou et al., 1994). N-terminal truncated analogs of PACAP exhibit antagonistic activity of PAC1-R, indicating that the N-terminal domain is required for receptor activation but is not essential for the recognition of the binding site. Gradual deletion of the N-terminal amino acid residues of PACAP27 and PACAP38 has shown that amino acid 6 to 38 of PACAP [PACAP(6-38)] is the most potent antagonist (Robberecht et al., 1992b). Paradoxically, shorter analogs such as PACAP(14-38) retain some adenylyl cyclase-stimulating potency (Vandermeers et al., 1992). Although both PACAP27 and PACAP38 are potent agonists on PACAP/VIP receptors, the C-terminal domain appears to play a facilitatory role in the recognition of the binding sites. For instance, N-terminal truncated or substituted analogs derived from PACAP38 exhibit higher activity than their PACAP27 counterparts (Fig. 7; Vandermeers et al., 1992). The fact that a chimeric peptide formed by adding the PACAP(28-38) sequence to the VIP moiety exhibits a 100-fold higher affinity than VIP for PAC1-R (Gourlet et al., 1996a, 1997b) provides additional evidence that the C-terminal region of PACAP38 reinforces the binding efficacy of the peptide. Concurrently, the 28-38 extension may also be involved in the recognition of PACAP by specific binding proteins. In support of this notion, it has been found that one such potential binding protein, ceruloplasmin, can bind PACAP38 but not PACAP27 (Tams et al., 1999).

View larger version (30K): [in this window] [in a new window]   Fig. 7.   Structure-activity relationships of various PACAP38-related peptides. The binding affinity of a series of truncated PACAP analogs and their potency to stimulate adenylyl cyclase are indicated. Amino acid substitutions are indicated in black. a, Gourlet et al., 1991b; b, Robberecht et al., 1991b; c, Schäfer et al., 1991; d, Robberecht et al., 1992a; e, Robberecht et al., 1992b; f, Vandermeers et al., 1992; g, Ciccarrelli et al., 1994; h, Hou et al., 1994; i, Ciccarelli et al., 1995; j, Van Rampelbergh et al., 1996.

A natural peptide called maxadilan has been characterized as a selective agonist of PAC1-R (Moro and Lerner, 1997). Maxadilan is a 61-amino acid peptide that was isolated from the salivary gland of the blood-feeding sand fly Lutzomia lingipalpis on the basis of its vasodilatory activity (Lerner et al., 1991). As maxadilan does not possess any significant sequence identity with PACAP, this is a unique example of functional convergence between two peptides that do not share structural similarity. A shortened maxadilan synthetic analog, termed M65, in which the amino acid sequence 25 to 41 has been deleted, acts as a specific antagonist of PAC1-R (Uchida et al., 1998; Moro et al., 1999).

Most type II receptor antagonists designed so far are N-terminal truncated or substituted VIP peptides (Pandol et al., 1986; Turner et al., 1986; Gozes et al., 1995; Gourlet et al., 1997a). Cyclic lactam analogs of PACAP behave as potent type II receptor antagonists (Bitar et al., 1994). A cyclic peptide, RO 25-1553, acts as a selective VPAC2-R agonist with respect to binding affinity and adenylyl cyclase-stimulating potency (O'Donnell et al., 1994; Gourlet et al., 1997c). Amino acid substitutions and addition of a fatty acyl moiety have led to the development of lipophilic VIP derivatives that exhibit enhanced potency and specificity for VPAC-R (Gozes and Fridkin, 1992; Gozes et al., 1995; Gourlet et al., 1998). These data suggest that several domains are involved in the binding of PACAP to its receptors, and demonstrate the possibility of developing powerful and selective agonists or antagonists with potential therapeutic value.

The CHO and NIH 3T3 cell lines, and the yeast Saccharomyces cerevisiae, which are naturally devoid of PACAP receptors, have been widely used for the pharmacological and functional characterization of each PACAP receptor subtype after transfection (Ciccarelli et al., 1994; Delporte et al., 1995; Gaudin et al., 1996; Gourlet et al., 1996b; Van Rampelbergh et al., 1996; Hansen et al., 1999). Concurrently, the CHO and COS-7 cell lines have been used to investigate the binding properties of chimeric PACAP/VIP receptors (Vilardaga et al., 1995, 1996; Van Rampelbergh et al., 1996; Hashimoto et al., 1997; Juarranz et al., 1999b; Lutz et al., 1999a).

The localization of PACAP binding sites and PACAP receptor mRNAs has been investigated thoroughly in the rat brain (Masuo et al., 1991, 1992; Schäfer et al., 1991; Hashimoto et al., 1996a; Nomura et al., 1996; Shioda et al., 1997a; Vertongen et al., 1997b; M. Basille, D. Vaudry, Y. Coulouarn, S. Jégou, I. Lihrmann, A. Fournier, H. Vaudry and B. J. Gonzalez, submitted). The distribution and relative density of type I (PACAP-specific) and type II (PACAP/VIP) binding sites are compared in Table 4.