Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP-27, but not PACAP-38) degradation by the neutral endopeptidase EC 3.4.24.11

VIP (vasoactive intestinal polypeptide) and PACAP (pituitary adenylate cyclase-activating polypeptide), which are potent relaxing agents in the airways, were submitted to in vitro degradation by the neutral endopeptidase EC 3.4.24.11 (NEP), one of the most active peptidase in the lung, to test their relative resistance to proteolysis. Both VIP and PACAP(1-27) were cleaved by NEP, but PACAP(1-38) was not. The main fragments produced were VIP(1-22) and VIP(1-25), and PACAP(1-22) and PACAP(1-25), respectively. The degradation of VIP(1-27), PACAP(6-27), and PACAP(13-27) was also hindered by extending their C-terminal ends with the (28-38) sequence of PACAP(1-38). The sensitivity to enzyme degradation was gradually reduced when the C-terminal extension was increased from PACAP(1-27) to PACAP(1-29), PACAP(1-32) and PACAP(1-38). The biological activities of the degradation products were evaluated on the three classes of PACAP/VIP receptors, with VIP(1-25) and PACAP(1-25) retaining an important part of their activities on the VIP1 receptor. Thus, the degradation of VIP and PACAP(1-27) by the neutral endopeptidase 24.11 might produce a VIP1 receptor-selective active metabolite, provided that very high VIP or PACAP(1-27) concentrations are achieved in the receptor vicinity.     

Regulation of interleukin-6 (IL-6) secretion in primary cultured rat astrocytes: synergism of interleukin-1 (IL-1) and pituitary adenylate cyclase activating polypeptide (PACAP)

Adjuvant-induced arthritis (AA) in specific strains of rats is an immunologically mediated inflammatory disease which is also characterised by activation of the endocrine system. To further investigate the effects of AA on processing of the pro-opiomelanocortin (POMC) precursor in rat immune tissues, we utilised radioimmunoassays for adrenocorticotrophin (ACTH), beta-endorphin and alpha-melanocyte-stimulating hormone (alpha-MSH) to measure these peptides in the spleen and thymus. 14 days following adjuvant injection, spleen levels of ACTH were elevated in the AA group (4.47 +/- 1.04 ng/g tissue, n = 9) compared to controls (2.42 +/- 0.4 ng/g) and exacerbation of the disease by removal of circulating glucocorticoids through bilateral adrenalectomy (ADX) resulted in further elevation of spleen ACTH (5.11 +/- 1.22 ng/g). beta-Endorphin levels in both the AA (10.60 +/- 1.61 ng/g) and AA/ADX (13.37 +/- 2.36 ng/g) groups were higher than controls (5.57 +/- 0.65 ng/g). Conversely, alpha-MSH spleen levels were decreased in the AA (2.89 +/- 0.22 ng/g) and AA/ADX (2.22 +/- 0.33 ng/g) groups compared to controls (4.62 +/- 0.45 ng/g) and were also decreased following adrenalectomy. In the thymus, ACTH levels were elevated in the AA group (8.95 +/- 1.41 ng/g) compared to controls (5.79 +/- 0.63 ng/g), and the same pattern was evident for thymic alpha-MSH (0.64 +/- 0.08 ng/g in AA animals compared to control levels of 0.35 +/- 0.03 ng/g). Following G50 gel filtration, ACTH and beta-endorphin immunoreactivities (ir) were present in both spleen and thymus as two peaks, one which eluted near the void volume and one which eluted in a lower molecular mass position than the standards.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gene expression of pituitary adenylate cyclase activating polypeptide (PACAP) in the rat hypothalamus

Pituitary adenylate cyclase activating polypeptide (PACAP) isolated from ovine hypothalamus is considered to be a member of the vasoactive intestinal peptide/glucagon/secretin/growth hormone-releasing hormone family of peptides. Two forms of PACAP, PACAP38 and PACAP27, have been demonstrated in the rat hypothalamus. The PACAP precursor contains another peptide called PACAP-related peptide (PRP), but so far no information on this peptide in tissue exists. We have developed three radioimmunoassays specific for PACAP38, PACAP27 and PRP and demonstrate that all three preproPACAP peptides are expressed in the rat hypothalamus, the PACAP38/PACAP27 ratio being around 60 and the PACAP38/PRP ratio being around 10. HPLC analysis of hypothalamic extract showed that PACAP38 and PACAP27 are found in only one form corresponding to the respective synthetic peptides, whereas PRP eluted in two peaks, the predominant form corresponding to synthetic PRP1-29. The cellular distribution of PACAP38, PACAP27, and PRP and corresponding mRNA in the hypothalamus was determined with immunohistochemistry and in situ hybridization histochemistry. PACAP- and PRP-immunoreactive neuronal perikarya were observed in the medial parvocellular part of the paraventricular nucleus (PVN) in colchicine pretreated rats. Some cell bodies of magnocellular variety were found in the PVN. PACAP mRNA containing cells were observed in moderate numbers in the same parts of the paraventricular nucleus. PACAP- and PRP immunoreactive fibres and varicosities were distributed in the PVN and in the periventricular nucleus. These data show that PACAP38, PACAP27 and PRP are expressed in the parvocellular part of the PVN, implying roles as hypothalamic regulatory peptides.     

Vasoactive intestinal polypeptide (VIP)--immunoreactive nerve fibres in the mammary gland of the pig

Immunohistochemical studies have been performed to investigate the coexistence of VIP with dopamine-beta-hydroxylase (D(beta)H), vesicular acetylcholine transporter (VAChT), somatostatin (SOM) or neuropeptyd Y (NPY) within nerve fibres supplying the immature mammary gland in the pig. Generally, a moderate number of the VIP-immunoreactive (VIP-IR) nerve fibres were located in the nipple and parenchyma of the gland. VIP-IR fibres surrounded smooth muscle cells (SMC), blood vessels (BV) and lactiferous ducts (LD). Double-labelling immunohistochemistry revealed that some of VIP-IR nerve fibres also contained immunoreactivity to D(beta)H. VIP/D(beta)H-IR nerves were associated with BV and SMC and single fibres were observed around the LD in both nipple and parenchyma of the gland. VIP/VAChT-IR nerve fibres were not observed. The majority of VIP-IR fibres associated with SMC were also SOM-IR. Less numerous VIP/SOM-IR fibres supplied the BV and were located around the LD of the gland. A small number of VIP-IR nerves also displayed immunoreactivity to NPY. VIP/NPY-IR nerve fibres supplied the BV of the gland.     

Stage-specific expression of pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA in the rat seminiferous tubules

We have used in situ hybridization and Northern blot analysis with oligonucleotide probe to characterize the site of pituitary adenylate cyclase-activating polypeptide (PACAP) synthesis in the rat testis. We observed strong hybridization signal in one third of the cross-sections of the seminiferous tubules, whereas some tubules were devoid of hybridization signal, thus suggesting that PACAP mRNA is expressed in a stage-specific manner. More detailed analysis showed that PACAP mRNA was present in round spermatids at stages III-VII of the cycle. Northern blot hybridization to RNAs extracted from samples of seminiferous tubules at different stages of the epithelial cycle confirmed that expression of PACAP mRNA is restricted to specific stages of the cycle. The highest amount of PACAP mRNA was detected at stages V to early -VII of the cycle, whereas very low levels of mRNA were present at stages I-II and IX-XIV. The present results demonstrate that PACAP mRNA is expressed in the developing germ cells. This suggests that PACAP may function as a paracrine or autocrine regulatory factor for the Sertoli and germ cells, with a specific function during early spermiogenesis, shortly before the onset of nuclear elongation, at the last period of haploid gene activity.     

Vasoactive intestinal polypeptide (VIP) innervation of rat spleen, thymus, and lymph nodes

In the thymus, VIP-positive (+) fibers were found in the capsular/septal system, cortex, and medulla. In the spleen, VIP+ nerves coursed along large arteries and central arterioles, and in the white pulp, venous/trabecular system, and red pulp. Splenic VIP innervation was more robust in Long-Evans hooded rats than in Fischer 344 rats. VIP+ nerves in mesenteric lymph nodes were found in the cortex, and along the cortical vasculature and medullary cords. No VIP innervation was observed in popliteal lymph nodes. Immunocytes also were VIP+, suggesting that both neural and cellular synthesis of VIP contributes to VIP concentration in lymphoid organs. Surgical sympathectomy did not alter splenic or thymic VIP content, respectively, and VIP innervation of these organs was not altered, suggesting an origin for VIP+ nerves other than the sympathetic nervous system.     

IL-13 protects mice from lipopolysaccharide-induced lethal endotoxemia: correlation with down-modulation of TNF-alpha, IFN-gamma, and IL-12 production

IL-13 is a potent down-modulator of macrophage proinflammatory activity in vitro, similar in this context to the anti-inflammatory cytokines IL-4 and IL-10. Since IL-10 effectively confers protection to mice from LPS-induced lethal endotoxemia through inhibition of proinflammatory cytokine production, we investigated whether IL-13 may also be capable of providing protection in this experimental model of endotoxic shock. A single injection of recombinant murine IL-13 (rmIL-13; 0.5-10 microg) significantly increased survival in a dose-dependent manner when a lethal i.p. injection of endotoxin was administered to BALB/c mice. This effect appeared to be IL-13 specific, since survival was not affected in mice that received heat-inactivated rmIL-13. rmIL-13 provided significant protection to mice even when given 30 min after LPS injection; however, this protection decreased in a time-dependent manner as the administration of rmIL-13 was delayed by 1, 2, and 5 h following LPS injection. The protective effect of IL-13 was correlated with significant decreases in the production of the inflammatory mediators TNF-alpha, IFN-gamma, and IL-12 as well as a decrease in the anti-inflammatory mediator IL-10. Our data suggest that IL-13 provides protection from LPS-induced lethal endotoxemia in a manner that is similar to but independent from that of IL-10, and therefore can be added to the list of cytokine immunomodulators that might be beneficial in the treatment of septic shock.     

PACAP (pituitary adenylate cyclase-activating peptide)-like immunoreactivity in the gill arch of the goldfish, Carassius auratus: distribution and comparison with VIP

Pituitary adenylate cyclase-activating peptide (PACAP) is a novel vasoactive intestinal peptide (VIP)-like peptide isolated from ovine hypothalamus. It is present in neuronal elements of a number of peripheral organs. We have examined whether PACAP occurs in the gill arch of Carassius auratus L. in which our recent studies have shown the presence of VIP-like peptide. Immunohistochemistry has revealed PACAP-like immunoreactivity in the anterior branches of the post-trematic glossopharyngeal and vagus nerves. PACAP-immunoreactive nerve cell bodies and fibers are present in connective tissue on the oral side of the gill arch. Colocalization studies carried out by the application of double immunofluorescence show that a PACAP-like peptide coexists with VIP in the same nerve cell bodies and fibers. The localization pattern of PACAP in the gill arch of goldfish suggests its possible involvement in the regulation of secretory activities.     

Isolation and structural characterization of pituitary adenylate cyclase activating polypeptide (PACAP)-like peptide from the brain of a teleost, stargazer, Uranoscopus japonicus

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel neuropeptide consisting of 38-residue (PACAP 1-38) and a truncated form with 27 residues (PACAP 1-27) that plays several roles in tetrapods. We isolated a highly purified PACAP-like peptide from the brain of a teleost, the stargazer, by extracting of acetone-dried powder with acetic acid followed by high-performance liquid chromatography (HPLC) on gel-filtration, cation-exchange, and reverse-phase columns. Purification was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis using an anti-PACAP 1-27 antiserum. The PACAP-like peptide thus obtained had a molecular mass of 4,623, determined by mass spectrometry, and its amino acid sequence showed 89 and 87% identity with those of ovine and frog PACAPs, respectively. These results indicate that a PACAP-like peptide, which is a highly homologous with tetrapod PACAP, is present in the teleost brain.     

Expression of pituitary adenylate cyclase activating polypeptide (PACAP) in the postnatal and adult rat cerebellar cortex

In adult rat, immunostaining for pituitary adenylate cyclase activating polypeptide (PACAP) was demonstrated in the soma and dendrites of Purkinje cells and in nerve fibres around granule cells. PACAP in the Purkinje cells was confirmed by the presence of mRNA. The concentration of PACAP-38 was high after birth and declined to adult levels within a few weeks. At birth PACAP-immunoreactive nerve fibres and a few cells were present in the Purkinje cell layer, but already at postnatal day 7 an adult PACAP immunostaining pattern was found. The findings suggest that PACAP could be a neurotransmitter in the adult cerebellum and provide a morphological correlate for the reported in vitro anti-apoptotic PACAP effects on cerebellar granule cells from 1-week-old rats.     

The effect of pituitary adenylate cyclase activating polypeptide (PACAP) on amylase secretion from guinea pig pancreatic acini

Beta-lactamase production is one of the major mechanisms of resistance amongst bacteria especially the enteric bacilli. The purpose of this study is to assess the in-vitro activity of Sulperazon, a combination of cefoperazone and an irreversible beta-lactamase inhibitor, sulbactam, against the cefoperazone resistant isolates of aerobic gram-negative bacilli. A total of 92 such strains were tested. It was found that at a concentration of < or = 8 mg/l of sulbactam added to cefoperazone 82% of Klebsiella spp, 100% of E. coli, 100% of Enterobacter spp, 33% of Pseudomonas aeruginosa, 67% of Pseudomonas spp and 62% of Acinetobacter spp that were resistant to cefoperazone alone were susceptible to the combination. Hence it is concluded that the addition of sulbactam to cefoperazone does expand the spectrum of the in-vitro activity of cefoperazone.     

Antinociceptive effect of intrathecally administered pituitary adenylate cyclase activating polypeptide (PACAP) on the rat formalin test

Diclofenac (0.5-2 mM) dose- and time-dependently reduces the viability of isolated hepatocytes. This effect cannot be counteracted by the calcium channel blockers diltiazem (0.05-0.1 mM) and verapamil (0.05-0.5 mM), the calmodulin antagonist calmidazolium (0.01 mM) or Quin 2-AM (0.1 mM), an intracellular calcium chelating agent. On the contrary, verapamil even accentuates the toxic effects of diclofenac. It is concluded from these results, that diclofenac causes cell damage by other mechanisms than calcium overload.     

Vasoactive intestinal peptide, but not pituitary adenylate cyclase-activating peptide, modulates the responsiveness of the gonadotroph to LHRH in man

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) are hypothalamic peptides sharing considerable sequence homology which are postulated to be hypophysiotrophic releasing factors. When infused into man, PACAP has no effect on anterior pituitary hormone levels, while VIP causes a significant increase in circulating prolactin concentrations. However, PACAP has recently been shown to augment the release of LH and FSH in response to LHRH in rat anterior pituitary cell culture. In order to ascertain if either peptide has a similar effect in man, PACAP and VIP were infused at 3.6 pmol/kg per min into six healthy male volunteers, and an LHRH test was performed 30 min after the infusion was commenced. Infusion of PACAP did not alter the gonadotrophin response to LHRH significantly. However, VIP augmented the release of LH significantly, both during the infusion and for 30 min thereafter, although there was no effect on FSH release. Thus VIP, but not PACAP, potentiates the release of LH after LHRH injection in man.     

Purification and primary structure of pituitary adenylate cyclase activating polypeptide (PACAP) from the brain of an elasmobranch, stingray, Dasyatis akajei

Pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from ovine hypothalami and found to exist as two amidated forms with 38 (PACAP 38) and 27 (PACAP 27) residues. The amino acid sequences of PACAPs isolated from the vertebrates, such as a bird, a frog and teleost fish, appear to be well conserved. In the present study, we attempted to isolate PACAP from the brain of an elasmobranch fish, Dasyatis akajei (stingray), which belongs to the Chondrichthyes (cartilaginous fish), by extraction of the acetone-dried powder with acetic acid, followed by successive high-performance liquid chromatography (HPLC) on a gel-filtration, a cation-exchange and two reverse-phase columns. Purification was monitored by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting analysis using an anti-PACAP 27 serum. The PACAP thus obtained consisted of 44 residues. The amino acid sequence of the comparable portion of its N-terminal 38 residues showed 92%, 89%, 89%, and 82% identity with those of mammalian, chicken, frog and teleost PACAPs with 38 residues, respectively. The extra six C-terminal residues of the stingray resembled those of tetrapod and teleost PACAP precursors which were deduced from the respective cDNAs. These results indicate that PACAP, which has an amino acid sequence showing high similarity with those of tetrapod and teleost PACAPs, is present in the elasmobranch brain.