Organ distribution and characterization of porcine peptides (VIP, CGRP and PHI) that increase cAMP in rat platelets.

Using an assay for rat platelet cAMP, we investigated the organ distribution of peptides that increase cAMP in rat platelets in porcine tissues. Marked activity was observed in the duodenum, pancreas and brain. By analysis with reverse phase high performance liquid chromatography (HPLC), three major peaks of activity were observed in porcine tissues. The first peak was vasoactive intestinal polypeptide (VIP), and the second peak was calcitonin gene-related peptide (CGRP). The third peak of activity was isolated from porcine duodenum. By analysis with a gas phase sequencer and with an amino acid analyzer, this peptide was identified as peptide histidine isoleucine (PHI). In a glucagon-secretin family of neuropeptides, pituitary adenylate cyclase activating polypeptide (PACAP) significantly increased platelet cAMP levels in a dose-dependent manner; however, glucagon did not. These results suggest that not only VIP and CGRP but also PHI and PACAP act upon platelets, as well as vascular tissues.     

Isolation and partial sequencing of a pancreatic polypeptide-like neuropeptide from the liver fluke, Fasciola hepatica.

1. A pancreatic polypeptide (PP)-immunoreactive neuropeptide has been isolated and partially sequenced from the liver fluke, Fasciola hepatica. 2. Gel permeation chromatography of an acid ethanol extract of cattle flukes showed that the peptide is similar in size to mammalian (bovine) PP. 3. The Fasciola peptide was purified to homogeneity by means of reverse-phase HPLC, employing different column chemistries. 4. The purified peptide was sequenced using automated gas-phase Edman degradation and the first 24 amino acid residues determined.     

Isolation and partial sequencing of a pancreatic polypeptide-like neuropeptide from the liver fluke,Fasciola hepatica

1. A pancreatic polypeptide (PP)-immunoreactive neuropeptide has been isolated and partially sequenced from the liver fluke, Fasciola hepatica.2. Gel permeation chromatography of an acid ethanol extract of cattle flukes showed that the peptide is similar in size to mammalian (bovine) PP.3. The Fasciola peptide was purified to homogeneity by means of reverse-phase HPLC, employing different column chemistries.4. The purified peptide was sequenced using automated gas-phase Edman degradation and the first 24 amino acid residues determined.     

Biosynthetic Processing of Preprovasoactive Intestinal Polypeptide in Parasympathetic Neurons of the Sphenopalatine Ganglion

Abstract: The precursor for rat vasoactive intestinal polypeptide (preproVIP) is processed by proteolytic cleavage into a signal peptide and five further functional domains: preproVIP 22–79, peptide histidine isoleucine (PHI), preproVIP 111–122, VIP, and preproVIP 156–170. To investigate the biosynthetic processing of preproVIP in peripheral parasympathetic neurons, the sphenopalatine ganglion and one of its projection areas, the nasal mucosa, were used. By immunohistochemistry it was shown that in the sphenopalatine ganglion, preproVIP-derived peptides are localized mainly in neuronal cell bodies, whereas in the nasal mucosa immunoreactivity was found only in nerve fibers and terminals. The peptides were quantified and characterized by radioimmunoassay, HPLC, and gel chromatography using antisera specific for the different precursor products. In the rat sphenopalatine ganglion, the different peptides were found in approximately equimolar amounts, with the exception of PHI and its C-terminally extended variant, PHV, which were present at considerably lower concentrations. However, in the nasal mucosa there was a preferential accumulation of VIP to at least three times the concentration of any of the other peptides. Our results suggest that all preproVIP-derived peptides are present and processed in the sphenopalatine ganglion but that there is a selective accumulation of VIP in the nerve terminals. This indicates that VIP is physiologically the most important transmitter among the preproVIP-derived peptides in parasympathetic nerves originating in the sphenopalatine ganglion.     

Pancreatic polypeptide-like material in nerves and endocrine cells of the rat

A region-specific antiserum (AbS11) raised against the carboxyl-terminal hexapeptide of pancreatic polypeptide has been employed to measure rat pancreatic polypeptide specifically and to demonstrate apparent immunoreactivity in nerves and in endocrine cells outside the pancreas. The concentration of pancreatic polypeptide in the head of the rat pancreas measured with AbS11 (176 +/- 47 pmol/g) was 750 fold higher than that measured with a conventional anti-bPP antiserum (0.23 +/- 0.08 pmol/g). Column chromatographs of rat pancreatic extracts demonstrated two peaks of immunoreactivity both eluting after the porcine pancreatic polypeptide standard. AbS11 also detected specific immunoreactivity in rat brain (470 fmol/g) which went undetected in convention assays. Although immunohistochemical studies with AbS11 and human pancreatic polypeptide antiserum demonstrated immunoreactivity in the same population of pancreatic endocrine cells, immunoreactive nerve fibres and enteroglucagon cells were only demonstrable with AbS11. These studies demonstrate that the carboxyl terminus of rat pancreatic polypeptide is immunochemically similar to that of higher mammals. Furthermore, neural and extrapancreatic endocrine variants of this peptide share an immunochemical determinant contained within the carboxyl-terminal hexapeptide.     

Immunohistochemical studies on glucagon, glicentin and pancreatic polypeptide in human stomach: normal and pathological conditions

Summary Endocrine-like cells containing glucagon, glicentin or pancreatic polypeptide immunoreactivity in human foetal and adult stomach, with or without disease, were studied with the indirect immunoperoxidase method and mirror sectioning technique. In foetal and neonatal oxyntic mucosae, there were endocrine-like cells with glucagon and glicentin immunoreactivities and argyrophilia. Cells containing glicentin immunoreactivity alone were detected earlier than glucagon cells during foetal development, and were also distributed throughout foetal to neonatal life. Bovine pancreatic polypeptide immunoreactivity coexisted in a subpopulation of the glucagon-glicentin cells. These cells were absent from normal oxyntic mucosa in the postneonatal period and from normal antral mucosa throughout life. Hamartomatous polyp in adult oxyntic mucosa, hyperplastic oxyntic mucosa in Menetrier's disease and atrophic oxyntic mucosa in a remnant stomach with cancer showed scattered glucagon-glicentin cells, but few or no cells containing bovine pancreatic polypeptide. Intestinalized mucosa showed plentiful glicentin cells with occasional glucagon and/or bovine pancreatic polypeptide immunoreactivity. Some gastric cancer cells of both diffuse and adenoplastic types contained immunoreactive glicentin and, less frequently, glucagon. Bovine pancreatic polypeptide immunoreactivity was detected in a few adenoplastic cancer cells, but not in diffuse type cells. Three different anti-pancreatic polypeptide sera against bovine, porcine or human pancreatic polypeptide detected basically the same cells mentioned above, but pancreatic polypeptide cells lacking human pancreatic polypeptide immunoreactivity were also present in foetal oxyntic mucosa. Immunoabsorption tests revealed that the bovine pancreatic polypeptide immunoreactivity was remote from peptide YY and neuropeptide Y.     

Mammalian regulatory peptide immunoreactivity in the trematode parasite Haplometra cylindracea and the lung of its frog host, Rana temporaria: comparative chromatographic characterisation using reverse-phase high-performance liquid chromatography.

1. Extracts of Haplometra cylindracea and lung tissues of its host, Rana temporaria, were subjected to radioimmunoassay using antisera to nine mammalian regulatory peptides. 2. In these extracts, immunoreactivity was measured to pancreatic polypeptide, substance P, neurokinin A, gastrin-releasing peptide and glucagon. The levels of each peptide varied considerably with some marked differences between those demonstrable in parasite and host (notably pancreatic polypeptide). 3. Reverse-phase HPLC fractionation of extracts revealed general chemical differences between parasite and host peptides, with some peptides present in more than one molecular form.     

Localization of vasopressin-, vasoactive intestinal polypeptide-, peptide histidine isoleucine-and somatostatin-mRNA in rat suprachiasmatic nucleus

Summary Messenger RNAs (mRNA) coding for vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), somatostatin and vasopressin were localized in the suprachiasmatic nucleus (SCN) of the rat hypothalamus using in situ hybridization histochemistry. Specific mRNA coding for each of these peptides was distributed in areas coextensive with the immunohistochemical localization of the appropriate peptide. The autoradiographic signal produced with probes to VIP and PHI created dense concentrations of silver grains over neuronal perikarya in the ventrolateral SCN, and the coextensive distribution of both VIP-and PHI-mRNAs suggests that both peptides are synthesized within the same neurons. The distribution of somatostatin-mRNA was distinct from that of VIP and PHI. Labeled neurons are observed at the interface of the two SCN subdivisions and the distribution of these neurons is identical to those shown to contain somatostatin immunoreactivity. Vasopressin-mRNA is also differentially concentrated within neurons in the dorsomedial subdivision of the SCN in an area that is coextensive with vasopressin-immunoreactive perikarya. The discrete pattern of hybridization for each of these mRNAs indicates that each of these peptides are synthesized in SCN neurons and reaffirms the differential distribution of each of these chemically defined cell populations within cytoarchitecturally distinct subdivisions of the nucleus.     

Corticotropin releasing factor (CRF)-like immunoreactivity in the hypothalamus and posterior pituitary of the goat, bovine, rat, monkey and human

Goat hypothalamic extract prepared by HCl extraction and chromatographed on a Sephadex G-50 column showed two immunoreactive CRF peaks. Most of the immunoreactivity coeluted with synthetic ovine CRF, and a small peak eluted near the void volume. Bovine, monkey, rat and human hypothalamic extracts prepared by acid-acetone or acid-methanol extraction showed three immunoreactive peaks. Most of the immunoreactivity coeluted with ovine CRF, and other smaller peaks eluted near the void volume and slightly before arginine vasopressin. Goat hypothalamic extract showed the highest cross-reactivity with anti-ovine CRF serum, followed by bovine hypothalamic extract. Less cross-reactivity was found in human, rat and monkey hypothalamic extracts. CRF immunoreactivity in goat hypothalamic extract coeluted with ovine CRF on reversed phase high performance liquid chromatography (HPLC) and main CRF immunoreactivity in human and rat hypothalamic extracts eluted slightly later than ovine CRF. These results suggest that there is a heterogeneity among the CRF molecules in these species and that goat CRF may be more similar to that of sheep CRF and the amino acid sequence or molecular weight of other animals CRF may be different from that of sheep CRF. The monkey posterior pituitary and rat neurointermediate lobe showed similar elution patterns of CRF immunoreactivity to their hypothalamic extracts on Sephadex gel filtration and HPLC. These results indicate that the posterior pituitary contains a similar CRF to hypothalamic CRF.     

The distribution of putative neurotransmitters in the nervous system of the dipteran Chironomus tentans insect larva: An immunohistochemical study using antisera to 5-hydroxytryptamine,tyrosine hydroxylase,methionine-enkephalin,proctolin and bombesin

Using the indirect immunofluorescence technique, the localization and distribution of transmitters, transmitter-related enzymes and neuropeptides was studied in the larvae of the dipteran species Chironomus tentans. Immunoreactivity could be seen for 5-hydroxytryptamine, tyrosine hydroxylase (the rate-limiting enzyme in the catecholamine synthesis), and the neuropeptides methionine-enkephalin (met-enk), proctolin and bombesin. The immunoreactivity was confined both to cell bodies as well as to nerve fibers within ganglia and along the alimentary canal. Furthermore, tyrosine hydroxylase immunoreactivity could also be seen in epithelial cells locally distributed along a short, middle part of the alimentary tract. These latter cells were regarded as endocrine-like cells. No immunoreactivity could be found with certainty for the enzyme phenylethanolamine-N-methyltransferase (PNMT) nor for the peptides vasoactive intestinal polypeptide (VIP), dynorphin, substance P, somatostatin, thyrotropin releasing hormone (TRH), neuropeptide Y (NPY), peptide histidine isoleucine amide (PHI), neurotensin, galanin and cholecystokinin (CCK).     

Brain motilin-like peptides are localized within the cell nucleus

The subcellular localization of motilin-like immunoreactive (MLIP) peptides in comparison to vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine amide-27-like peptide (PLP) was investigated in rat brain applying different subcellular fractionation techniques. Unlike VIP or PLP [17], motilin-like peptides were not located in synaptosomes, but in the cell nucleus. This is the first report of a non-vesicular localization of this neuropeptide and is suggestive of a possible non-neurotransmitter role of MLIP. Previous developmental studies point to a possible role for motilin-like peptides as trophic or developmental factors. These results open the possibility that brain motilin-like peptides may operate by binding to chromatin and regulating gene expression.     

Peptide histidine isoleucine (PHI) immunoreactivity in the rat retina: identification and characterisation by radioimmunoassay, immunohistochemistry and high-performance liquid chromatography

The presence of peptide histidine isoleucine immunoreactivity (PHI-IR) in the retinae of male Wistar rats has been demonstrated using a specific radioimmunoassay (RIA), immunohistochemistry (IHC) and high performance liquid chromatography (HPLC). PHI levels (mean +/- S.E.M.) of 16.68 +/- 1.61 ng/g wet weight of normal retinal tissue were measured by RIA. IHC showed PHI-IR to be localized to a population of amacrine cells distributed along the proximal aspect of the inner nuclear layer and to processes in laminae I and IV of the inner plexiform layer. PHI-IR in retinal extracts was shown to coelute with a synthetic porcine PHI standard on a reverse phase HPLC system. Additionally levels of PHI-IR were significantly increased (P less than 0.001) with light adaptation.     

Hymenolepis diminuta: changes in the levels of certain intestinal regulatory peptides in infected C57 mice.

The levels of 10 regulatory peptides in acid-alcohol extracts of three regions of the small intestine (0-20%, 30-60%, and 70-100%, with respect to distance from the pylorus) have been monitored radioimmunometrically in sham-infected male (6-8 week old) C57 mice and mice given a 5-cysticercoid infection of the rat tapeworm Hymenolepis diminuta and autopsied 10 days postprimary infection and 5 days postsecondary infection (administered 28 days postprimary infection). The regulatory peptides examined were gastrin, gastrin-releasing peptide (GRP), glucagon (= enteroglucagon), motilin, neurotensin (NT), pancreatic polypeptide (PP), peptide histidine isoleucine (PHI), somatostatin (SRIF), substance P (SP), and vasoactive intestinal peptide (VIP). Statistical analyses revealed significant deviations from control values of five of the peptides (enteroglucagon and SP, both elevated; NT, PHI and VIP, all lowered) in intestinal tissue from infected mice; measurement of the same peptides in colonic extracts revealed no significant differences between infected and sham-infected mice. Parallel changes in peptide levels between normal infected and immunosuppressed infected mice were not evident, although elevations in the tissue levels of enteroglucagon and SP were found in infected Wistar rats (normal host). Results are discussed with respect to a peptidergic involvement in the pathology and host immune response to an intestinal tapeworm.     

Presence of islet amyloid polypeptide in rat islet B and D cells determines parallelism and dissociation between rat pancreatic islet amyloid polypeptide and insulin content.

The islet amyloid polypeptide (IAPP) immunoreactivity of the adult rat pancreas is located in insulin-containing B cells as well as in somatostatin-containing D cells. In both cell types, the IAPP immunoreactivity is identical to rat synthetic IAPP in terms of its elution position after reversed phase HPLC and its binding to IAPP antibodies. The IAPP content per 10(6) B-cells is more than 100 fold lower than the corresponding insulin content, but comparable to the IAPP content of D cells. After induction of diabetes by streptozotocin, pancreatic IAPP seems predominantly located in somatostatin-containing cells. In normal rats, pancreatic insulin and IAPP content increase 20 fold from birth to 12 weeks of age; beyond week 12, the further rise in pancreatic insulin was not paralleled by an increase in IAPP content.     

Characterization of vasopressin and oxytocin immunoreactivity in the sheep and rat pineal gland: absence of vasotocin and detection of a vasopressin-like peptide

The presence of arginine-vasopressin (VP), arginine-vasotocin (VT) and oxytocin (OT) were studied in sheep and rat pineal gland by the combination of HPLC and radioimmunoassays. Three immunoreactive substances, which had the same retention time as OT, VP and VT, were detected in sheep and rat pineal extracts after HPLC separation. Parallelism of dilution curve and standard curve demonstrated that two of them were identical to OT and VP respectively. The third component was distinct from VT on basis of its immunological reaction with two different antibodies; it resembled the immunoreactivity of VP. This substance was also detected in the hypothalamus, brain cortex and pituitary gland, but not in the hippocampus and adrenal gland. The results support the notion that VT is absent in the adult mammalian pineal gland and point to the existence of another, possibly related peptide.     

Peptide-immunoreactive nerves in the mammalian female genital tract

Summary Vasoactive intestinal polypeptide, substance P, neuropeptide Y and peptide histidine isoleucine immunoreactivities have been demonstrated in the female genitalia of rat, cat, mouse and guinea-pig using immunocytochemistry and radioimmunoassay. They were localized to nerves. Each type of immunoreactive nerve showed a distinct pattern of distribution, though all were associated to some degree with blood vessels and smooth muscle. Vasoactive intestinal polypeptide-immunoreactive and neuropeptide Y-immunoreactive nerves were the most abundant. Higher concentrations of peptides were detected in the female genitalia of the mouse than those of the other species studied. Vasoactive intestinal polypeptide-immunoreactive nerves were particularly concentrated in the cervix (89.1±17.2 pmol/g, mean±S.E.M.) and the uterus (57.4±14.8 pmol/g) of the mouse, while neuropeptide Y immunoreactivity was more abundant in the Fallopian tube of the mouse (31.6±11.8 pmol/g) and the vagina of the rat (38.6±4.8 pmol/g) than in other regions. Separate populations of ganglion cells in the paracervical ganglia were found to contain vasoactive intestinal polypeptide and neuropeptide Y immunoreactivities. Peptide histidine isoleucine-immunoreactive and vasoactive intestinal polypeptide-immunoreactive nerves were similarly distributed, but the former were much less frequent. Substance P-immunoreactive nerves were seen mainly beneath the epithelium of the vagina and were, in general, more numerous in the guinea-pig than in other species. The significance of these peptide-immunoreactive nerves in the female genital organ remains to be determined.Dr. Wang is on leave from The Institute of Acupuncture, The Academy of Chinese Traditional Medicine, Peking, China.     

Isolation and characterization of circulating low molecular weight peptides in steer, sheep and rat portal and peripheral blood.

1. Low mol. wt peptides in plasma were isolated by reverse-phase HPLC from steer and sheep carotid arterial and rat heart blood and portal blood from all three species. 2. Elution profiles for peptide fractions were similar but the concentration of peptide-bound amino acids (PBAA) in fractions corresponding to different mol. wt peptides was not constant across species. 3. PBAA contributed between 65 and 78% to the plasma amino acid pool in steer and sheep but only 52% in the rat (P less than 0.05). 4. The percentage of many individual amino acids present in either free amino acid (FAA) or PBAA pools was different for ruminant compared with rat plasma but it was similar for steer and sheep apart from branch-chain amino acids (P less than 0.05).     

Vascular control of the pig nasal mucosa: distribution and effect of somatostatin in relation to noradrenaline and neuropeptide Y

By means of immunohistochemistry and radioimmunoassay (RIA), we have investigated the possible occurrence of somatostatin (SOM)-like immunoreactivity (-LI) in the autonomic innervation of the pig nasal mucosa. SOM-immunoreactive (-IR) fibres were present around nasal arteries, arterioles and venous sinusoids. Double-labelling experiments revealed that SOM-LI was co-localized with the noradrenaline (NA) markers tyrosine hydroxylase and dopamine-β-hydroxylase as well as with neuropeptide Y (NPY) in a subpopulation of neurons in the superior cervical sympathetic ganglion and in perivascular nerve terminals. Furthermore, SOM-LI was also present in perivascular fibres containing vasoactive intestinal polypeptide (VIP) and NPY of presumably parasympathetic origin. The parasympathetic fibres that were associated with glands contained peptide histidine isoleucine (PHI), VIP and NPY but not SOM, suggesting that in the nasal mucosa SOM-IR is restricted to perivascular nerves. As revealed by RIA, the content of SOM-LI in biopsies of both nasal mucosa and superior cervical sympathetic ganglion was about 12 pmol/g and the reverse phase HPLC characterisation of SOM-LI shown two separate peaks for SOM-28 and SOM-14.     

Neuropeptides of the primary sensory neurones in rat skin: an ontogenic study.

Cutaneous primary sensory neurones contain a number of biologically-active peptides, including substance P (SP), neurokinin A (NKA) and calcitonin gene-related peptide (CGRP). However, little information is available on ontogenic changes in the tissue concentrations of these neuropeptides. In this study, the concentrations of these neuropeptides have been assessed in dorsal and ventral abdominal rat skin at various stages of development from foetal, early neonatal, late neonatal, weaner to adult, using sensitive and specific radioimmunoassays. In addition, the levels of peptide histidine isoleucine (PHI), a peptide found in non-sensory cutaneous nerves, were assessed to control the study. The levels of PHI and NKA immunoreactivity did not change significantly at any stage of development. However, the levels of SP and CGRP immunoreactivity were significantly elevated in the early neonate with CGRP remaining elevated in the late neonate. The levels of both SP and CGRP were not significantly different between other developmental groups. Significant elevations in cutaneous SP and CGRP concentrations in early neonatal life in the rat, at a time when the pups are blind and naked, may be related to control of cutaneous sensitivity, which during this period of development, has positive survival value for the pups.     

A novel form of the polypeptide PHI isolated in high yield from bovine upper intestine. Relationships to other peptides of the glucagon-secretin family

A novel form of the polypeptide termed PHI (peptide HI with N-terminal histidine and C-terminal isoleucine amide) has been isolated from bovine upper intestine. This bovine peptide was obtained in a 40 times higher yield than the corresponding polypeptide isolated from porcine intestine. Bovine PHI is, like porcine PHI, composed of 27 amino acid residues. The complete amino acid sequence of the bovine peptide is His-Ala-Asp-Gly-Val-Phe-Thr-Ser-Asp-Tyr-Ser-Arg-Leu-Leu-Gly-Gln-Leu-Ser- Ala- Lys-Lys-Tyr-Leu-Glu-Ser-Leu-Ile-NH2. This sequence differs from porcine PHI at position 10 and from human PHI at positions 10, 12 and 27. The amino acid residue exchange between porcine and bovine PHI makes the latter more similar to the vasoactive intestinal polypeptide (VIP), gastric inhibitory polypeptide (GIP), glucagon and the growth-hormone-releasing factor (GRF).