Mammalian Neuropeptide Research Articles

Survey of neuropeptide-like immunoreactivity in the lamprey spinal cord

The distribution of neural elements immunoreactive to several peptides with a possible messenger role was studied in the lamprey spinal cord by using an indirect immunofluorescence method. Different patterns of immunoreactive (IR) fibers were detected by antisera raised against cholecystokinin, metorphamide, bombesin, galanin, corticotropin-releasing factor, somatostatin, peptide YY, neurotensin, calcitonin and FMRFamide. Somatostatin-IR cell bodies were located around the central canal. All immunoreactivity could be abolished by preabsorption with the corresponding peptide. The results suggest that the lamprey spinal cord has several fiber systems containing peptides sharing immunogenic properties with mammalian neuropeptides.

Pharmacologic characterization and autoradiographic distribution of binding sites for iodinated tachykinins in the rat central nervous system

P-type, E-type, and K-type tachykinin binding sites have been identified in the mammalian CNS. These sites may be tachykinin receptors for which the mammalian neuropeptides substance P, neuromedin K, and substance K are the preferred natural agonists, respectively. In the present investigation, we have compared the pharmacology and the autoradiographic distribution of CNS binding sites for the iodinated ( 125I-Bolton-Hunter reagent) tachykinins substance P, eledoisin, neuromedin K, and substance K. Iodinated eledoisin and neuromedin K exhibited an E-type binding pattern in cortical membranes. Iodinated eledoisin, neuromedin K, and substance K each labeled sites that had a similar distribution but one that was considerably different from that of sites labeled by iodinated substance P. CNS regions where there were detectable densities of binding sites for iodinated eledoisin, neuromedin K, and substance K and few or no sites for iodinated substance P included cortical layers IV–VI, mediolateral septum, supraoptic and paraventricular nuclei, interpeduncular nucleus, ventral tegmental area, and substantia nigra pars compacta. Binding sites for SP were generally more widespread in the CNS. CNS regions where there was a substantial density of binding sites for iodinated substance P and few or no sites for iodinated eledoisin, neuromedin K, and substance K included cortical layers I and II, olfactory tubercle, nucleus accumbens, caudate-putamen, globus pallidus, medial and lateral septum, endopiriform nucleus, rostral thalamus, medial and lateral preoptic nuclei, arcuate nucleus, dorsal raphe nucleus, dorsal parabrachial nucleus, parabigeminal nucleus, cerebellum, inferior olive, nucleus ambiguus, retrofacial and reticular nuclei, and spinal cord autonomic and somatic motor nuclei. In the brainstem, iodinated substance P labeled sites in both sensory and motor nuclei whereas iodinated eledoisin, neuromedin K, and substance K labeled primarily sensory nuclei. Our results are consistent with either of two alternatives: (1) that iodinated eledoisin, neuromedin K, and substance K bind to the same receptor site in the rat CNS, or (2) that they bind to multiple types of receptor sites with very similar distribution.

Structure and gene organization of bovine neuromedin K precursor.

cDNA and genomic DNA clones for the precursor of a mammalian neuropeptide tachykinin, neuromedin K, have been isolated and characterized by molecular cloning and sequence analysis. The deduced amino acid sequence indicates that the bovine neuromedin K precursor (preprotachykinin B) consists of 126 amino acid residues including a putative signal peptide. There are two preprotachykinin B mRNAs that differ only at the 5' extremity of the untranslated regions. The major mRNA species is encoded by seven exon sequences, while the minor species includes two extra 5' exon sequences and lacks the 5' terminus of the first exon sequence for the major mRNA species. The above gene organization for the major preprotachykinin B mRNA closely resembles that for the preprotachykinin A mRNA encoding the precursor for substance P and substance K. This structural resemblance strongly suggests that the two preprotachykinin genes have evolved from a common ancestor gene. Furthermore, we have found that the preprotachykinin A and B mRNAs markedly differ in their major expression sites. The results described thus indicate that the diversity of the mammalian tachykinin system has been acquired through various cellular mechanisms including gene duplication and differential expression of duplicated genes.

Characterization of a neurokinin B receptor site in rat brain using a highly selective radioligand.

We have recently characterized a tachykinin receptor subtype (SP-N) whose preferred ligand is the mammalian neuropeptide, neurokinin B (Laufer, R., Wormser, U., Friedman, Z. Y., Gilon, C., Chorev, M., and Selinger, Z. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7444-7448). To investigate this novel tachykinin receptor, we have now prepared a radiolabeled peptide, N alpha-[( 125I]desamino-3-iodotyrosyl)-[Asp5,6, N-methyl-Phe8]substance P (5-11) heptapeptide (125I-BH-NH-Senktide), which selectively interacts with the SP-N receptor subtype. The binding of 125I-BH-NH-Senktide to rat cerebral cortex membranes was studied under conditions that minimized nonspecific binding. Unlike other tachykinin receptor probes, this radioligand is not degraded during the binding experiment. Binding of 125I-BH-NH-Senktide is reversible, saturable, and of high affinity (KD = 0.9 nM). The radioligand labels a single class of binding site (122 fmol binding sites/mg of protein), as indicated by a linear Scatchard plot and a Hill coefficient close to unity (nH = 1.05). The pharmacological specificity of this binding site corresponds to that of the neuronal SP-N receptor in guinea pig ileum myenteric plexus, which was determined by a functional bioassay. Among various rat brain regions, the highest binding was observed in the cerebral cortex, olfactory bulb, hypothalamus, and hippocampus. These results suggest the existence and specific distribution of a neurokinin B receptor site of the SP-N type in rat brain. 125I-BH-NH-Senktide is the first selective and potent probe for this receptor and is thus an important tool for further studies of its distribution, regulation, and functional role.

Immunoreactive helodermin-like peptides in rat: A new class of mammalian neuropeptides related to secretin and VIP

Helodermin is a peptide from the venom of the lizard Heloderma Suspectum (Gila Monster) showing a high degree of sequence similarity with VIP, PHI and secretin in its N-terminal moiety. The present data support the presence of peptide(s) closely related to helodermin in the brain, gut and salivary glands of rat. In our radioimmunoassays, we routinely used one of the three specific antisera obtained from rabbits that were immunized against lizard helodermin coupled to bovine serum albumin with carbodiimide. Heat- and acid-stable immunoreactive helodermin-like material was more abundant in striatum, hippocampus and anterior pituitary than in cerebral cortex and hypothalamus. High levels of helodermin-like material were also present in salivary glands, duodenum and jejunum. When submitted to gel permeation chromatography on a TSK-G 2000 SW column, the apparent molecular radius of most of the immunoreactive material ranged from 6 to 12 KDa.

Rohdei-litorin: a new peptide from the skin of Phyllomedusa rohdei

The bombesin-litorin family of peptides is characterized by the common amino acid sequence -Gly-His-X-Met-NH 2 at the C-terminus, where X is a hydrophobic or aromatic residue. A new member of this family, rohdei-litorin, has been isolated from amphibian skin and its structure shown to be: Glp-Leu-Trp-Ala-Thr-Gly-His-Phe-Met-NH 2. This new peptide displayed a greater affinity than other members of the family for rat urinary bladder receptors. A litorin-like peptide, with high affinity for this kind of receptor, has already been described in mammalian spinal cord and named neuromedin B. Rohdei-litorin shares with neuromedin B the entire C-terminal octrapeptide and may be considered the amphibian counterpart of this mammalian neuropeptide. Rohdei-litorin Amino acid sequencing Amphibian skin Neuromedin

Neuropeptide immunocytochemistry in protostomian invertebrates, with special reference to insects and molluscs

In some molluscs (Aplysia and Fusitriton) and insects (silkworm and cricket), occurrence and distribution of neuropeptides in the nervous system and gut were studied with following results: (1) in these invertebrates and also in planaria, PP-like immunoreactivity is extensively distributed in neurons and (in insects) in gut endocrine paraneurons. These cells are negative for NPY, the mammalian neuropeptide related to PP in molecular structure. (2) PHI-like immunoreactivity is widely distributed in the neurons of those invertebrates; it occurs also in gut endocrine paraneurons in insects. The PHI-immunopositive cells are immunonegative for VIP and the coexistence of both peptides due to the common precursor in mammals cannot be recognized in these invertebrates. (3) Immunoreactivity for urotensin I, the neuropeptide derived from teleostean urophysial neurons, is widely distributed in the neurons of the invertebrates. In insects (cricket) it occurs in gut endocrine cells.

Tissue-specific generation of two preprotachykinin mRNAs from one gene by alternative RNA splicing.

A novel mammalian neuropeptide, the tachykinin substance K, is specified by a discrete genomic segment. Alternative RNA splicing generates two distinct mRNAs encoding the neuropeptide substance P alone or with substance K from a single preprotachykinin gene. Relative amounts of the mRNAs vary in different tissues, suggesting that the substance K-encoding sequence is regulated in a tissue-specific manner.

Immunohistochemical localization of (neuro)peptide hormones in endocrine cells and nerves of the gut of a stomachless teleost fish, Barbus conchonius (Cyprinidae).

Enteroendocrine cells containing glucagon-, substance P-, neurotensin- and VIP-like substances have been demonstrated immunocytochemically in the gut of Barbus conchonius. Mainly based on the distribution of the immunoreactive endocrine cells in this and a previous study, at least eight different enteroendocrine cell types appear to be present in this stomachless fish: C-terminal-gastrin-immunoreactive cells, predominantly present in the upper parts of the folds of the proximal part of the intestinal bulb. Metenkephalin-immunoreactive cells, basally located in the folds of the first segment. Pancreatic polypeptide (PP)-immunoreactive cells, mainly present in the first half of the first segment. Glucagon-like-immunoreactive (GLI) cells that are basally located in the folds of the first segment and that contain a different polypeptide (possibly glicentin) than pancreatic glucagon cells. Substance P-immunoreactive cells, present in the upper parts of the folds throughout the gut. C-terminal-neurotensin-immunoreactive cells, basally located in the folds throughout the first segment. Vasoactive intestinal polypeptide (VIP)-immunoreactive cells, present in small numbers in the proximal part of the intestinal bulb. Nonspecifically-immunoreactive cells, found throughout the intestinal bulb. Many VIP-immunoreactive nerves have been demonstrated in the smooth muscle layer and myenteric plexus of the gut; furthermore some of them are peptide histidine-isoleucine (PHI)-immunoreactive. Substance P-, somatostatin-, neurotensin- and met-enkephalin-immunoreactive nerves are also found. Thus, at least partial sequences of four different mammalian neuropeptide hormones (VIP, substance P, neurotensin, met-enkephalin) occur both in endocrine cells and enteric nerves of the gut of B. conchonius.

Cholecystokinin turnover in brain

Cholecystokinin octapeptide (CCK-8) occurs in relatively large amounts in some neurons of the cerebral cortex. Unlike most other mammalian neuropeptides, this compound contains a sulfate ester. We injected radiolabelled inorganic sulfate ([ 35S]sulfate) into rat cerebral cortex and measured the formation and elimination of radiolabelled CCK-8 using HPLC. The data allow the first calculation of the turnover rate of a putative transmitter neuropeptide in brain. The turnover of CCK-8 (half-life = 16 h) is considerably slower than that of the biogenic amines and amino acid neurotransmitters (half-lives less than 4 h).

The effect of the mammalian neuropeptide, gastrin-releasing peptide (GRP), on gastrointestinal and pancreatic hormone secretion in man.

Gastrin-releasing peptide, a newly isolated mammalian peptide similar in its structure and actions to the amphibian peptide, bombesin, has recently been localized to nerves in the brain, gut and pancreas. The present study investigates its effects on gut and pancreatic peptides in man. Intravenous infusion of 0.7 and 2.9 pmol min-1 kg-1 produced significant elevation of plasma gastrin, cholecystokinin-like immunoreactivity and neurotensin. It was found also to potentiate glucose-dependent insulin secretion. Its specific location in nerve fibres in the proximal gut and pancreas and its selective effect on gastroenteropancreatic peptides may favour its role as a physiological regulatory neuropeptide.

FMRFamide-like immunoreactivity in rat brain: Development of a radioimmunoassay and its application in studies of distribution and chromatographic properties

A radioimmunoassay is described for the molluscan neuropeptide, Phe Met Arg Phe NH 2 (FMRFamide). The antibody used is C-terminal-specific and shows slight but significant (1–2%) cross-reactivity with chicken pancreatic polypeptide (APP). The assay has been used to identify in rat brain extracts a pair of molecules that may represent mammalian counterparts FMRFamide. Their concentrations were highest in spinal cord and hypothalamus (10pmol·g −1) and lowest in cerebellum and striatum (< 3.5pmol·g −1). The two immunoreactive peptides were separated on CM ion-exchange chromatography where they appeared to be less basic than FMRFamide. On Sephadex G50 gel filtration one eluted in a similar position to FMRFamide and the other slightly earlier suggesting it may be of higher molecular weight. The rat immunoreactive components do not correspond to previously described neuropeptides or hormones, and may be members of a new group of mammalian neuropeptides with transmitter or modulatory functions.

Amino acid sequences of three bombesin-like peptides from canine intestine extracts.

Amino acid sequences of three canine bombesin-like peptides were determined after sequential purification of an extract obtained from 820 g of intestinal muscle. These three peptides contained 27, 23, and 10 amino acid residues. The sequences of the two shorter forms were identical to the corresponding carboxyl-terminal sequence of the heptacosapeptide. The sequence of the largest peptide is H2N-Ala-Pro-Val-Pro-Gly-Gly-Gln-Gly-Thr-Val-Leu-Asp-Lys-Met-Tyr-Pro-Arg-Gly-Asn - His(Trp-Ala-Val-Gly-His-Leu-Met-CONH2). The sequence of the 23-residue peptide is H2N-Gly-Gly-Gln-Gly-Thr-Val-Leu-Asp-Lys-Met-Tyr-Pro-Arg-Gly-Asn-His-Trp-Ala- Val-Gly(His-Leu-Met-CONH2). The sequence of the decapeptide is: H2N-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-CONH2. Comparison of the 27-residue peptide with the known structure of porcine gastrin-releasing peptide, another bombesin-like heptacosapeptide, reveals four amino acid substitutions: canine bombesin-like peptide had Pro 4, Gly 5, Gln 7, Asp 12, whereas porcine gastrin-releasing peptide had Ser 4, Val 5, Gly 7, Ala 12. Radioimmunoassay of brain extracts after similar purification revealed the presence of similar large and small forms of immunoreactive bombesin peptides, but with apparent tissue concentrations of about 4% of those present in intestinal muscle. Canine bombesin-like peptides represent another example of mammalian neuropeptides existing in more than one biologically active molecular form.

Arginine vasotocin, an endogenous neuropeptide of Aplysia, suppresses the gill withdrawal reflex and reduces the evoked synaptic input to central gill motor neurons.

The superfusion (15 min) of arginine vasotocin (AVT; 10(-9)--10(-12) M) over the abdominal ganglion of Aplysia californica suppressed the amplitude of the gill withdrawal reflex evoked by tactile stimulation of the siphon, increased the rate of gill reflex habituation, and decreased the evoked synaptic activity to central gill motor neurons. The suppressive effects of AVT on gill reflex behaviors were not due to toxic effects of the hormone since the effects were completely reversible following washout and 3 h rest. The results obtained with AVT were similar to those previously found using the mammalian neuropeptide arginine vasopressin. AVT may act by increasing the activity of central neurons which exert suppressive control over both gill reflex behaviors and evoked activity to central gill motor neurons.