Peptides (v.21, #10)

A cell-permeable peptide inhibits activation of PKR and enhances cell proliferation by Sergei Nekhai; Donald P Bottaro; Girma Woldehawariat; Amy Spellerberg; Raymond Petryshyn (1449-1456).
The double-stranded RNA dependent protein kinase (PKR) is a negative regulator of cell proliferation and thus itself a target for modulation. We show that a cell-permeable peptide (PRI), containing a conserved double-stranded RNA binding motif found in PKR, inhibits activation of the kinase and activity to phosphorylate its substrate. Further, the PRI-peptide localizes to the cytoplasm of murine embryonic fibroblasts and ablates cellular PKR activation. The PRI-peptide enhances cell proliferation compared to treatment with a variant control peptide, resulting in cultures with increased cell density. We conclude that peptides that interfere with PKR may be useful tools for regulating cell proliferation.
Keywords: PKR; Cell-permeable peptide; eIF-2α phosphorylation; Regulation of cell proliferation;

We report the discovery of a linear lead antagonist for the insect pheromone biosynthesis activating neuropeptide (PBAN) which inhibits sex pheromone biosynthesis in the female moth Heliothis peltigera. Two approaches have been used in attempting to convert PBAN agonists into antagonists. The first involved omission of the C-terminal amide and reduction of the sequence from the N-terminus in a linear library based on PBAN 1–33NH2. The second involved replacement of L amino-acids by the D hydrophobic amino acid D-Phe in a linear library based on PBAN28–33NH2. Screening of the two libraries for pheromonotropic antagonists resulted in the disclosure of one compound out of the D-Phe library (Arg-Tyr-Phe-D-Phe-Pro-Arg-Leu-NH2) which inhibited sex pheromone production by 79 and 64% at 100 pmol in two moth colonies and exhibited low agonistic activity. Omission of the C-terminal amide in PBAN 1–33NH2 and its shorter analogs did not lead to the discovery of an antagonistic compound.
Keywords: PBAN; Pheromone biosynthesis; Insect neuropeptides; Heliothis peltigera; Neuropeptide antagonists;

Uroguanylin, a well-known ligand of guanylyl cyclase C receptor in the gastrointestinal tissue, has recently been reported to have pulmonary effects. We investigated the inhibitory effects of uroguanylin against leukotriene C4-induced bronchoconstriction and airway microvascular leakage. Anesthetized guinea pigs, ventilated via a tracheal cannula in a plethysmograph box, were measured by pulmonary mechanics for 10 min after i.v. administering 2 μg/kg leukotriene C4. Airway microvascular leakage was assessed by extravasation of Evans blue dye into airway tissues. Both inhalant and i.v. pretreatment of uroguanylin significantly inhibited leukotriene C4-induced pulmonary changes in a dose-dependent manner, suggesting its effectiveness against an asthmatic condition.
Keywords: Uroguanylin; Leukotriene C4; Inhalation; Bronchoconstriction; Airway microvascular leakage; Guanylyl cyclase C receptor; Guinea-pig;

NF-κB activation is inhibited in human pulmonary epithelial cells transfected with α-melanocyte-stimulating hormone vector by Takashi Ichiyama; Kuniyuki Okada; Iain L. Campbell; Susumu Furukawa; James M. Lipton (1473-1477).
α-Melanocyte-stimulating hormone (α-MSH) modulates inflammation. We investigated the influence of α-MSH on NF-κB activation in human pulmonary epithelial cells (A549) using a plasmid vector encoding α-MSH (pCMV-ssMSH). Electrophoretic mobility shift assays demonstrated that NF-κB activation induced by lipopolysaccharide was inhibited in A549 cells transfected with pCMV-ssMSH. Western blot analysis revealed that this inhibition was linked to preservation of expression of IκBα protein. Chloramphenicol acetyltransferase assay indicated that NF-κB-dependent reporter gene expression was suppressed in A549 cells transfected with pCMV-ssMSH. The findings indicate that anti-inflammatory actions are exerted via modulation of NF-κB activation by preservation of IκBα protein in human pulmonary epithelial cells transfected with α-MSH vector. We showed a possibility of gene therapy for chronic inflammatory lung diseases.
Keywords: α-Melanocyte-stimulating hormone; A549 cell; NF-κB; IκBα protein; Gene therapy;

Leptin resistance in obesity is characterized by decreased sensitivity to proopiomelanocortin products by Huiqing Lu; Anne Buison; K.-L.Catherine Jen; Joseph C. Dunbar (1479-1485).
Obesity in normal animals has been demonstrated to be associated with a decrease in sensitivity to leptin especially as it relates to leptin’s capacity to increase sympathetic nerve activity and enhance cardiovascular dynamics. In normal animals leptin has been demonstrated to exert significant regulatory responses by its capacity to increase proopiomelanocortin (POMC) expression and especially the increase in alpha melanocyte stimulating hormone (αMSH). These responses to leptin are blocked by a melanocortin-4 (MC-4) receptor antagonist. In this study we investigated the responsiveness of the sympathetic nervous system and cardiovascular system of high fat fed obese animals to the intracerebroventricular (ICV) administration of the POMC products αMSH and β-endorphin (β-END). We further investigated these responses in obese animals following leptin administration in the presence of MC-4 receptor and opioid receptor blockade. The ICV administration of leptin resulted in an increase in lumbar sympathetic nerve activity (LSNA) and mean arterial pressure (MAP) in normals but decreased it in the obese. The ICV administration of αMSH increased the LSNA and MAP in normal animals but to a lesser degree in obese animals. On the other hand β-endorphin decreased the LSNA and MAP in normal animals but increased it in obese animals. Additionally ICV leptin administration in obese animals in the presence of MC-4 or opioid receptor blockade resulted in an increase in sympathetic activity and a pressor response. From these studies we conclude that obesity in high fat fed animals is characterized by a decreased sensitivity to αMSH and a paradoxical response to β-endorphin and this altered responsiveness may be a factor in the altered leptin resistance characteristic of obese animals.

To test if elevated CRH and decreased NPY might account for pituitary-adrenal activity and hypophagia in dietary protein deprivation, rats received normal or protein-free diet, or were food-or weight-restricted to match effects of protein deprivation. Protein or food restriction increased plasma ACTH. However, hypothalamic CRH mRNA was unchanged by protein deficiency and significantly decreased by food restriction when protein intake was > 50% of normal. Arcuate nucleus NPY mRNA increased in rats given protein-free diet, correlating with leptin rather than decreased feeding. We conclude that CRH and NPY gene expression does not explain adrenocortical axis or feeding activity in protein-deprived rats.
Keywords: Malnutrition; Anorexia; Food intake; Corticotropin-releasing hormone; Neuropeptide Y; Hypothalamus; Weight loss;

NPY receptors and opioidergic system are involved in NPY-induced feeding in goldfish by Nuria de Pedro; Marcos A López-Patiño; Ana I Guijarro; Ma Luisa Pinillos; Ma Jesús Delgado; Mercedes Alonso-Bedate (1495-1502).
The present study evaluated the effects of both intraperitoneal (i.p.) and intracerebroventricular administration of selective Y1 [(Leu31, Pro34)-NPY] and Y2 [(Pro13, Tyr36)-NPY (13–36)] receptor agonists on food intake in satiated goldfish. Food intake (FI) was significantly increased by central administration of the Y1 agonist (1 μg), but not by the Y2 agonist, at 2 h postinjection. The feeding increase induced by (Leu31, Pro34)-NPY was in a similar magnitude to that obtained after ICV injection of the neuropeptide Y, and both feeding stimulations were reversed by the NPY (27–36), a general NPY antagonist. The i.p. administration of the agonists either did not significantly modify (Y2 agonist) or decreased (Y1 agonist) food intake in goldfish. These data indicate that it is the Y1-like (similar to Y1 and/or Y5) receptor, and not Y2, that is involved in the central modulation of the feeding behavior in goldfish. We also investigated the possible involvement of opioid peptides as mediators of the NPY stimulatory action on food intake in goldfish. The ICV administration of naloxone (10 μg), a general opioid antagonist, blocked the NPY-induced feeding in goldfish, suggesting that the opioidergic system is involved in feeding regulation by NPY.
Keywords: NPY, neuropeptide Y; Fish; Goldfish; NPY receptors; Agonists; Antagonists; Food intake; Feeding; Opioid receptors; Naloxone;

Neuropeptides modulate the function of classic neurotransmitters in the regulation of body function. The role of neuropeptides in the regulation of endocrine secretion from the pancreas of diabetic rat is poorly understood. This study examined the pattern of distribution of neuropeptide-Y (NPY) and substance P (SP) in normal and diabetic rat pancreases. In addition to this, the effect of NPY and SP on glucagon secretion was also examined in the pancreases of normal and diabetic rats. Four weeks after the induction of diabetes, the pancreaseses of normal and diabetic rats were removed and processed for immunohistochemistry and glucagon secretion. The pattern of distribution of glucagon in the pancreas of diabetic rat was conspicuously deranged after the onset of diabetes. The pattern of distribution of NPY and SP was, however, similar in the pancreases of both normal and diabetic rats. Stimulation of normal rat pancreatic tissue with NPY (10−12 and 10−9 M) evoked large and significant (P < 0.001) increases in glucagon secretion compared to basal. In contrast to this, NPY inhibited glucagon secretion from the pancreas of diabetic rat. Treatment of pancreatic tissue fragments of normal rat with 10−9 M SP resulted in significant (P < 0.03) increases in glucagon secretion. SP inhibited glucagon secretion from diabetic rat pancreas. In conclusion, NPY and SP stimulated glucagon secretion from the pancreas of normal rat. In contrast, NPY and SP inhibited glucagon secretion from diabetic rat pancreas.
Keywords: Glucagon secretion; Neuropeptide Y; Substance P; Radioimmunoassay; Immunohistochemistry; Neuropeptides; Pancreas; Rat;

Potential targets for glucagon-like peptide 2 (GLP-2) in the rat: distribution and binding of i.v. injected 125I-GLP-2 by Jesper Thulesen; Bolette Hartmann; Cathrine Ørskov; Palle Bekker Jeppesen; Jens Juul Holst; Steen Seier Poulsen (1511-1517).
Glucagon-like peptide 2 (GLP-2) is a 33-amino acid (1–33) intestinotrophic peptide. In this study, the distribution and binding of i.v. injected radiolabeled GLP-2 (1–33) were investigated in rats using autoradiography in order to target possible binding sites. The major part of 125I-GLP-2 (1–33) was distributed to kidneys, liver, and the gastrointestinal tract. In the small intestine, a high density of grains was localized in the epithelium with a predominance in the luminal part of the villus. The saturability of 125I-GLP-2 (1–33) was investigated by administration of excess amounts of non-radioactive GLP-2 (1–33) or the primary metabolite of GLP-2 degradation, GLP-2 (3–33). In the small intestine, 125I-GLP-2 was displaced both by non-radioactive GLP-2 (1–33) and (3–33), suggesting that the uptake of GLP-2 (1–33) in the small intestine is receptor-specific and that the metabolite GLP-2 (3–33) may interact with the GLP-2 receptor.
Keywords: Proglucagon-derived peptides; Glucagon-like peptide 2; Metabolism; Autoradiography; Rat;

Structure, antihyperglycemic activity and cellular actions of a novel diglycated human insulin by F.P.M. O’Harte; A.C. Boyd; A.M. McKillop; Y.H.A. Abdel-Wahab; H. McNulty; C.R. Barnett; J.M. Conlon; P. Højrup; P.R. Flatt (1519-1526).
Human insulin was glycated under hyperglycemic reducing conditions and a novel diglycated form (Mr 6135.1 Da) was purified by RP-HPLC. Endoproteinase Glu-C digestion combined with mass spectrometry and automated Edman degradation localized glycation to Gly1 and Phe1 of the insulin A- and B-chains, respectively. Intraperitoneal (i.p.) administration of diglycated insulin to mice alone or in combination with glucose (7 nmol/kg) resulted in a 43–61% and 11–34% reduction in glucose lowering activity, respectively, compared with native insulin. Consistent with these findings, diglycated insulin (10−9 to 10−7 mol/liter) was 22–38% less effective (P < 0.001) than native insulin in stimulating glucose uptake, glucose oxidation and glycogen production in isolated mouse abdominal muscle.
Keywords: Glycation; Insulin; Diglycated insulin; Glucose transport and metabolism; Glucose homeostasis; Mass spectrometry;

Caseinomacropeptide specifically stimulates exocrine pancreatic secretion in the anesthetized rat by Nathalie Ledoux-Rolf Pedersen; Claire Nagain-Domaine; Sylvain Mahé; Jacques Chariot; Claude Rozé; Daniel Tomé (1527-1535).
The effect of caseinomacropeptide (CMP) (the [106–169] fragment of κ-casein produced during digestion of milk protein), was studied in anesthetized rats using bile diversion for a pure pancreatic juice collection system. Intraduodenal administration of CMP induced a dose-related specific stimulation of pancreatic secretion which was nearly abolished by devazepide, atropine, hexamethonium, vagotomy or perivagal capsaicin pretreatment. Moreover, CMP did not inhibit in vitro trypsin activity. These results demonstrate that CMP is more likely to stimulate pancreatic secretion specifically through cholecystokinin release and activation of a vago-vagal cholinergic reflex loop than by inhibition of luminal trypsin, in anesthetized rats.
Keywords: Bioactive peptide; Casein; CMP; CCK; Pancreatic secretion;

The role of sensory neurons in the antiulcer effect of centrally injected amylin in rat by F. Guidobono; F. Pagani; V. Sibilia; A. Soglian; D. Rapetti; C. Netti (1537-1541).
Central administration of amylin (2.2 μg/rat, i.c.v.) reduces (from a minimum of 67% to 83%) indomethacin (Indo, 20 mg Kg−1, orally) induced ulcers in rats. The anti-ulcer effect of the peptide is not removed by the administration of prokinetic drugs like domperidone or neostigmine but it is reduced by 35% in rats treated with capsaicin or with the CGRP antagonist, CGRP8–37. These data indicate that amylin gastroprotection involves capsaicin-sensitive nerve fiber leading to CGRP-dependent gastric vasodilatory effect. Additional mechanisms could involve noradrenergic α2 receptors as the peptide gastroprotective activity is reduced from 67% to 20% by the α2 antagonist yohimbine.
Keywords: Amylin; Ulcers; Indomethacin; Capsaicin; Yohimbine; CGRP;

Development of selective agonists and antagonists for the human vasoactive intestinal polypeptide VPAC2 receptor by D. Moreno; P. Gourlet; P. De Neef; J. Cnudde; M. Waelbroeck; P. Robberecht (1543-1549).
Ro 25–1553 is a cyclic VIP derivative with a high affinity for the VPAC2 receptor subtype. Our goal was to identify the modifications that support its selectivity for VPAC2 receptors, and to develop a VIP or Ro 25–1553 analog behaving as a high affinity, VPAC2 selective antagonist. The selectivity of Ro 25–1553 for the human receptor was supported mainly by the acetylation of the amino-terminus, by the introduction of a lysine residue in position 12, and by the carboxyl-terminal extension. The lactam bridge created between positions 21 and 25 contributed to the affinity of the compound for the VIP receptors but participated only marginally to its selectivity. Deletion of the first five aminoacid residues led to a low affinity antagonist with a low selectivity. Introduction of a D-Phe residue in position 2 reduced the affinity, the selectivity and the intrinsic activity, the compound being a partial agonist. Myristoylation of the amino-terminus of [K12]VIP(1–26) extended carboxyl-terminally with the -K-K-G-G-T sequence of Ro 25–1553 led to a high affinity, selective VPAC2 receptor antagonist. This molecule represents the first selective human VPAC2 receptor antagonist described to date.
Keywords: Vasoactive intestinal peptide; VPAC1 receptor; VPAC2 receptor; Selective agonist; Selective antagonist; Ro 25–1553; Lipophilic VIP analog;

Regional distribution of immunoreactive prolactin-releasing peptide in the human brain by Kazuhiro Takahashi; Ayako Yoshinoya; Zenei Arihara; Osamu Murakami; Kazuhito Totsune; Masahiko Sone; Hironobu Sasano; Shigeki Shibahara (1551-1555).
Regional distribution of prolactin-releasing peptide (PrRP) in the human brain was studied by radioimmunoassay. The antiserum raised against human PrRP-31 in a rabbit was used in the assay, which showed 100% cross reaction with PrRP-20 and no significant cross reaction with other peptides. The highest concentrations of immunoreactive-PrRP were found in hypothalamus (912 ± 519 fmol/g wet weight, n = 6, mean ± SEM), followed by medulla oblongata (496 ± 136 fmol/g wet weight) and thalamus (307 ± 117 fmol/g wet weight). On the other hand, immunoreactive-PrRP was not detected in frontal lobe or temporal lobe (<50 fmol/g wet weight). Sephadex G50 column chromatography of the immunoreactive-PrRP in the hypothalamus and medulla oblongata showed three immunoreactive peaks; one peak eluting in the position of PrRP-20, one eluting in the position of PrRP-31 and one eluting earlier. Reverse phase high-performance liquid chromatography (HPLC) of these brain tissue extracts showed a peak eluting in the position of PrRP-20 and PrRP-31. The present study has shown for the first time the presence of immunoreactive-PrRP in the human brain. The immunoreactive-PrRP levels in the human hypothalamus were, however, lower than the levels of other neuropeptides with prolactin-releasing activity, such as thyrotropin-releasing hormone and vasoactive intestinal polypeptide.
Keywords: Prolactin-releasing peptide; Hypothalamus; Brain; Radioimmunoassay;

Evidence of NPY Y5 receptor involvement in food intake elicited by orexin A in sated rats by Michael G Dube; Tamas L Horvath; Pushpa S Kalra; Satya P Kalra (1557-1560).
Intracerebroventricular (icv) injections of orexin A stimulate feeding in sated rats. Since neuropeptide Y is a potent orexigenic peptide and orexin-containing neurons are morphologically linked with NPY-producing neurons in the hypothalamus, we evaluated the functional relationship between the two orexigenic peptides. The results show that whereas it was ineffective on its own, a selective NPY Y5 receptor antagonist, injected icv 15 min. before orexin A significantly suppressed orexin A-induced feeding. Since previous investigations demonstrated that an NPY Y1 receptor antagonist also inhibits feeding induced by orexin A, the current results further underscore the existence of a functional link between orexin and NPY producing neurons as the orexin network appears to be capable of influencing NPYergic signaling through Y1 and Y5 receptors to stimulate feeding.
Keywords: Obesity; Y1 antagonist; Hypothalamus;

Release of distal gut peptide YY (PYY) by fat in proximal gut depends on CCK☆ by Henry C. Lin; William Y. Chey; Xiao-Tuan Zhao (1561-1563).
We tested the hypothesis that the release of PYY by fat confined to the proximal small intestine is dependent on CCK. Using a multi-fistulated model, plasma PYY levels were compared in 6 dogs after 60 mM oleate was perfused into the proximal one-half of the small intestine following i.v. administration of saline or devazepide, a CCK-A antagonist. Plasma PYY increased with fat (P < 0.05), but plasma PYY level was lower following devazepide at 60 min and 90 min (P < 0.05). We conclude that CCK serves as a foregut signal linking fat in the proximal gut with the release of distal gut PYY.
Keywords: Gastrointestinal motility; Gut peptides;

Peptidergic regulation of gastrointestinal motility in rodents by Mineko Fujimiya; Akio Inui (1565-1582).
Peptides involved in the endocrine and enteric nervous systems as well as in the central nervous system exert concerted action on gastrointestinal motility. Mechanical and chemical stimuli which induce peptide release from the epithelial endocrine cells are the earliest step in the initiation of peristaltic activities. Gut peptides exert hormonal effects, but peptide-containing stimulatory (Ach/substance P/tachykinin) and inhibitory (VIP/PACAP/NO) neurons are also involved in the induction of ascending contraction and descending relaxation, respectively. The dorsal vagal complex (DVC), located in the medulla of the brainstem, constitutes the basic neural circuitry of vago-vagal reflex control of gastrointestinal motility. Several gut peptides act on the DVC to modify vagal cholinergic reflexes directly (PYY and PP) or indirectly via afferent fibers in the periphery (CCK and GLP-1). The DVC is also a primary site of action of many neuropeptides (such as TRH and NPY) in mediating gastrointestinal motor activities. The identification over the last few years of a number of neuropeptide systems has greatly changed the field of feeding and body weight regulation. By exploring the brain and gut systems that employ recently identified peptidergic molecules, it will be possible to elaborate on the central and peripheral pathways involved in the regulation of gastrointestinal motility.