Peptides (v.22, #4)

Recognition and oxidative metabolism of cyclodipeptides by hepatic cytochrome P450 by Marcel Delaforge; Geneviève Bouillé; Maryse Jaouen; Christopher K Jankowski; Christine Lamouroux; Claude Bensoussan (557-565).
Possible recognition of peptide derivatives by hepatic cytochrome P450 3A has been suggested by binding and metabolism of numerous pseudopeptidic compounds such as ergot derivatives and cyclosporin.Natural linear or cyclic dipeptides containing hydrophobic amino acids produced by microorganisms and present in mammals are able to interact with the P450 active site through either iron-amine interactions (Type II) or hydrophobic Type I interactions. P450 3A from dexamethasone-treated rats or yeast-expressed P450 human 3A4 are the most potent in such interactions, which are particularly strong with peptides containing a histidyl residue.Some cyclodipeptides are rapidly transformed by rat cytochrome P450 3A to mono- or dihydroxylated metabolites, with turnovers around 3 nmoles min−1 P450−1. Linear peptides are poorly transformed in these conditions. This metabolism of cyclodipeptides occurs in 8 species including man.Such interactions and metabolism have only minor consequences in terms of P450 3A binding and metabolism of classical P450 3A substrates. These data reinforce the concept that, in addition to their effect on the regulation of P450 neosynthesis, naturally occurring endogenous peptides are also substrates of P450 3A. The physiological activities of these peptides may be modulated by their metabolism.
Keywords: cytochrome P450 3A; amino acids; cyclopeptides; diketopiperazine; peptides; active site binding;

Solution NMR structure of a model class A (apolipoprotein) amphipathic α helical peptide☆ by Vinod K. Mishra; Mayakonda N. Palgunachari; G.M. Anantharamaiah; Martin K. Jones; Jere P. Segrest; N.Rama Krishna (567-573).
To better understand the structural determinants of the physical-chemical and the biological properties of Ac-18A-NH2 (acetyl-AspTrpLeuLysAlaPheTyrAspLysValAlaGluLysLeuLysGluAlaPhe-amide), we have determined its structure in 50% (v/v) trifluroethanol (TFE-d3)/water mixture (5 mM potassium phosphate, pH 5.5, 310K) using two-dimensional proton NMR spectroscopy. Stereospecific assignments have been made for CβH protons (all the residues except Ala and Val) and γCH3 (Val) groups. Nuclear Overhauser effects are observed between the nonpolar side chains spaced at (i) and (i + 4) position in the primary sequence, e.g., Trp2 and Phe6, and Phe6 and Val10. This suggests that in addition to N-terminal acetyl and C-terminal amide groups, the amphipathic α helical structure of Ac-18A-NH2 is further stabilized by interactions between the hydrophobic residues on the nonpolar face of the helix.
Keywords: Amphipathic α helix; Peptide; NMR; Apolipoproteins; Trifluoroethanol;

Functional analysis of glucose-dependent insulinotropic polypeptide fusion proteins by Ke-Hong Ding; Carlos M. Isales; Qing Zhong; Roni J. Bollag (575-582).
To generate functional fluorescently tagged glucose-dependent insulinotropic polypeptide (GIP), a series of GIP expression constructs were devised. These included G1 (complete preprohormone), G2 (lacking the C-terminal extension), G3 (lacking both N- and C-terminal extensions), G4 (G2 fused to green fluorescent protein, GFP), and G5 (G3 fused to GFP). Expression of G5 in bacteria generated immunopositive GIP together with GFP fluorescence, while G4 generated only fluorescence without immunoreactivity. Transfection of NIH3T3 cells with cDNAs of G1, G3, G5, but not G2, G4, and EGFP, resulted in immunologically detectable GIP formation, although fluorescence could be detected in the latter two. GIP as well as GIP-GFP secreted by NIH3T3 cells significantly stimulated intracellular cAMP accumulation and Ca2+ mobilization in SaOS2 cells. The GIP receptor antagonist GIP(7-30) abolished these responses. These results suggest that a GIP-GFP fusion protein seven times larger than the native peptide retains function and may be used as an in vivo probe to detect GIP receptor distribution and to explore GIP’s biological roles.
Keywords: Glucose-dependent insulinotropic polypeptide (GIP); Green fluorescent protein (GFP); Signal transduction; Fusion protein; Peptide secretion;

Peripheral administration of urocortin suppresses operant responding for food reward by Jefferson W. Kinney; Brandi Scruggs; David D. Avery (583-587).
The effects of peripheral systemic administration of urocortin on operant responding to obtain food were investigated using three separate concentrations. The drug was administered intraparitoneally at a concentration of 10 μg/ml/Kg, 5 μg/ml/Kg, and 0 μg/ml/Kg suspended in saline at a volume of 1 ml/Kg to Sprague-Dawley rats fifteen minutes prior to being exposed to an operant bar press task. Eleven subjects were used, each receiving a single injection of each concentration on separate days with the order of treatment counterbalanced. The results indicated that the administration of urocortin in a dose dependent manner reduced responding of food deprived subjects for a food reward in a thirty minute session. These data indicated that the peripheral administration of urocortin reduced the motivation of food deprived subjects to respond.
Keywords: Urocortin; Corticotropin-releasing factor; Satiety; Operant responding;

Proadrenomedullin N-terminal 20 peptide (PAMP) inhibits food intake and gastric emptying in mice by Kousaku Ohinata; Akio Inui; Akihiro Asakawa; Keiji Wada; Etsuko Wada; Masaaki Yoshikawa (589-595).
We found that proadrenomedullin N-terminal 20 peptide (PAMP) decreased dose-dependently (3–30 nmol/mouse) food intake after intra-third cerebroventricular administration in fasted ddY mice. Gastric emptying also was delayed after central injection of PAMP. In our previous study, PAMP was demonstrated to elicit hyperglycemia via bombesin (BN) receptor. Then, we examined whether the effects of PAMP on feeding and gastric emptying were induced through BN receptor. Surprisingly, PAMP-induced reductions in feeding and gastric emptying rate were not blocked by a BN antagonist, [d-Phe6, Leu-NHEt13, des-Met14]-BN (6–14). PAMP suppressed feeding in mice lacking gastrin-releasing peptide receptor or BN receptor subtype-3. These results indicate that centrally administered PAMP inhibits food intake, involving the delayed gastric emptying, not through BN receptors but through selective PAMP receptor.
Keywords: Proadrenomedullin N-terminal 20 peptide (PAMP); Food intake; Gastric emptying; Bombesin;

Human pheochromocytomas, but not adrenal medulla, express glucagon-receptor gene and possess an in vitro secretory response to glucagon by Giovanna Albertin; Francesco Aragona; Lucia Gottardo; Ludwik K. Malendowicz; Gastone G. Nussdorfer (597-600).
Glucagon-receptor mRNA was detected by reverse transcription-polymerase chain reaction in three human pheochromocytomas, but not in four normal adrenal medullas. Quantitative autoradiography demonstrated the presence of abundant [125I-Thyr10]glucagon binding sites in pheochromocytomas, which were displaced by both cold glucagon and the glucagon receptor antagonist Des-His1[Glu9]glucagon amide (GR-A). Adrenal medulla was weakly labeled, and the binding was not displaced by GR-A. Glucagon enhanced epinephrine and norepinephrine release by pheochromocytoma slices, minimal and maximal effective concentrations being 10−8 M and 10−6 M. Adrenomedullary slices evidenced a weak catecholamine response only to 10−5 M glucagon. GR-A abolished the secretory response to glucagon of pheochromocytomas, but not of adrenal medullas. Collectively, these findings indicate that human pheochromocytomas, but not adrenal medulla, express glucagon receptors and possess a marked secretory response to glucagon, thereby providing the rationale to explain the specificity of the glucagon provocative test in the diagnosis of pheochromocytoma.
Keywords: Pheochromocytoma; Adrenal medulla; Glucagon; Glucagon receptors;

Effects of naltrexone and CCK on estrous behavior and food intake in Syrian hamsters by Juli E. Jones; Eric S. Corp; George N. Wade (601-606).
Food deprivation inhibits estrous behavior in several species of rodents, but little is known about the neurotransmitter systems mediating this phenomenon. We determined whether partial blockade of opioid receptors by continuous infusion of naltrexone and/or acute peripheral injection of cholecystokinin (CCK) administration would overcome the suppressive effects of food deprivation on estrous behavior in Syrian hamsters. Contrary to expectation, naltrexone produced a slight suppression of estrous behavior, and systemic CCK administration had no effect. This dose of naltrexone was sufficient to reduce in vivo binding of [3H]naloxone in the brain, and both compounds affected other parameters such as food intake and body weight gain. Thus, the doses of CCK and naltrexone that were used were physiologically effective. These findings suggest that neither peripheral CCK nor opioid systems are likely to play a major role in the suppression of hamster estrous behavior by food deprivation.
Keywords: Naltrexone; CCK; Estrous behavior; Lordosis; Hamster; Food intake; Opioids;

Naloxone blocks ‘anxiolytic’ effects of neuropeptide Y by Karen Thatcher Britton; Scott Southerland (607-612).
Intracerebroventricular injection of neuropeptide Y (NPY) produces potent ‘anxiolytic’ effects in animal models of anxiety. Administration of opioid receptor antagonists suppresses NPY-induced food intake and thermogenesis. The present study examined whether the opiate antagonist naloxone would also suppress the ‘anxiolytic’ effects of neuropeptide Y. Following training and stabilization of responding in an operant conflict model of anxiety, rats were injected with either NPY or diazepam. Both NPY (veh., 2, 4, 6 μg, icv) and chlordiazepoxide (veh., 2, 4, 6 mg/kg, i.p.) produced a dose-dependent increase in punished responding in the conflict test. The ‘anxiolytic’ effects of NPY were not blocked by the administration of flumazenil (3, 6, 12 mg/kg, ip). The administration of naloxone (0.25–2.0 mg/kg, s.c) antagonized the effects of NPY. Central administration of the selective mu opiate antagonist CTAP (1 μg, icv) partially blocked NPY-induced conflict responding. These results support the hypothesis that NPY may play an important role in experimental anxiety independent of the benzodiazepine receptor and further implicate the opioid system in the behavioral expression of anxiety.
Keywords: Neuropeptide Y; Naloxone; Conflict test; Anxiety;

In vitro metabolism of opioid tetrapeptide agonists in various tissues and subcellular fractions from rats by E Krondahl; H von Euler-Chelpin; A Orzechowski; G Ekström; H Lennernäs (613-621).
The metabolism of three μ-selective opioid tetrapeptide agonists, Tyr-d-Arg-Phe-Nva-NH2 (TArPN), Tyr-d-Arg-Phe-Phe-NH2 (TArPP), and Tyr-d-Ala-Phe-Phe-NH2 (TAPP), was investigated in different rat tissues. High metabolic activity (<20% peptide remaining after 30 min) was found against the three peptides in the kidney homogenate and against TArPN in spleen homogenate. Low metabolic activity (>80% peptide remaining after 30 min) was found for all peptides in brain homogenate and plasma, and for TArPN and TArPP in blood. The other tissue homogenates, prepared from the small and large intestine, liver and lung, all exhibited intermediate metabolic activity (20–80% peptide remaining after 30 min) against the peptides. In all tissues investigated, the tetrapeptides were metabolized at the C-terminal amide by deamidation.A further in depth metabolic investigation was performed in subcellular fractions isolated from three tissues (small intestine, liver and kidney). In the liver, the deamidation was predominantly localized to the mitochondrial/lysosomal fraction, while hydrolysis at the N-terminal Tyr residue was the major metabolic pathway in the microsomal/brush-border membrane fraction from the kidney and small intestine.
Keywords: Opioid peptides; Peptide hydrolysis; Metabolism; Homogenate; Subcellular fractions; Blood; Plasma; Deamidation;

[125I]EYF: a new high affinity radioligand to neuropeptide FF receptors by Christine Gouardères; Catherine Mollereau; Jean A.M. Tafani; Honoré Mazarguil; Jean-Marie Zajac (623-629).
[125I]EYF ([125I]EYWSLAAPQRFamide), a new radioiodinated probe derived from a peptide present in the rat Neuropeptide FF precursor (EFWSLAAPQRFamide, EFW-NPSF) was synthesized and its binding characteristics investigated on sections of the rat spinal cord and on membranes of mouse olfactory bulb. In both tissues, [125I]EYF binding was saturable and revealed a very high affinity interaction with a single class of binding sites in rat and mouse (KD = 0.041 and 0.019 nM, respectively).Competition studies showed that [125I]EYF bound to one class of binding sites exhibiting a high affinity for all the different peptides the precursor could generate (NPA-NPFF, SPA-NPFF, NPFF, EFW-NPSF, QFW-NPSF) with the exception of NPSF which displayed a low affinity.Autoradiographic studies demonstrated that [125I]EYF binding sites were fully inhibited by a synthetic Neuropeptide FF agonist (1DMe) in all areas of the rat brain. The density of [125I]EYF binding sites was high in the intralaminar thalamic nuclei, the parafascicular thalamic nucleus and in the superficial layers of the dorsal horn.Non specific binding reached 5–10% of the total binding in all brain areas. Similarly, in mouse brain experiments, the non-specific binding was never superior to 10%.These findings demonstrate that putative neuropeptides generated by the Neuropeptide FF precursor and containing the NPFF or NPSF sequences should bind to the same receptor. Furthermore, these data indicate that [125I]EYF is a useful radiolabeled probe to investigate the NPFF receptors; its major advantages being its high affinity and the very low non-specific binding it induces.
Keywords: NPFF receptors; Radioligand binding; Autoradiography;

Opioid peptides attenuate blood pressure increase in acute respiratory failure by Fiorella Fontana; Pasquale Bernardi; Lucia Tartuferi; Stefano Boschi; Rosanna Di Toro; Santi Spampinato (631-637).
Plasma opioid peptides, norepinephrine, atrial natriuretic factor (ANF) and blood pressure (BP) were assessed in 24 chronic obstructive pulmonary disease patients with acute respiratory failure. Hypoxemic-hypercapnic patients had high BP, β-endorphin, Met-enkephalin and dynorphin B, whereas hypoxemic-normocapnic and hypoxemic-hypocapnic patients showed normal BP, high β-endorphin, and normal Met-enkephalin and dynorphin B. Norepinephrine and ANF were high in all patients, particularly in hypoxemic-hypercapnic patients. Infusion with the opioid antagonist naloxone hydrochloride significantly increased systolic blood pressure (SBP) in hypoxemic-hypercapnic (182.0 ± 3.2 versus 205.1 ± 3.0 mmHg; P < 0.01), hypoxemic-normocapnic (149.3 ± 1.8 versus 169.1 ± 2.2 mmHg; P < 0.01) and hypoxemic-hypocapnic (147.3 ± 1.3 versus 166.8 ± 2.2 mmHg; P < 0.01) patients, norepinephrine in hypoxemic-hypercapnic patients (3583.2 ± 371.8 versus 5371.3 ± 260.0 fmol/ml; P < 0.01), and reduced ANF in hypoxemic-normocapnic (18.3 ± 0.8 versus 11.9 ± 1.0 fmol/ml; P < 0.05) and hypoxemic-hypocapnic (18.1 ± 1.2 versus 12.1 ± 2.1 fmol/ml; P < 0.05) patients. These results indicate that the endogenous opioid system attenuates SBP responses in acute respiratory failure by affecting norepinephrine or ANF release.
Keywords: Acute respiratory failure; Hypoxemia; Hypercapnia; β-endorphin; Met-enkephalin; Dynorphin B; Norepinephrine; Atrial natriuretic factor;

Mechanisms underlying increased release of endothelin-1 from aorta in diabetic rats by Ayako Makino; Shu-Ichi Oda; Katsuo Kamata (639-645).
To clarify the mechanism underlying increased endothelin-1 release in diabetic rats, we examined its release from thoracic aortas obtained from streptozotocin-induced diabetic rats. The methoxamine-induced contraction was significantly inhibited by BQ-123 plus BQ-788 (specific antagonists for ETA and ETB receptors) in diabetic, but not control rats. Preincubation with phosphoramidon also inhibited the methoxamine-induced contraction in diabetic but not control rats. The expression of prepro endothelin-1 mRNA was significantly enhanced in aortas from streptozotocin-induced diabetic rats. These results suggest that the increases in the basal and α-agonist-induced release of endothelin-1 in the diabetic state may be due to an overexpression of the mRNA for prepro endothelin-1.
Keywords: Endothelin-1; Methoxamine; Diabetes; Thoracic aorta; PreproET-1 mRNA; Streptozotocin; Rats;

About 8- and ∼84-h rhythms in endotheliocytes as in endothelin-1 and effect of trauma by G.S Katinas; F Halberg; G Cornélissen; D Hawkins; M.V Bueva; D.E Korzhevsky; L.R Sapozhnikova; N Rhodus; E Schaffer (647-659).
Population densities (PD) of capillaries (C) and endotheliocytes (E) were determined in pinnal dermis of C57BL mice before and after trauma. Moving (and overall) least-squares spectra before trauma detected in EPD (versus CPD) pronounced 3.5-day (circasemiseptan) and 8-h oscillations corresponding to components of the endothelin-1 chronome in human blood plasma reported earlier. Circadians were more pronounced in CPD. After trauma, circasemiseptan oscillations appeared also in CPD; their period gradually shortened and in two weeks split into about 2.5- and about 4.5-day oscillations; and circadian components became very pronounced. The pre-traumatic chronome was not restored within three weeks following trauma.
Keywords: Circasemiseptan; Circadian; Chronome; ET-1; Endotheliocytes; Capillaries; Population density; Regeneration; Trauma;

Neutrophil apoptosis is an important event in the resolution of inflammation. The role of substance P (SP) in neutrophil apoptosis has not been previously investigated. We found that substance P delays apoptosis in neutrophils. Human neutrophils were isolated and cultured up to 24 hours. Apoptosis was detected by light and electron microscopy, as well as DNA-fragmentation assays. Substance P delayed the spontaneous apoptosis of neutrophils at 6, 12, 18 and 24 hours in a dose-dependent fashion in the range of 10–100 μM. Whereas the both peptide neurokinin-1 (NK-1) receptor antagonists [D-Pro2, D-Trp7,9]-SP and GR 82334 inhibited the substance P effect on neutrophils, the nonpeptide NK1 receptor antagonist L-703.606 itself, an analogue of CP-96,345, induced apoptosis of neutrophils. Surprisingly, the effect of L-703.606 could be prevented by substance P. Western blotting results showed that the neuropeptide substance P inhibited the spontaneous apoptosis-associated caspase-3 activation in the same concentration range as described above. Parallel the inhibition of cleavage of focal adhesion kinase (FAK), a substrate of caspases could be observed by substance P. In conclusion, our results extend the range of biological effects of the neuropeptide substance P and provide new insight to the role of this tachykinin in the modulation of the inflammatory response by the nervous system.
Keywords: Substance P; Apoptosis; Neutrophils; Caspase-3;

Liposomal VIP potentiates DNA synthesis in cultured oral keratinocytes by Israel Rubinstein; Sumeet Dagar; Varun Sethi; Aparna Krishnadas; Hayat Önyüksel (671-675).
The purpose of this study was to determine whether association of vasoactive intestinal peptide with sterically stabilized liposomes (VIP on SSL) amplifies DNA synthesis evoked by the peptide in cultured chemically transformed hamster oral keratinocytes (HCPC-1) and, if so, whether this response in mediated, in part, by SSL-induced inactivation of neutral endopeptidase 24.11 (NEP; EC and angiotensin I-converting enzyme (ACE; EC, two ectoenzymes that modulate HCPC-1 cell growth, in these cells. We found that VIP (10−9–10−6 M) alone elicited a modest, albeit significant, concentration-dependent increase in DNA synthesis in HCPC-1 cells that was maximal after 48–72-h incubation (p < 0.05). VIP on SSL potentiated DNA synthesis in these cells relative to VIP alone. The magnitude of VIP on SSL-induced responses was 1.2–1.6-fold higher than that of VIP alone with maximal effects observed at 10−9 M and 10−6 M after 72- and 48-h incubation, respectively. Empty SSL had no significant effects on DNA synthesis. Empty SSL and VIP on SSL had no significant effects on NEP 24.11 and ACE activity in HCPC-1 cells. Collectively, these data indicate that association of VIP with SSL potentiates DNA synthesis in cultured oral keratinocytes relative to VIP alone and that this response is not related to non-specific effects of SSL.
Keywords: Oral mucosa; Epithelium; HCPC-1 cells; Cell proliferation; BrdU; Neuropeptide; Hamster; NEP; ACE;

Phorbol ester differentially regulates oxytocin receptor binding activity in hypothalamic cultured neurons and astrocytes by Marie-Thérèse Strosser; Marie-Elisabeth Evrard; Christophe Breton; Dominique Guenot-Di Scala (677-683).
Hypothalamic cultured neurons and astrocytes were used to investigate the cellular mechanisms underlying the oxytocin receptor-mediated downregulation through a possible involvement of protein kinase C (PKC). For this purpose, the effects of PKC activators, inhibitor and of OT on OT receptor binding activity were compared in both cultures. In neurons, phorbol-myristate-acetate (PMA), a potent PKC activator, increased the binding of an OT receptor antagonist whereas in astrocytes, a decrease was observed. Pre-treatment of the cells with bisindolylmaleimide (10−4 M), a PKC inhibitor, prevented the PMA-induced up- and downregulation. In contrast, receptor downregulation resulting from treatment of both cells with OT (10−9 M) was not affected by the PKC inhibitor. On the other hand, when PMA (10−7 M) was tested along with OT (10−9 M), a subsequent decrease in ligand binding was observed in astrocytes. In neurons, PMA attenuated the OT-induced downregulation. Structural analysis of neuron and astrocyte OT receptor mRNA by RT-PCR, subcloning and sequencing, demonstrated identical sequence to rat uterine receptor. In conclusion, these data suggest that activation of PKC has opposite effect on OT receptor binding activity in neurons and astrocytes but they do not support the involvement of PKC in the OT-induced downregulation.
Keywords: Protein kinase C; Oxytocin receptor; Neurons; Astrocytes; Phorbol ester;

Beta endorphin-like immunoreactivity in human aqueous humor and crystalline lens by Tamás Bender; Irén Török; István Barna; Pál Géher (685-687).
We measured the concentration of β-endorphin (β-End) in plasma, as well as in aqueous humor and crystalline lens removed during cataract surgery. β-End was detected both in the aqueous humor and in the crystalline lens. The concentration of β-End in the aqueous humor corresponded to almost the half of the plasma level (2.18 fmol/l and 4.55 fmol/l). Endogenous β-End is presumed to enter the intraocular structures by passive diffusion.
Keywords: Beta-endorphin; Aqueous humor; Crystalline lens; Transport;

Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are frequently expressed by cancers of the gastrointestinal tract, breast, lung, and prostate. Most studies have found that GRP and its amphibian homologue bombesin act to increase tumor cell proliferation, leading to the hypothesis that this peptide hormone is a mitogen important for the growth of various cancers. Yet GRP/GRP-R co-expression in cancer promotes the development of a well-differentiated phenotype; while multiple studies suggest that the presence of these 2 proteins confer a survival advantage. Along with recent reports showing that GRP and its receptor critically regulate aspects of colon and lung organogenesis, we argue that these proteins do not function primarily as mitogens when aberrantly expressed in cancer. Rather, we postulate that GRP/GRP-R are onco-fetal antigens that function as morphogens, with their effect on tumor cell proliferation being a component property of their ability to regulate differentiation. Thus aberrant GRP/GRP-R expression in cancer recapitulates, albeit in a dysfunctional manner, their normal role in development.
Keywords: FAK, focal adhesion kinase; GI, gastrointestinal; GRP, gastrin-releasing peptide; GRP-R, GRP receptor; SCCL, small cell cancer of the lung;