Peptides (v.32, #2)

Peptidomic analysis of skin secretions from the bullfrog Lithobates catesbeianus (Ranidae) identifies multiple peptides with potent insulin-releasing activity by Milena Mechkarska; Opeolu O. Ojo; Mohammed A. Meetani; Laurent Coquet; Thierry Jouenne; Yasser H.A. Abdel-Wahab; Peter R. Flatt; Jay D. King; J. Michael Conlon (203-208).
▶ A comprehensive analysis using peptidomics methodology of the insulin-releasing peptides present in skin secretions of the American bullfrog Lithobates catesbeianus. ▶ Characterization of five newly described peptides (ranatuerin-1CBb, ranatuerin-2CBc, and -CBd, palustrin-2CBa, and temporin-CBf). ▶ Identification of ranatuerin-2CDb as a potent, non-toxic insulin-releasing peptide with therapeutic potential for treatment of patients with Type 2 diabetes.Using a combination of reversed-phase HPLC and electrospray mass spectrometry, peptidomic analysis of norepinephrine-stimulated skin secretions of the American bullfrog Lithobates catesbeianus Shaw, 1802 led to the identification and characterization of five newly described peptides (ranatuerin-1CBb, ranatuerin-2CBc, and -CBd, palustrin-2CBa, and temporin-CBf) together with seven peptides previously isolated on the basis of their antimicrobial activity (ranatuerin-1CBa, ranatuerin-2CBa, brevinin-1CBa, and -1CBb, temporin-CBa, -CBb, and -CBd). The abilities of the most abundant of the purified peptides to stimulate the release of insulin from the rat BRIN-BD11 clonal β cell line were evaluated. Ranatuerin-2CBd (GFLDIIKNLGKTFAGHMLDKIRCTIGTCPPSP) was the most potent peptide producing a significant stimulation of insulin release (119% of basal rate, P  < 0.01) from BRIN-BD11 cells at a concentration of 30 nM, with a maximum response (236% of basal rate, P  < 0.001) at a concentration of 3 μM. Ranatuerin-2CBd did not stimulate release of the cytosolic enzyme, lactate dehydrogenase at concentrations up to 3 μM, indicating that the integrity of the plasma membrane had been preserved. Brevinin-1CBb (FLPFIARLAAKVFPSIICSVTKKC) produced the maximum stimulation of insulin release (285% of basal rate, P  < 0.001 at 3 μM) but the peptide was cytotoxic at this concentration.
Keywords: Frog skin; Insulin-releasing peptide; Lithobates; Ranatuerin-2;

Ghrelin inhibits insulin resistance induced by glucotoxicity and lipotoxicity in cardiomyocyte by Cuiping Cao; Ying Chen; Wei Wang; Ying Liu; Guoliang Liu (209-215).
▶ These results indicate that the direct damage of high glucose and high palmitate on cardiomyocyte might be through insulin resistance (IR). ▶ Ghrelin may inhibit the effect in cardiomyocyte through PI3K/AKT pathway in dose-dependent manner. ▶ Ghrelin also increased the mRNA levels of glucose transporter 4 (GLUT4), peroxisome proliferators (PPARr) and AMP activated protein kinase (AMPK) genes in insulin signal transduction pathway.Ghrelin has wide effects on cardiovascular and endocrine system. The aims of this study are to investigate the direct damage effect of high glucose and high palmitate on cardiomyocyte, and to study the effect of ghrelin on insulin resistance induced by glucotoxicity/lipotoxicity in cardiomyocyte and the possible mechanism underlying the cardioprotective activities of ghrelin. The changes of [3H]-2-deoxy-d-glucose (3H-G) intake rates were detected by isotope tracer method and the gene expressions in insulin signal transduction pathway were detected by real-time PCR and Western blot assay. The 3H-G intake rate significantly reduced in high glucose (25 mmol/l) or high palmitate (0.5 mmol/l) treated primary rat ventricular myocytes. After the treatment of ghrelin (10−7  mol/l), the 3H-G intake rate recovered to the normal level. In addition, the phosphorylation of AKT occurred in 10 min and was the highest in 30 min after the stimulation with ghrelin, which can be blocked by phosphoinositide 3-kinase (PI3K) inhibitor, LY2940002. Ghrelin also increased the mRNA levels of glucose transporter 4 (GLUT4), peroxisome proliferators (PPARr) and AMP activated protein kinase (AMPK) genes in insulin signal transduction pathway. These results indicate that the direct damage of high glucose and high palmitate on cardiomyocyte might be through insulin resistance (IR). Ghrelin can inhibit gluco/lipotoxicity induced insulin resistance by PI3K/AKT pathway. This may provide a clue for therapy for myocardial disease in diabetes mellitus.
Keywords: Ghrelin; Glucose; Palmitate; Insulin resistance; PI3-kinase/AKT pathway; Cardiomyocyte;

▶ VIP and VPAC1 agonist ameliorate STZ-induced type 1 diabetes. ▶ VIP and VPAC1 agonist exerted anti-oxidative and anti-inflammatory effects in diabetic mice. ▶ VPAC1 agonist displayed more effective influence in ameliorating diabetes than VIP.Vasoactive intestinal peptide (VIP) is a pleiotropic neuropeptide with potent anti-inflammatory properties, and its receptor, VPAC1, mediates most of the anti-inflammatory effects of VIP. Diabetes mellitus is characterized by increased oxidation and inflammation due to persistent hyperglycemia. This research was performed to investigate the effects of VIP and a VPAC1 agonist on streptozotocin (STZ)-induced type 1 diabetic mice. Intraperitoneal injection of VIP and VPAC1 agonist (50 nmol/kg/day in saline) over a 28-day period (1) decreased food intake, (2) increased body weight, (3) improved visceral index, (4) increased the fasting plasma insulin levels, (5) decreased the fasting plasma glucose, (6) improved the glucose tolerance, (7) decreased pancreas H2O2 and malondialdehyde (MDA) and (8) increased total antioxidant activity (T-AOC) in the liver, spleen and pancreas. The results of histopathological and immunohistochemical analysis showed that VIP and the VPAC1 agonist improved the structure and cellularity of islets and ameliorated the insulin-secreting activity of islets. Additionally, administration of VIP or the VPAC1 agonist not only significantly decreased the plasma TNFα and CRP and promoted IL-10 in diabetic mice but also blocked the increased NF-κB activity of pancreatic tissue in diabetic mice. Furthermore, the VPAC1 agonist displayed stronger effects than VIP. These results show that both VIP and VPAC1 agonist ameliorated STZ-induced diabetes and protected mice against oxidative stress and inflammation associated diabetes, with VPAC1 being the receptor most responsible for these positive effects in diabetic mice.
Keywords: Vasoactive intestinal peptide (VIP); VPAC1 agonist; Anti-inflammatory; Antioxidant; STZ-induced diabetes;

Exendin-4 improves hepatocyte injury by decreasing proliferation through blocking NGF/TrkA in diabetic mice by Selda Gezginci-Oktayoglu; Ozlem Sacan; Refiye Yanardag; Ayse Karatug; Sehnaz Bolkent (223-231).
▶ The data presented here outline the link between NGF/its receptors and hepatocyte proliferation or apoptosis, and biological effects of exendin-4 on these systems. ▶ Using immunohistochemical technique we have demonstrated for the first time that NGF and its high affinity receptor TrkA are expressed by hepatocytes and they are related with proliferation during experimental diabetes. ▶ We have shown attenuator effect of exendin-4 on hepatocyte proliferation by decreasing NGF and TrkA. ▶ With treatment of exendin-4 to the diabetic mice lipid peroxidation (LPO) level an oxidative injury marker, was decreased while activity of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione (GSH) levels were increased. ▶ To the best of our knowledge, this is the first study demonstrating the antiproliferative effect of exendin-4 on hepatocytes, NGF and its receptors and oxidative stress in liver of diabetic mice.The hepatocytes express nerve growth factor (NGF) and its high affinity receptor tyrosine kinase A (TrkA). However, the link between NGF/TrkA system and hepatocyte proliferation in diabetic animals and the effects of exendin-4, a glucagon like peptide-1 (GLP-1) receptor agonist, on this system are not known. BALB/c male mice were divided into four groups. The first group was given citrate buffer only, the second group was administered exendin-4 alone, the third group received streptozotocin (STZ), and the fourth group was given both STZ and exendin-4. Exendin-4 (3 μg/kg) was administered by subcutaneous injection daily for 30 days after the animals were rendered diabetic by administration of STZ (200 mg/kg). With treatment of exendin-4 to the diabetic mice the following results were noted (i) NGF, TrkA and proliferating cell nuclear antigen positive hepatocytes were decreased; (ii) p75 neurotrophin receptor and caspase-3 positive hepatocyte could not be detected; (iii) liver alanine transaminase and aspartate transaminase activities, lipid peroxidation, protein carbonyl and myeloperoxidase levels were decreased; (iv) liver catalase, superoxide dismutase, glutathione peroxidase activities and glutathione levels were increased. These data suggest that exendin-4 might exerts its anti-proliferative action through blocking NGF/TrkA system and stimulating oxidative defense system in liver of diabetic mice.
Keywords: Exendin-4; Hepatocyte; NGF; TrkA; p75NTR; Oxidative stress;

In vitro leptin treatment of rainbow trout hypothalamus and hindbrain affects glucosensing and gene expression of neuropeptides involved in food intake regulation by Ariel J. Aguilar; Marta Conde-Sieira; Marcos A. López-Patiño; Jesús M. Míguez; José L. Soengas (232-240).
▶ Leptin treatment directly activates glucosensor system in hypothalamus and hindbrain of rainbow trout in a way dependent on glucose concentration. ▶ Leptin treatment inhibits NPY mRNA levels in hypothalamus without affecting POMC, CART and CRF mRNA levels. ▶ Leptin treatment does not affect NPY, POMC, CART and CRF mRNA levels in hindbrain. ▶ The anorexic effect of leptin in fish can be mediated by reduced expression of NPY in hypothalamus and may be related to activation of the glucosensing system.The aim of the present study was to evaluate in hypothalamus and hindbrain of rainbow trout in vitro the effect of leptin treatment on glucosensing capacity and the expression of orexigenic and anorexigenic peptides involved in the control of food intake. In a first experiment, the response of parameters involved in glucosensing (GK, PK and GSase activities; GK expression and glucose; glycogen and DHAP levels) and the expression of orexigenic (NPY) and anorexigenic (POMC, CART, CRF) peptides was assessed in hypothalami and hindbrain incubated for 1 h with 2, 4 or 8 mM d-glucose alone (controls) or with 10 nM leptin, or with 10 nM leptin plus inhibitors of leptin signaling pathways (50 nM wortmannin and 500 nM AG490). Leptin treatment increased levels in parameters involved in glucosensing. Leptin treatment decreased NPY mRNA levels in hypothalamus without affecting the expression of the other peptides assessed. Leptin effects were reverted in the presence of inhibitors for all parameters assessed suggesting the involvement of JAK/STAT and IRS-PI(3)K pathways. In a second experiment, we observed time-dependent (1–3 h) and dose (10, 20 and 50 nM)- effects of leptin treatment in decreasing NPY mRNA levels without affecting expression of the other peptides assessed. Considering the orexigenic action of NPY in fish, it seems that the anorexic effect of leptin can be mediated by reduced expression of NPY occurring in hypothalamus, and that change can be related to the activation of the glucosensing system occurring simultaneously.
Keywords: Trout; Glucosensing; Leptin; Hypothalamus; Hindbrain; CART; POMC; CRF; NPY;

Gastrin releasing peptide-29 evokes feeding responses in the rat by Martha C. Washington; Susan A. Wright; Ayman I. Sayegh (241-245).
▶ GRP-29 may evokes feeding responses consistent with a possible role in satiety. ▶ We measured meal size, IMI and SR by GRP-29, 27 or 10. ▶ GRP-29 and 27 reduced the size of the first meal, and increased IMI and SR. ▶ The order of potency was GRP-29 = GRP-27 > GRP-10. ▶ There is a role for GRP-29 in the short-term regulation of food intake.In mammals, gastrin releasing peptide (GRP) 10 and 27 reduce food intake. In the current work, we test the hypothesis that GRP-29, the large molecular form of GRP in the rat, also evokes feeding responses consistent with a possible role in satiety. Here, we measured three feeding responses, size of first meal, intermeal interval (IMI, time between first and second meal) and satiety ratio (SR, satiation period for every unit of food consumed in the first meal), in overnight food deprived rats following GRP-10, 27 or 29 (0, 0.3, 1.0, 2.1, 4.1, 10.3, 17.2 nmol/kg) intraperitoneally and presentation of a 10% sucrose test diet. GRP-29 and GRP-27 reduced the size of the first meal, prolonged IMI and increased SR, but GRP-10 failed to exhibit similar feeding responses. The order of potency was GRP-29 = GRP-27 > GRP-10. The current data support a role for GRP-29 in the short-term regulation of food intake.
Keywords: Satiety; Satiation; Meal size; Intermeal interval; Satiety ratio;

▶ ORX-B predominantly excites the NAcSh neurons tested, whereas DA excites half of the neurons and inhibits one-fourth. ▶ DA-induced excitation is attenuated by SCH23390 or sulpiride, whereas DA-induced inhibition is suppressed by sulpiride. ▶ In two-thirds of the NAcSh neurons that respond with excitation to ORX-B, simultaneous application of ORX-B and DA increases their firing rate to two times greater than that obtained by ORX-B. ▶ These results suggest that an interaction between the ORXergic and DAergic systems occurs in the NAcSh.Orexin (ORX) plays a critical role in reward-seeking behavior for natural rewards and drugs of abuse. The mesolimbic dopamine (DA) pathway that projects into the nucleus accumbens (NAc) from the ventral tegmental area is deeply involved in the neural mechanisms underlying reward, drug abuse and motivation. A recent study demonstrated that ORX-immunopositive fibers densely project into the shell of the NAc (NAcSh), suggesting that the NAcSh might be a site of the interaction between the ORXergic and DAergic systems for reward-seeking behavior. Therefore, the electrophysiological effects of ORX-B and DA on NAcSh neurons were examined extracellularly in rat brain slice preparations. ORX-B excited approximately 78% of neurons tested and inhibited 4%, whereas DA excited 50% and inhibited 22% of NAcSh neurons. These excitations and inhibitions persisted during synaptic blockade in a low-Ca2+/high-Mg2+ solution. DA-induced excitation was attenuated by SCH23390 or sulpiride, whereas DA-induced inhibition was suppressed by sulpiride. Of the neurons that were excited by ORX-B, 71% and 18% were excited and inhibited by DA, respectively. In 63% of neurons that were excited by ORX-B, the simultaneous application of ORX-B and DA increased the firing rate to two times greater than ORX-B alone, whereas, the simultaneous application significantly decreased the neuronal firing rate by 73% in the remaining 37% compared to ORX-B. These results suggest that an interaction between the ORXergic and DAergic systems occurs in the NAcSh and that the NAcSh is involved in the neural mechanisms in which ORX participates in the regulation of reward-seeking behavior.
Keywords: Orexin/hypocretin; Dopamine; Nucleus accumbens shell; Reward-seeking behavior; Drug addiction; SCH23390; Sulpiride;

Change in plasma copeptin level after acute spontaneous basal ganglia hemorrhage by Xiao-Qiao Dong; Man Huang; Wen-Hua Yu; Zu-Yong Zhang; Qiang Zhu; Zhi-Hao Che; Quan Du; Hao Wang (253-257).
▶ Plasma copeptin level increased in patients with intracerebral hemorrhage. ▶ Copeptin could possibly serve as a novel biomarker in hemorrhagic brain injury. ▶ Copeptin may be a good prognostic factor for mortality in patients with intracerebral hemorrhage.High plasma copeptin levels are associated with mortality after intracerebral hemorrhage (ICH). However, there is a paucity of data available on whether copeptin is an independent prognostic marker of mortality. Thus, we sought to furthermore evaluate this relation. Thirty healthy controls and 86 patients with acute ICH were included. Plasma samples were obtained on admission and at days 1, 2, 3, 5, and 7 after ICH. Its concentration was measured by enzyme-linked immunosorbent assay. After ICH, plasma copeptin level in patients increased during the 6-h period immediately, peaked in 24 h, decreased gradually thereafter, and was substantially higher than that in healthy controls during the 7-day period. A multivariate analysis showed plasma copeptin level was an independent predictor for 1-week mortality (odds ratio, 1.013; 95% confidence interval (CI), 1.003–1.023; P  = 0.009) and positively associated with hematoma volume (t  = 6.616, P  < 0.001). A receiver operating characteristic curve identified that a baseline plasma copeptin level >577.5 pg/mL predicted 1-week mortality with 87.5% sensitivity and 72.2% specificity (area under curve (AUC), 0.873; 95% CI, 0.784–0.935). The AUC of the copeptin concentration was similar to those of Glasgow Coma Scale (GCS) scores and hematoma volumes (P  = 0.136 and 0.280). However, copeptin did not statistically significantly improve the AUCs of GCS scores and hematoma volumes (P  = 0.206 and 0.333). Hence, increased plasma copeptin level is associated with hematoma volume and an independent prognostic marker of mortality after ICH.
Keywords: Copeptin; Intracerebral hemorrhage; Prognosis; Stroke; Biomarkers;

▶ Non-specific binding of NPY, PYY and PP is typically in 6:2:1 ratio. ▶ This relates to acidic pairs with aspartate (2 in NPY, 1 in PYY, 0 in PP) and proline-13 in NPY. ▶ The above should also be important in agonist selectivity of Y receptor subtypes. ▶ The differences should also be important for partial agonism and cross-reactivity.Non-specific binding of Y receptor agonists to intact CHO cells, and to CHO cell or rat brain particulates, is much greater for human neuropeptide Y (hNPY) compared to porcine peptide Y (pPYY), and especially relative to human pancreatic polypeptide (hPP). This binding of hNPY is reduced by alkali cations in preference to non-ionic chaotrope urea, while the much lower non-specific binding of pPYY is more sensitive to urea. The difference could mainly be due to the 10–16 stretch in 36-residue Y agonists (residues 8–14 in N-terminally clipped 34-peptides), located in the sector that contains all acidic residues of physiological Y agonists. Anionic pairs containing aspartate in the 10–16 zone could be principally responsible for non-specific attachments, but may also aid the receptor site binding. Two such pairs are found in hNPY, one in pPYY, and none in hPP. The hydroxyl amino acid residue at position 13 in mammalian PYY and PP molecules could lower conformational plasticity and the non-selective binding via intrachain hydrogen bonding. The acidity of this tract could also be important in agonist selectivity of the Y receptor subtypes. The differences point to an evolutionary reduction of promiscuous protein binding from NPY to PP, and should also be important for Y agonist selectivity within NPY receptor group, and correlate with partial agonism and out-of group cross-reactivity with other receptors.
Keywords: Neuropeptide Y; Peptide YY; Pancreatic polypeptide; Low-affinity binding; Partial agonism;

Guinea pig ileum motility stimulation elicited by N-formyl-Met-Leu-Phe (fMLF) involves neurotransmitters and prostanoids by Mariantonella Colucci; Marica Mastriota; Francesco Maione; Amalia Di Giannuario; Nicola Mascolo; Maura Palmery; Cinzia Severini; Mauro Perretti; Stefano Pieretti (266-271).
Display Omitted▶ The formyl peptide receptor agonist N-formyl-Met-Leu-Phe-OH (fMLF) induces a dose-dependent contraction of guinea pig ileum (GPI). ▶ fMLF contracts GPI through the release of the excitatory neurotransmitters acetylcholine and substance P from myenteric motorneurons. ▶ fMLF also induces the release of prostanoids such as PGE2 probably from inflammatory cells that are in close proximity to the myenteric plexus and intestinal smooth muscle cells.In guinea-pig ileum (GPI), the chemotactic peptide N-formyl-Met-Leu-Phe-OH (fMLF) possesses spasmogenic properties through the activation of formyl peptide receptors (FPRs). Despite this, the mediators involved remain to be elucidated. fMLF (1 nM–1 μM) induced a dose-dependent contraction of GPI (EC50  = 24 nM), that is blocked by pre-treatment with the FPRs antagonist Boc2. The pre-treatment with tetrodotoxin (TTX) atropine or with SR140333 reduced the fMLF-induced contraction, whereas with hexamethonium, MEN10627, SB222200, mepyramine, cimetidine, thioperamide or methysergide did not produce any effect. With DuP697 pre-treatment, but not with piroxicam, reduced the fMLF-induced contraction. After stimulation with 24 nM fMLF, a strong increase in the PGE2 levels was observed. Finally, the concomitant blocking of the NK1 receptor, the muscarinic receptors and COX-2 abolished the GPI contractions induced by fMLF.fMLF induced a concentration-dependent contraction of guinea-pig jejunum (EC50  = 11 nM), proximal colon (EC50  = 3.5 nM) and distal colon (EC50  = 2.2 nM), with a time-course similar to that observed in GPI. In these preparations as well, the co-administration of atropine, SR140333 and DuP697 abolished the contractions induced by fMLF. Intraperitoneal injection of fMLF (0.1 or 1 μmol/kg) enhanced the gastrointestinal motility in mice, abolished by the co-administration of atropine, SR140333 and DuP697. In conclusion, we showed that fMLF exerts spasmogenic actions on guinea-pig intestine both in vitro and in vivo through the release of acetylcholine and substance P from myenteric motorneurons and through prostanoids, probably from the inflammatory cells of the enteric immune system.
Keywords: FPR; Formyl peptide receptors; GPI; Guinea-pig intestine; fMLF; Gastrointestinal transit;

Cholecystokinin-8 activates myenteric neurons in 21- and 35-day old but not 4- and 14-day old rats by Martha C. Washington; Candace R. Murry; Shannon J. Raboin; Allison E. Roberson; Mahmoud M. Mansour; Carol S. Williams; Ayman I. Sayegh (272-280).
▶ Cholecystokinin (CCK) activates the myenteric neurons in rats. ▶ We determined the ontogeny of this activation. ▶ CCK activated myenteric neurons of 21- and 35-day old rats but not 4 and 14. ▶ CCK activated the dorsal vagal complex of all age groups. ▶ This delayed activation reflects a delayed role for these neurons.Cholecystokinin (CCK) activates the myenteric neurons of adult rats. The goal of this work is to determine the ontogeny of this activation by CCK-8 in the myenteric plexus of the duodenum (2 cm immediately following the pyloric sphincter aborally) and compare it with that of the dorsal vagal complex (DVC) – which occurs in 1-day old pups. Despite the existence of both of the CCK receptors, CCK1 and CCK2, in 4, 14, 21 and 35 day old rats, CCK-8 (0, 5, 10, 20 and 40 μg/kg, i.p.) increased Fos-like immunoreactivity (Fos-LI, a marker for neuronal activation) in the myenteric neurons of 21- and 35-day old rats but in the DVC of all age groups. As such, this belated activation of myenteric neurons by CCK-8 compared to the DVC may reflect a delayed role for these neurons in CCK-related functions.
Keywords: Fos; Pups; CCK1 receptor; CCK2 receptor; Myenteric neurons;

▶ Rapakinin, Arg–Ile–Tyr, derived from rapeseed napin, showed anti-opioid activity. ▶ The anti-opioid activity of rapakinin was mediated by cholesystokinin (CCK) and the CCK2 receptor. ▶ The anti-opioid activity of rapakinin was also mediated by prostaglandin I2 and the IP receptor. ▶ CCK–CCK2 system was activated downstream of the PGI2–IP receptor system. ▶ Rapakinin shows anti-opioid activity via the activation of the PGI2–IP receptor system followed by the CCK–CCK2 receptor system.Rapakinin, Arg–Ile–Tyr, is a vasorelaxing, anti-hypertensive and anorexigenic peptide derived from rapeseed napin. In this study, we found that rapakinin intracerebroventricularly administered to mice inhibited the analgesic effect of morphine, evaluated by the tail-pinch test. The anti-opioid activity of rapakinin was blocked by LY225910, an antagonist of the cholecystokinin (CCK) CCK2 receptor, but not by lorglumide, an antagonist of the CCK1 receptor. The anti-opioid activity of rapakinin was also blocked by CAY10441, an antagonist of the prostaglandin (PG) IP receptor. These results suggest that the anti-opioid activity of rapakinin is mediated by the CCK2 and IP receptors. The anti-opioid activity induced by ciprostene, an IP receptor agonist, was blocked by LY225910, while that of CCK-8 was not blocked by CAY10441. Thus, it is demonstrated that the CCK–CCK2 system was activated downstream of the PGI2–IP receptor system. Taken together, rapakinin shows anti-opioid activity via the activation of the PGI2–IP receptor system followed by the CCK–CCK2 receptor system.
Keywords: Anti-opioid activity; Rapakinin; Prostaglandin I2; IP receptor; Cholecystokinin; CCK2 receptor;

▶ GRP immunoreactive nerve fibers were found in mouse skin. ▶ RC-3095, a GRPR antagonist, does not inhibit GNTI-induced scratching. ▶ [d-Phe6]bombesin(6-13) methyl ester, a GRPR antagonist, has no marked effect on GNTI-induced scratching. ▶ McN-A-343, a muscarinic M1 receptor antagonist, attenuates GNTI-induced scratching.Gastrin-releasing peptide (GRP) has been implicated in the itch-scratch cycle. We investigated if this gut–brain–skin peptide plays a role in the compulsive, hindleg scratching of the neck of mice by 5′-guanidinonaltrindole (GNTI), the kappa opioid receptor antagonist, and in the antipruritic activity of nalfurafine, the kappa opioid agonist. Previously, we showed that GNTI (0.03–1 mg/kg, s.c.) elicits dose-related scratching and that nalfurafine (0.001–0.02 mg/kg, s.c.) inhibits this behavior in mice. Utilizing immunohistochemistry, GRP positive nerve fibers were detected in mouse skin and superficial layer of the dorsal horn of the spinal cord as well as GRP positive cells in the dorsal root ganglion. Pretreating mice with either a pseudopeptide GRP receptor antagonist, RC-3095 (10–30 mg/kg, s.c. at −15 min), or a peptide GRP receptor antagonist, [d-Phe6]bombesin(6-13) methyl ester (2–100 nmol, i.t. at −10 min), did not suppress GNTI-induced scratching. However, pretreating mice with either antagonist inhibited scratching precipitated by the GRP receptor agonist, GRP18–27 (2 nmol, i.t.). Pretreating mice with a muscarinic M1 receptor agonist, McN-A-343 (1.5–15 μg/5 μl, i.t. at −10 min) antagonized GNTI-induced scratching. Norbinaltorphimine (20 mg/kg, i.p. at −18 to −20 h), a kappa opioid antagonist, countered the antiscratch activity of nalfurafine. We conclude that (a) the GRP receptor system does not mediate GNTI-induced scratching and (b) the kappa opioid system is involved, at least in part, in the scratch suppressing activity of nalfurafine.
Keywords: Itch; Gastrin-releasing peptide; GNTI; Nalfurafine; Kappa opioid receptor; Muscarinic receptor;

Synthesis and antinociceptive effects of endomorphin-1 analogs with C-terminal linked by oligoarginine by Chang-lin Wang; Chao Guo; Yi-qing Wang; Ying Zhou; Qian Li; Jing-man Ni; Rui Wang (293-299).
▶ The oligoarginine vectors can enhance intracellular and the BBB delivery. ▶ EM-1 analogs with C-terminal linked by oligoarginine attenuated μ-opioid affinity. ▶ Analogs 4, 7–9 showed central antinociception following peripheral injection. ▶ The antinociception is due in part to an improvement in brain delivery.Endomorphins (EMs) cannot be delivered into the central nervous system (CNS) in sufficient quantity to elicit antinociception when given systemically because they are severely restricted by the blood–brain barrier (BBB). In the present study, we investigated herein a series of EM-1 analogs with C-terminal linked by oligoarginine in order to improve the brain delivery and antinociception after systemic administration. Indeed, all these analogs decreased the opioid receptor affinity and in vitro pharmacological activity. Moreover, analogs 4, 7–9 produced a less potent antinociceptive activity after intracerebroventricular (i.c.v.) administration, with the ED50 values about 11- to 13-fold lower potencies than that of EM-1. Nevertheless, our results revealed that EM-1 failed to induce any significant antinociception at a dose of 50 μmol/kg after subcutaneous (s.c.) administration, whereas equimolar dose of these four analogs produced a little low but significant antinociceptive effects. Naloxone (10 nmol/kg, i.c.v.) significantly blocked the antinociceptive effects, indicating an opioid and central mechanism. These results demonstrated that C-terminal of EM-1 linked to oligoarginine improved the brain delivery, eliciting potent antinociception following peripheral administration.
Keywords: Endomorphin-1 analogs; BBB; μ-Opioid receptor; CPPs; Oligoarginine; Antinociception;

Structure–function relationship of conotoxin lt14a, a potential analgesic with low cytotoxicity by Dandan Sun; Zhenghua Ren; Xiayun Zeng; Yuwen You; Wuguang Pan; Maojun Zhou; Lei Wang; Anlong Xu (300-305).
▶ Determined the solution structure of conotoxin lt14a by NMR. ▶ An analog [K7A]-lt14a exhibited higher activity than lt14a as long-lasting analgesic. ▶ Both lt14a and [K7A]-lt14a had low toxicity to human cells. ▶ It is assumed that hydrophobic residues play a key role in binding processing of lt14a.A novel conotoxin lt14a containing 13 amino acid residues with an amidated C-terminus derived from Conus litteratus, belongs to C–C–C–C cysteine pattern. As the smallest peptide of conotoxin framework 14, lt14a could inhibit nicotinic acetylcholine receptor and suppress pain. To elucidate structure–function relationship, we determine the solution structure by NMR and find that lt14a comprises a short duple β-strand region and β-turn motif. An analog [K7A]-lt14a of Ala substitution for Lys in position 7 is designed. Interestingly, [K7A]-lt14a exhibits higher activity than lt14a as long-lasting analgesic in the hotplate pain model in mice. Additionally, MTT assay reveals that the two peptides have low toxicity to human cells. The studies suggest that positively charged residue may not be involved in the blocking mechanism. However, due to the Ala substitution, hydrophobic residues’ patch expansion strengthens the binding ability. A hypothesis is given that in conotoxin lt14a, hydrophobic residues rather than charged residues play a key role during target binding.
Keywords: Conotoxin; lt14a; Solution structure; NMR; Analgesic;

Intraspecies variability and conopeptide profiling of the injected venom of Conus ermineus by Jose A. Rivera-Ortiz; Herminsul Cano; Frank Marí (306-316).
▶ We analyzed the intraspecies variability of the injected venom of Conus ermineus. ▶ We found extreme variability in the peptide composition in the venom. ▶ Over 800 unique peptides were identified in C. ermineus. ▶ These results expand the potential for prospecting neuroactive peptides from Conus. ▶ The differential expression of venom components is a new neurochemical paradigm.The venom of cone snails (ssp. Conus), a genus of predatory mollusks, is a vast source of bioactive peptides. Conus venom expression is complex, and venom composition can vary considerably depending upon the method of extraction and the species of cone snail in question. The injected venom from Conus ermineus, the only fish-hunting cone snail species that inhabits the Atlantic Ocean, was characterized using nanoNMR spectroscopy, MALDI-TOF mass spectrometry, RP-HPLC and nanoLC–ESI-MS. These methods allowed us to evaluate the variability of the venom within this species. Single specimens of C. ermineus show unchanged injected venom mass spectra and HPLC profiles over time. However, there was significant variability of the injected venom composition from specimen to specimen, in spite of their common biogeographic origin. Using nanoLC–ESI-MS, we determined that over 800 unique conopeptides are expressed by this reduced set of C. ermineus specimens. This number is considerably larger than previous estimates of the molecular repertoire available to cone snails to immobilize prey. These results support the idea of the existence of a complex regulatory mechanism to express specific venom peptides for injection into prey. These intraspecies differences can be a result of a combination of genetic and environmental factors. The differential expression of venom components represents a neurochemical paradigm that warrants further investigation.
Keywords: Cone snails; Conus ermineus; Injected venom; Intraspecies variability; Molecular fingerprint; NanoNMR; LC–ESI-MS; Conopeptides;

Evidence for the participation of cocaine- and amphetamine-regulated transcript peptide (CART) in the fluoxetine-induced anti-hyperalgesia in neuropathic rats by Manoj A. Upadhya; Manoj P. Dandekar; Dadasaheb M. Kokare; Praful S. Singru; Nishikant K. Subhedar (317-326).
▶ Sciatic nerve ligation induced the neuropathic pain leading hyperalgesia. ▶ CART as well as fluoxetine treatment attenuated the hyperalgesic response. ▶ CART pre-treatment significantly potentiated fluoxetine-induced anti-hyperalgesia. ▶ The CART-immunoreactivity increased in VLPAG, DRD and LC following neuropathy. ▶ Fluoxetine treatment in neuropathic rats restored immunoreactivity to control.Cocaine- and amphetamine-regulated transcript peptide (CART) has a role in chronic pain, and also in the actions of selective serotonin reuptake inhibitors (SSRIs) employed in the treatment of neuropathic pain. Herein, we test the hypothesis that CART may mediate the anti-hyperalgesic effect of the SSRI, fluoxetine, in neuropathic rats. Sciatic nerve in the right hind paw of rat was ligated to induce neuropathic pain, and the paw withdrawal latency was evaluated using Hargreaves apparatus. Fluoxetine [5–25 mg/kg, intraperitoneal (ip)] or CART (54–102) [0.1–1.5 μg/rat, intracerebroventricular (icv)] dose-dependently attenuated the hyperalgesic response observed in neuropathic rats, indicating anti-nociceptive properties of each agent. The anti-hyperalgesic effect of fluoxetine was potentiated by the subeffective dose of CART, and attenuated by CART-antibody (1:500 dilution; 5 μl/rat, icv); CART-antibody had no effect per se. Isobolographic analysis showed a significant synergism between fluoxetine and CART, and antagonism between fluoxetine and CART-antibody. Immunocytochemical labeling with monoclonal antibodies against CART showed drastic increase in CART-immunoreactive fibers in the ventrolateral periaqueductal gray (VLPAG; 116%), dorsal subdivision of dorsal raphe nucleus (DRD; 176%), and locus coeruleus (LC; 733%) of neuropathic animals. Fluoxetine treatment significantly reduced the immunoreactivity in these areas. However, CART-immunoreactive cells and fibers in the arcuate nucleus did not respond to neuropathy or fluoxetine treatments. We suggest that the CART innervation of DRD, LC and VLPAG may be involved in the (i) central processing of neuropathic pain and (ii) fluoxetine-induced anti-hyperalgesic effect in neuropathic pain.
Keywords: Cocaine- and amphetamine-regulated transcript peptide; Neuropathic pain; Fluoxetine; Anti-hyperalgesia; Hargreaves apparatus; Immunocytochemistry;

Distribution of Substance P (SP) and Vasoactive Intestinal Peptide (VIP) in pseudocapsules of uterine fibroids by Antonio Malvasi; Andrea Tinelli; Carlo Cavallotti; Manrico Morroni; Daniel Alberto Tsin; Camran Nezhat; Michael Stark; Liselotte Mettler (327-332).
▶ Substance P (SP) and Vasoactive Intestinal Peptide (VIP) are present in the pseudocapsule of uterine fibroids. ▶ This quantitative distribution is similar to theirs distribution in the myometrium. ▶ The fibroid removal by intracapsular method is neurotransmitter sparing at the hysterotomy site. ▶ This method preserves SP and VIP fibers in the uterus, allows an optimal healing of myometrium and maintains its functionality for future pregnancies and labor.The authors examined the presence of Substance P (SP) and Vasoactive Intestinal Polypeptide (VIP) and their related fibers in the pseudocapsule of uterine fibroids (PUF) and in normal myometrium (NM) during myomectomies in 57 non-pregnant women. 4 samples were removed from the normal myometrium (NM) and from PUF. The samples were sent for histological and immune-fluorescent investigations. SP and VIP values were found non-significantly higher in PUF than in NM: SP values were 10.2 ± 0.1 conventional units (C.U.) in PUF at the fundus of the uterus (FU) vs. 8.1 ± 0.6 C.U. of NM in the FU (p  > 0.05), and SP values were 25.1 ± 0.9 C.U. in PUF in the uterine body (UB) compared to. 23.2 ± 1.4 C.U. of NM in the myometrium of the UB (p  > 0.05). VIP values were 11.5 ± 0.9 C.U. in the PUF in FU compared to 9.8 ± 1.4 C.U. of NM in the FU (p  > 0.05), and VIP values were 33.9 ± 3.9 C.U. in the PUF in the UB vs. 32.6 ± 4.8 C.U. of the NM in the UB (p  > 0.05). These findings show that SP and VIP neurofibers are present in the fibroid pseudocapsule, similar to the values in the normal myometrium of a non-pregnant uterus. An intracapsular myoma excision which respects the pseudocapsule permits a physiological healing process of the uterine scar, due to a neurotransmitter sparing at the hysterotomic site. In women planning pregnancy, the myomectomy should be preferably performed respecting the pseudocapsule in order to preserve the neurotransmission.
Keywords: Fibroid; Myoma; Myomectomy; Myoma pseudocapsule; Laparoscopy; Uterine rupture; Substance P; SP; Vasoactive intestinal polypeptide; VIP; Neurotransmitters; Neuropeptides;

▶ Antimicrobial peptide (AMP) tilapia hepcidin (TH)2-3 regulates cell differentiation in mouse macrophage cells. ▶ TH2-3-mediated morphological changes were similar to those of the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA). ▶ PMA and TH2-3 differentially regulated tumor necrosis factor (TNF)-α and cyclooxygenase (COX)-2. ▶ The TH2-3 peptide will be a useful tool in investigating the fish immune system and advancing study toward clinical applications of TH2-3.The antimicrobial and immunomodulatory functions of the antimicrobial peptide, tilapia hepcidin (TH)2–3, were previously studied. Herein, we report the differential modulation of protein kinase C (PKC)-associated proteins by TH2-3, and the PKC activator, phorbol 12-myristate 13-acetate (PMA), in RAW264.7 macrophages. Treatment with TH2-3 at 40 or 80 μg/ml did not affect the cell morphology, but TH2-3 at 120 μg/ml produced morphological changes similar to those after treatment with PMA in RAW264.7 cells. The coexistence of the PKC inhibitor, Ro-31-8220, prevented morphological changes induced by either PMA or 120 μg/ml TH2-3 in RAW264.7 cells. Since PMA is known to induce expression of the proinflammatory cytokine, tumor necrosis factor (TNF)-α, activation of the TNF-α promoter in response to TH2-3 and PMA treatments in lipopolysaccharide (LPS)-stimulated cells was compared. In LPS-stimulated RAW264.7 cells, TNF-α promoter activity was significantly suppressed by TH2-3, but not by PMA. In addition, PMA activated prostaglandin synthase-associated cyclooxygenase (COX)-2 proteins on the cell surface, while the presence of TH2-3 inhibited its expression. Western blotting demonstrated that the expressions of PKC-μ, phosphorylated (p)-PKCμ at serine (S)-744, and p-PKCδ were activated by PMA, but were suppressed by TH2-3. In addition, p-PKC at S-916 was activated by TH2-3 and inhibited by PMA. In conclusion, the differential regulation of PKC isoforms by PMA and TH2-3 may influence morphological changes and regulation of TNF-α in RAW264.7 cells.
Keywords: Antimicrobial peptide; Tilapia hepcidin 2-3; PMA; PKC; TNF-α; COX-2;

Tilapia (Oreochromis mossambicus) antimicrobial peptide, hepcidin 1–5, shows antitumor activity in cancer cells by Wang-Ting Chang; Chieh-Yu Pan; Venugopal Rajanbabu; Chun-Wen Cheng; Jyh-Yih Chen (342-352).
▶ Hepcidin 1–5 (TH1–5) induced cell membrane rupture like a lytic peptide. ▶ TH1–5 induced necrosis with high-concentration treatment and induced apoptosis with low-concentration treatment. ▶ TH1–5 inhibited tumor cell growth.The inhibitory function of tilapia hepcidin (TH)1–5, an antimicrobial peptide, was not examined in previous studies. In this study, we synthesized the TH1–5 peptide and tested TH1–5's antitumor activity against several tumor cell lines. We show that TH1–5 inhibited the proliferation of tumor cells and reduced colony formation in a soft agar assay. Scanning electron microscopy and transmission electron microscopy showed that TH1–5 altered the membrane structure similar to the function of a lytic peptide. Acridine orange/ethidium bromide staining, a wound-healing assay, and a flow cytometric analysis showed that TH1–5 induced necrosis with high-concentration treatment and induced apoptosis with low-concentration treatment. Inflammation is known to be closely associated with the development of cancer. TH1–5 showing anti-inflammatory effects in a previous publication induced us to evaluate the anti-inflammatory effects in cancer cell lines through the expressions of immune-related genes after being treated with the TH1–5 peptide. However, real-time qualitative RT-PCR indicated that TH1–5 treatment induced downregulation of the expressions of interleukin (IL)-6, IL-8, IL-12, IL-15, interferon-γ, CTSG, caspase-7, and Bcl-2, and upregulation of IL-2 and CAPN5 in HeLa cells, and upregulation of IL-8 and CTSG in HT1080 cells. These results suggest that TH1–5 possibly induces an inflammatory response in HeLa cells, but not in HT1080 cells. Overall, these results indicate that TH1–5 possesses the potential to be a novel peptide for cancer therapy.
Keywords: Antimicrobial peptide; Hepcidin 1–5; Antitumor activity;

Protective effects of antimicrobial peptide S-thanatin against endotoxic shock in mice introduced by LPS by Guoqiu Wu; Xiaofang Li; Xuepeng Deng; Xiaobo Fan; Shenglan Wang; Zilong Shen; Tao Xi (353-357).
▶ Different treatment methods of S-thanatin experimental conditions were designed. ▶ Tumor necrosis factor alpha (TNF-α) and endotoxin concentrations in plasma were measured. ▶ S-thanatin can reduce endotoxin and TNF-α level in plasma and result in a highest survival rates. ▶ Different dosages of S-thanatin administration in mice showed different inhibitory effects.Sepsis continues to be a major unresolved medical challenge of the present. Severe sepsis and septic shock are the leading causes of multiple organ failure and mortality in noncoronary intensive care units (ICUs). The primary reason of septic shock is the activation of host effecter cells by endotoxin and lipopolysaccharide (LPS) associated with cell membranes of gram-negative bacteria. For these reasons, the key point of treatment is removing LPS. S-thanatin (Ts), an analog of thanatin, was synthesized by substituting the 15th amino acid of threonine with serine, which showed a broad antimicrobial activity against gram-negative and gram-positive bacteria. We have reported its LPS-binding and -neutralizing activity in vitro. The aim of this study is to examine the LPS-neutralizing activities and the protective effects of S-thanatin in vivo. Every mice was injected intraperitoneally with LPS (from Escherichia coli O111:B4) 150 μg before injected intraperitoneally or vena caudalis with 3 mg/kg, 6 mg/kg and 12 mg/kg, and measured endotoxin and tumor necrosis factor alpha (TNF-α) concentrations in plasma, as well as lethality. The results showed that S-thanatin can significantly reduce endotoxin and TNF-α level in plasma, at the same time resulting in the highest survival rates.
Keywords: Thanatin; Antimicrobial peptide; Endotoxic shock; Lipopolysaccharide; TNF-α;

Relationship between total ghrelin and inflammation in hemodialysis patients by D. Mafra; N.E. Farage; J.C. Lobo; M.B. Stockler-Pinto; V.O. Leal; D.P. Carvalho; M. Leite (358-361).
▶ This study correlates the levels of ghrelin with TNF-α, IL-6, nutritional status and body composition in HD patients. ▶ Patients showed elevated plasma ghrelin levels when compared to healthy subjects. ▶ There was a positive correlation between ghrelin levels and TNF-α, IL-6. ▶ Plasma ghrelin levels are associated with the state of systemic inflammation.In hemodialysis (HD) patients studies have shown that plasma ghrelin is increased and it has been speculated that ghrelin levels might be related to systemic inflammation. The present study attempted to correlate the serum levels of total ghrelin with serum TNF-α and IL-6, and with nutritional status and body composition in HD patients. Forty-seven HD patients from a single dialysis unit (18 women, mean age 55.3 ± 12.2 yr; BMI 24.4 ± 4.2 kg/m2; % body fat 29.4 ± 7.4%) were studied and compared to 21 healthy subjects (12 women, 50.7 ± 15.7 yr and BMI 25.6 ± 4.0 kg/m2; % body fat 30.0 ± 5.7%). Biochemical data, serum total ghrelin, TNF-α and IL-6 levels were measured. The body composition was evaluated by dual energy X-ray absortiometry (DEXA) and energy and protein intake were evaluated. Patients showed elevated plasma ghrelin levels when compared to healthy subjects (1.14 ± 1.0 ng/mL vs 0.58 ± 0.4; p  < 0.001). There was a positive correlation between ghrelin levels and TNF-α (r  = 0.25; p  < 0.04), IL-6 (r  = 0.42; p  < 0.02), and a negative correlation between TNF-α and protein intake (r  = −0.28; p  < 0.03), and energy intake (r  = −0.34; p  < 0.01). No correlation was observed with any aspect of body composition. Plasma ghrelin levels are elevated in HD patients and associated with the state of systemic inflammation. We suggest that the inflammatory state may affect ghrelin bioactivity and metabolism in hemodialysis patients.
Keywords: Ghrelin; Inflammation; Nutrition; Hemodialysis;

Coronary vasoconstrictor effect of ghrelin is not mediated by growth hormone secretagogue receptor 1a type in dogs by Balazs Sax; Gyorgy L. Nadasy; Katalin Turi; Kristof Hirschberg; Dora Furjesz; Andrea Nagy; Bela Merkely; Gabor Szabo; Emil Monos; Violetta Kekesi (362-367).
▶ GHS-R1a and GHS-R1b receptors are present in the wall of canine coronary arterioles. ▶ Ghrelin constricts isolated canine coronary arterioles in a tone-dependent manner. ▶ This vasoconstrictor effect is mediated by a receptor other than GHS-R1a.Ghrelin (GHR) is a recently discovered endocrine regulatory peptide of gastrointestinal origin with multiple functions including cardiovascular effects. However, contradictory data are available on the vascular actions of GHR in different organs and species. The aim of this study was to characterize the direct effect of the peptide on the canine coronary bed and to evaluate the role of the growth hormone secretagogue receptor (GHS-R) in the effect of GHR on coronary arterioles. The presence of GHS-R1a and 1b subtypes in canine coronary arterioles was investigated using Western blotting and immunohistochemistry. Responses of coronary arterioles with spontaneous and elevated vascular tone (the latter evoked by the thromboxane mimetic agent U46619, 10−7–10−6  mol/l) to GHR (10−9–3 × 10−7  nmol/l) were recorded by video-microscopy as changes of vessel diameter. Positive immunostaining for both GHS-R subtypes was found in the wall of intramural arterioles. The microarteriographic study results showed that GHR alone could not elicit any significant effect on vessel diameter of arterioles with spontaneous tone. However, when vascular smooth muscle was preconstricted by the thromboxane mimetic agent U46619, administration of GHR induced further constriction (+31 ± 9% increase in contraction p  < 0.01). This was not abolished by the specific blockade of GHS-R1a by d-Lys3-GHRP-6 (5 × 10−6  mol/l). The results suggest that GHR induces tone-dependent constriction of canine coronary arterioles which is mediated by a receptor other than GHS-R1a.
Keywords: Ghrelin; Growth hormone secretagogue receptor; Coronary arterioles; Vasoconstriction; Canine;

Adrenomedullin level in the nasal discharge from allergic rhinitis cohort by Terumichi Fujikura; Kimihiro Okubo (368-373).
▶ The AM in the nasal discharge was significantly higher in the non-allergy group. ▶ AM-positive mast cells were found in the nasal mucosa. ▶ Reduction of AM may be associated with attenuation of immediate allergic responses. ▶ In the chronic phase, mast cells produce AM to inhibit inflammatory cell migration.Adrenomedullin (AM) is a potent hypotensive and vasodilatory peptide. AM may exert protective actions against the development of many diseases by modulating the blood circulation and body fluid balance. In addition to these functions, it has recently been reported to play important roles in the development of allergy and infections. The purpose of the present study was to demonstrate the existence of AM in the human nasal mucosa and to discuss whether AM might contribute to the pathogenesis of nasal congestion. We measured the total AM concentrations in the nasal discharge. The total AM concentration in the nasal discharge was significantly higher in the non-allergy group (72.1 ± 55.5 fmol/ml) than in the allergy group (37.1 ± 44.2 fmol/ml). By immunohistochemical examination, we identified AM-containing cells in the nasal mucosa from both subjects with and without nasal allergy, and also in nasal polyps. Moreover, those cells were positive for anti-tryptase antibody which recognizes mast cells. In nasal allergy, vasodilatation and increase in vascular permeability are characteristic features of the immediate phase response. Reduced AM levels in the nasal discharge may be associated with attenuation of both of these factors. On the other hand, immunohistochemical analysis demonstrated AM-immunoreactive cells in the chronic phase of rhinosinusitis. In the late and inflammatory phase, mast cells produce AM, which possibly acts as an inhibitor of inflammatory cell migration. In conclusion, AM may be actively secreted into the nasal discharge. AM in the nasal discharge may have protective and anti-inflammatory effects in the nasal mucosa.
Keywords: Nasal allergy; Mast cell; Nasal discharge; Neutral endopeptidase;

Endogenous angiotensin II suppresses stretch-induced ANP secretion via AT1 receptor pathway by Young-Bin Oh; Shan Gao; Amin Shah; Jong Hun Kim; Woo Hyun Park; Suhn Hee Kim (374-381).
▶ Perfusion of angiotensin II into isolated beating atria decreased the ANP secretion. ▶ The suppression of ANP secretion by angiotensin II was more prominent in high-stretched beating atria. ▶ Angiotensin II-induced suppression of ANP secretion was blocked by AT1 receptor blocker. ▶ Losartan augmented stretch-induced ANP secretion. ▶ In renal hypertensive atria, the responses of ANP secretion to angiotensin II and losartan were accentuated because of upregulation of AT1 receptor.Angiotensin II (Ang II) is released by stretch of cardiac myocytes and has paracrine and autocrine effects on cardiac myocytes and fibroblasts. However, the direct effect of Ang II on the secretion of atrial natriuretic peptide (ANP) is unclear. The aim of the present study is to test whether Ang II affects stretch-induced ANP secretion. The isolated perfused beating atria were used from control and two-kidney one-clip hypertensive (2K1C) rats. The volume load was achieved by elevating the height of outflow catheter connected with isolated atria from 5 cmH2O to 7.5 cmH2O. Atrial stretch by volume load caused increases in atrial contractility by 60% and in ANP secretion by 100%. Ang II suppressed stretch-induced ANP secretion and tended to increase atrial contractility whereas losartan stimulated stretch-induced ANP secretion. Neither PD123319 nor A779 had direct effect on stretch-induced ANP secretion. The suppressive effect of Ang II on stretch-induced ANP secretion was blocked by the pretreatment of losartan but not by the pretreatment of PD123319 or A779. In hypertrophied atria from 2K1C rats, stretch-induced ANP concentration attenuated and atrial contractility augmented. The response of stretch-induced ANP secretion to Ang II and losartan augmented. The expression of AT1 receptor protein and mRNA increased but AT2 and Mas receptor mRNA did not change in 2K1C rat atria. Therefore, we suggest that Ang II generated endogenously by atrial stretch suppresses stretch-induced ANP secretion through the AT1 receptor and alteration of Ang II effect in 2K1C rat may be due to upregulation of AT1 receptor.
Keywords: Hypertension; Hypertrophy; Atrial natriuretic peptide; Angiotensin II; Receptor; Atrial stretch;

Elevated expression of urotensin II and its receptor in diethylnitrosamine-mediated precancerous lesions in rat liver by Hongxia Wang; Kun Dong; Xiaowei Xue; Ping Feng; Xuejiang Wang (382-387).
▶ UII/UT system was up-regulated in liver precancerous lesion. ▶ UII derived from liver might induce the pathogenesis of precancerous lesion through PKC or ERK pathway as an autocrine/paracrine factor. ▶ UII induces cell proliferation of hepatic oval cells.Urotensin II (UII) is a somatostatin-like peptide involved in cell proliferation and in tumor biology. To explore the role of liver-derived UII in the pathogenesis of precancerous liver lesions in rat, we investigated the expression of UII and its receptor, UT, in diethylnitrosamine (DEN)-induced precancerous liver lesions and the effects of UII on cell proliferation by hepatic oval cells. Radioimmunoassay, RT-PCR, immunohistochemistry and western blot were used in this study. Compared with untreated controls, rats treated with DEN showed increased UII content by 47.7% in plasma and by 164.9% in liver tissue (all P  < 0.01). The expression of UII protein and of both UT mRNA and protein was significantly enhanced in the liver of treated rats. Western blot analysis revealed that the expression of phosphorylated protein kinase C (p-PKC) and phosphorylated extracellular signal-regulated kinase (p-ERK1/2) was increased in the liver of treated animals. Treatment with UII (10−10–10−6  M) for 24 h significantly increased number of cultured hepatic oval cells (at 10−9–10−8  M). However, during the pre-incubation with calphostin C (inhibitor of PKC) or PD98059 (inhibitor of MEK), the proliferation was decreased by 40.1% and 25.4% respectively (both P  < 0.05). In DEN-induced precancerous liver lesions, the UII/UT system was up-regulated, which may contribute to the pathogenesis of liver cancer through a PKC- or ERK1/2-dependent pro-mitogenic pathway in an autocrine/paracrine manner.
Keywords: Urotensin II; G-protein coupled receptor; Precancerous lesions; Liver;

Isolation, purification and characterization of antioxidant peptidic fractions from a bovine liver sarcoplasmic protein thermolysin hydrolyzate by Roberta Di Bernardini; Dilip K. Rai; Declan Bolton; Joseph Kerry; Eileen O’Neill; Anne Maria Mullen; Pádraigín Harnedy; Maria Hayes (388-400).
▶ Identification of peptides. ▶ LC–MS and protein databases. ▶ Novel antioxidants.Sarcoplasmic proteins isolated from bovine livers were hydrolyzed using the enzyme thermolysin at 37 ̊C for 2 h. The hydrolyzates were filtered through molecular weight cut off membranes (MWCO) and filtrates were obtained. The water activity (a w) of unhydrolysed sarcoplasmic protein, full hydrolyzates, 10-kDa and 3-kDa filtrates were below the limit necessary for microbial growth. The antioxidant activities of both filtrates and fractions were assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay, the ferric ion reducing antioxidant power (FRAP) assay and the Fe2+ chelating ability assay. RP-HPLC was used for purification of the full hydrolyzates, the 10-kDa and the 3-kDa filtrates. The peptidic content of the full hydrolyzates, the 10-kDa and the 3-kDa filtrates were assessed using the Dumas method and peptide contents of each fraction were characterized using electrospray quadrupole time-of-flight (ESI-Q-TOF) mass spectrometry with the resultant spectrum analysed using the software programmes Protein Lynx Global Server 2.4. and TurboSEQUEST. Similarities between the amino acid composition of characterized peptides from each fraction and previously reported antioxidant peptides were found. This study demonstrates that meat by-product such as liver can be utilised as raw material for the generation of bioactive peptides with demonstrated antioxidant activities in vitro using the enzyme thermolysin. It is significant as it presents a potential opportunity for meat processors to use their waste streams for the generation of bioactive peptides for potential functional food use.
Keywords: Bovine by-products; Thermolysin; Sacroplasmic proteins; Antioxidant peptides; DPPH radical scavenging activity assay; FRAP; Fe2+ chelating ability assay;

Novel vasoactive intestinal peptide derivatives with improved stability protect rat alveolar L2 cells from cigarette smoke-induced cytotoxicity and apoptosis by Shingen Misaka; Hideyuki Sato; Yosuke Aoki; Takahiro Mizumoto; Satomi Onoue; Shizuo Yamada (401-407).
▶ CSE induced apoptotic death of L2 cells. ▶ VIP and its derivatives exhibited significant attenuation of CSE-toxicity. ▶ New VIP derivative exceeded VIP in suppressing CSE-induced apoptotic death of L2 cells.Vasoactive intestinal peptide (VIP) has been thought to be a promising candidate for asthma/chronic obstructive pulmonary disease (COPD), and our group previously developed several long-lasting VIP derivatives. The objective of the present study was to clarify the therapeutic potential of new VIP derivatives with improved chemical and metabolic stability. Exposure of rat alveolar L2 cells to cigarette smoke extract (CSE) for 1 h led to release of lactate dehydrogenase (LDH) and decreased viability in a CSE concentration-dependent manner. There appeared to be marked induction of apoptosis after CSE exposure, as demonstrated by 59% elevation of caspase-3 activity and TUNEL staining. In contrast, a stabilized VIP derivative, [R15,20,21, L17]-VIP-GRR (IK312532), at a concentration of 10−7  M, exhibited 71% attenuation of LDH release and 85% decrease of the number of apoptotic cells. In addition to IK312532, new VIP derivatives also showed anti-apoptotic effects against CSE toxicity and marked reduction of nitric oxide production. In terms of cytoprotective effects, [R15,20,21, L17, A24,25, des-N28]-VIP-GRR was more effective than VIP and IK312532, possibly due to the improved stability. Thus, the present study is the first to demonstrate that novel stabilized VIP derivatives exert anti-apoptotic and cytoprotective effects on CSE-induced cytotoxicity.
Keywords: VIP; Cigarette smoking; Apoptosis; L2 cells;

The latest developments in synthetic peptides with immunoregulatory activities by Chun-lei Zhou; Rong Lu; Gang Lin; Zhi Yao (408-414).
▶ The class I and II MHC-derived peptides inhibit TCR recognition of antigen peptide–MHC complex. ▶ Rationally designed CD80 and CD154-binding peptides block interaction between cell surface costimulatory molecules on antigen-presenting cells (APCs) and T cells. ▶ Some peptides inhibit the activities of cell signal proteins, including JNK, NF-κB and NFAT. ▶ Some peptide antagonists competitively bind to important cytokines and inhibit their activities, such as TNF-α, TGF-β and IL-1β inhibitory peptides. ▶ Adhesion molecule ICAM-1 derived peptides block T cell adhesion and activation.In the past few years, many researches have provided us with much data demonstrating the abilities of synthetic peptides to impact immune response in vitro and in vivo. These peptides were designed according to the structure of some important protein molecules which play a key role in immune response, so they act with specific targets. The class I and II MHC-derived peptides inhibit the TCR recognition of antigen peptide–MHC complex. Rationally designed CD80 and CD154-binding peptides block the interaction between cell surface costimulatory molecules on antigen-presenting cells (APCs) and T cells. Some peptides were designed to inhibit the activities of cell signal proteins, including JNK, NF-κB and NFAT. Some peptide antagonists competitively bind to important cytokines and inhibit their activities, such as TNF-α, TGF-β and IL-1β inhibitory peptides. Adhesion molecule ICAM-1 derived peptides block the T cell adhesion and activation. These immunoregulatory peptides showed therapeutic effect in several animal models, including collagen-induced arthritis (CIA), autoimmune cystitis model, murine skin transplant model and cardiac allograft model. These results give us important implications for the development of a novel therapy for immune mediated diseases.
Keywords: Peptide; Immunosuppression; RDP-58; CD80–CAP1; pepJIP1; NBD peptide; VIVIT; RCAN; p17; p144; IL-1RacP; cIBR;

▶ Fish antimicrobial peptide protected diseases fish in the aquaculture. ▶ Fish antimicrobial peptide inhibited virus activity. ▶ Fish antimicrobial peptide inhibited tumor cell growth.Fish are a major component of the aquatic fauna. Like other organisms, fish secrete different kinds of antimicrobial peptides (AMPs), which are positively charged short amino-acid-chain molecules involved in host defense mechanisms. Environmental hazards and the greenhouse effect have led to increased evolution of drug- and vaccine-resistant pathogenic strains, and it is necessary to find new drugs with structural uniqueness to fight them. Aquatic sources contain thousands of fish species, and each secretes AMPs with structural differences which can be used by the pharmaceutical industry in its search for novel drugs to treat drug-resistant pathogens. Not only limited to antimicrobial functions, AMPs possess other desirable characteristics which may be exploited in the near future. In this review, we list fish AMPs available from published reports, and discuss application-oriented functions of these AMPs. Notably, the possibilities of using fish AMPs as antimicrobial agents, vaccine adjuvants, inactivated vaccines, and antitumor agents are discussed in this review.
Keywords: Fish; Antimicrobial peptide; Aquaculture; Anti-virus activity; Anti-tumor ractivity;

Dermorphin tetrapeptide analogs as potent and long-lasting analgesics with pharmacological profiles distinct from morphine by Hirokazu Mizoguchi; Giacinto Bagetta; Tsukasa Sakurada; Shinobu Sakurada (421-427).
▶ This review summarizes information about the endogenous/synthetic dermorphin analogs. ▶ Their potent and long-lasting analgesic effects. ▶ Their structure–activity relationship. ▶ Distinct antinociceptive profile of synthetic dermorphin tetrapeptide analogs. ▶ Adverse effects of synthetic dermorphin tetrapeptide analogs.Dermorphin (Tyr-d-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) is a heptapeptide isolated from amphibian skin. With a very high affinity and selectivity for μ-opioid receptors, dermorphin shows an extremely potent antinociceptive effect. The structure–activity relationship studies of dermorphin analogs clearly suggest that the N-terminal tetrapeptide is the minimal sequence for agonistic activity at μ-opioid receptors, and that the replacement of the d-Ala2 residue with d-Arg2 makes the tetrapeptides resistant to enzymatic metabolism. At present, only a handful of dermorphin N-terminal tetrapeptide analogs containing d-Arg2 have been developed. The analogs show potent antinociceptive activity that is greater than that of morphine with various injection routes, and retain high affinity and selectivity for μ-opioid receptors. Interestingly, some analogs show pharmacological profiles that are distinct from the traditional μ-opioid receptor agonists morphine and [d-Ala2,NMePhe4,Gly-ol5]enkephalin (DAMGO). These analogs stimulate the release of dynorphins through the activation of μ-opioid receptors. The activation of κ-opioid receptors by dynorphins is suggested to reduce the side effects of μ-opioid receptor agonists, e.g., dependence or antinociceptive tolerance. The dermorphin N-terminal tetrapeptide analogs containing d-Arg2 may provide a new target molecule for developing novel analgesics that have fewer side effects.
Keywords: Dermorphin analog; Analgesics; μ-Opioid receptor; κ-Opioid receptor; Dynorphins; Dependence;

▶ Microtubule dysfunctions are associated with schizophrenia. ▶ STOP (MAP6)-deficiency models aspects of schizophrenia in mice. ▶ ADNP is deregulated in schizophrenia. ▶ Davunetide (NAP) is a drug candidate derived from ADNP. ▶ Davunetide (NAP) enhances cognitive functions in STOP-deficient mice.NAP (davunetide) is an active fragment of activity-dependent neuroprotective protein (ADNP). ADNP and the homologous protein ADNP2 provide cell protection. ADNP is essential for brain formation, proper development and neuronal plasticity, all reported to be impaired in schizophrenia. ADNP haploinsufficiecy inhibits social and cognitive functions, major hallmarks in schizophrenia. Imbalance in ADNP/ADNP2 expression in the schizophrenia brain may impact disease progression. NAP treatment partly ameliorates ADNP haploinsufficiecy. The microtubule, stable tubule-only polypeptide (STOP)-deficient mice were shown to provide a reliable model for schizophrenia. Daily intranasal NAP treatment significantly decreased hyperactivity in STOP-deficient mice and protected visual memory, supporting further clinical development.
Keywords: Schizophrenia; Stable tubule-only polypeptide (STOP); Tau; Activity-dependent neuroprotective protein (ADNP); ADNP2; NAP (davunetide); Hyperactivity; Visual memory;