Peptides (v.35, #1)

Actions of incretin metabolites on locomotor activity, cognitive function and in vivo hippocampal synaptic plasticity in high fat fed mice by David Porter; Emilie Faivre; Peter R. Flatt; Christian Hölscher; Victor A. Gault (1-8).
► Comprehensive study investigating actions of GLP-1(9-36)amide and GIP(3-42) on locomotor activity, memory and learning and hippocampal long-term potentiation in high-fat fed mice. ► Truncated incretin metabolites did not alter general locomotor activity or learning ability of high-fat fed mice during an object recognition test. ► High-fat mice demonstrated impairment in hippocampal synaptic plasticity which was not affected by incretin metabolites. ► Changes in circulating concentrations of incretin metabolites are unlikely to have a negative impact on cognitive function.The incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) improve markers of cognitive function in obesity–diabetes, however, both are rapidly degraded to their major metabolites, GLP-1(9-36)amide and GIP(3-42), respectively. Therefore, the present study investigated effects of GLP-1(9-36)amide and GIP(3-42) on locomotor activity, cognitive function and hippocampal synaptic plasticity in mice with diet-induced obesity and insulin resistance. High-fat fed Swiss TO mice treated with GLP-1(9-36)amide, GIP(3-42) or exendin(9-39)amide (twice-daily for 60 days) did not exhibit any changes in bodyweight, non-fasting plasma glucose and plasma insulin concentrations or glucose tolerance compared with high-fat saline controls. Similarly, locomotor and feeding activity, O2 consumption, CO2 production, respiratory exchange ratio and energy expenditure were not altered by chronic treatment with incretin metabolites. Administration of the truncated metabolites did not alter general behavior in an open field test or learning and memory ability as recorded during an object recognition test. High-fat mice exhibited a significant impairment in hippocampal long-term potentiation (LTP) which was not affected by treatment with incretin metabolites. These data indicate that incretin metabolites do not influence locomotor activity, cognitive function and hippocampal synaptic plasticity when administered at pharmacological doses to mice fed a high-fat diet.
Keywords: Cognitive function; Glucose-dependent insulinotropic polypeptide (GIP); GIP(3-42); Glucagon-like peptide-1 (GLP-1); GLP-1(9-36)amide; Long-term potentiation (LTP);

CCK-B receptor gene and response to cholecystokinin-tetrapeptide in healthy volunteers by Diana Koszycki; Zoe Prichard; Alexandra J. Fiocco; Jakov Shlik; James L. Kennedy; Jacques Bradwejn (9-13).
► CCKBR (CT) polymorphism alleles 26 and 27 are associated with panic disorder (PD). ► We determined whether these alleles modulated panicogenic sensitivity to CCK-4 in healthy volunteers. ► We could not demonstrate that these PD risk alleles enhanced sensitivity to CCK-4. ► However, carriers of the alleles were more anxiety sensitive and introverted and they tended to experience higher pre-challenge levels of anxiety.Recent investigations suggest that genes that confer risk for panic disorder (PD) may moderate response to panicogenic agents in healthy volunteers. Given the potential role of the central cholecystokinin receptor (CCKBR) (CT) polymorphism alleles 26 and 27 in PD, the present study attempted to discern if these alleles moderated panicogenic sensitivity to the CCKBR agonist, CCK-tetrapeptide (CCK-4), in healthy volunteers. The study group consisted of 92 men and women with no personal or family history of psychiatric illness. Participants provided blood samples for genotyping of the CCKBR alleles and they received a 25 μg bolus injection of CCK-4. Behavioral, cardiovascular and hormonal responses to the peptide were assessed and analyzed with adjusted linear regression models. Carriers of the CCKBR alleles tended to have higher levels of pre-challenge anxiety and significantly higher levels of anxiety sensitivity and introversion than those without the alleles. However, they did not exhibit an enhanced panicogenic response to CCK-4. Overall, our findings do not demonstrate a role of these alleles in modulating CCK-4's panicogenicity. The significant association between the risk alleles and anxiety-related personality traits is intriguing and further exploration of this association is merited.
Keywords: CCK gene polymorphism; Cholecystokinin; CCK-B receptors; CCK-4; Panic attacks; Panic disorder;

► Satiation induces Fos in the ventral part of hypothalamic dorsomedial nucleus (DM). ► GLP-1 axon terminals overlap with refeeding-activated Fos neurons in the DM. ► Caudal pontine knife cuts eliminate GLP-1-containing axon terminals from the DM. ► Fos activation is reduced ipsilateral to the transection of ascending GLP-1 fibers. ► Fos activated cells in the ventral part of the DM express GLP-1 receptor mRNA.A high number of neurons express c-fos in response to unlimited food intake in fasted rats in the ventral subdivision of the hypothalamic dorsomedial nucleus (DMHv). We report here, that in same conditions, limited food consumption failed to induce Fos expression in DMHv neurons suggesting that satiation should be one of the important signals that activate these neurons. The possible origin of fibers conducting satiation signals to the DMHv could be in the lower brainstem, especially glucagon-like peptide-1 (GLP-1)-containing neurons in the nucleus of the solitary tract (NTS). We demonstrate that GLP-1-immunoreactive fibers and fiber terminals topographically overlap with activated Fos-positive neurons in the DMHv in refed rats. Using immunocytochemistry and in situ hybridization histochemistry, we demonstrated GLP-1 receptors in Fos-expressing neurons of the DMH. Unilateral transections of ascending GLP-1-containing fibers from the NTS inside the pons in refed rats (unlimited food consumption) resulted in a dramatic decrease in the density of GLP-1 fibers and in the number of Fos-immunoreactive neurons in the DMHv, but only on the side of the transection. Contralateral to the transection, neither the GLP-1 fiber density nor the number of Fos-positive cells changed significantly. Meanwhile, the density of GLP-1 immunoreactivity was markedly accumulated in transected nerve fibers caudal to the cuts, as a consequence of the interruption of the ascending GLP-1 transport route. These findings suggest that the solitary-hypothalamic projections may represent the neuronal route through GLP-1 neurons of the NTS activate DMHv neurons via GLP-1 receptors by conveying information on satiety.
Keywords: Surgical transections; Food intake; Glucagon-like peptide-1; c-fos activation; Satiety;

► DIO rats had down-regulated LKB1–AMPK signaling and consequently reduced expression of NPY in the hypothalamus. ► POMC expression was decreased in hypothalamus of DIO rats, which is not regulated by AMPK activation. ► DIO rats had elevated body weight, particularly in the size of abdominal adipose tissue depots and impaired LKB1–AMPK signaling in adipose tissue. ► LKB1 protein content and pAMPKα in skeletal muscle of DIO rats were not different from those in the muscles of chow-fed and DR rats. ► Although DR rats were fed with the same HF diet as the DIO rats, they kept normal appetite and visceral fat mass without abnormalities in AMPK cascade.AMPK not only acts as a sensor of cellular energy status but also plays a critical role in the energy balance of the body. In this study, LKB1–AMPK signaling was investigated in diet-induced obese (DIO) and diet resistant (DR) rats. In hypothalamus, DIO rats had lower level of LKB1, AMPKα and pAMPKα than chow-fed or DR rats. Both orexigenic peptide NPY and anorexigenic peptide POMC expression were reduced in hypothalamus of DIO rats. i.c.v. injection of AICAR, an activator of AMPK, increased NPY expression but did not alter POMC expression in DIO rats. In periphery, LKB1 protein content and pAMPKα level were lower in the adipose tissue of DIO rats compared to chow-fed and DR rats. Moreover, pAMPKα and LKB1 protein levels obtained from epididymal fat pad were inversely correlated with epididymal fat mass. LKB1 protein content and pAMPKα in skeletal muscle of DIO rats were not different from those in the muscles of chow-fed and DR rats. In summary, DIO rats, but not DR rats, have impaired LKB1–AMPK signaling in hypothalamus and adipose tissue, suggesting the disturbed energy balance observed in DIO rats is related with abnormalities of AMPK signaling in a tissue specific manner.
Keywords: Diet-induced obesity; AMPK; LKB1; NPY; POMC;

Intracerebroventricular administration of neuronostatin delays gastric emptying and gastrointestinal transit in mice by Shu-Fang Su; Ai-Min Yang; Shao-Bin Yang; Ning-Bo Wang; Song-Song Lu; Hui-Hui Wang; Qiang Chen (31-35).
► Intracerebroventricular administration of neuronostatin delayed gastric emptying and gastrointestinal transit in mice. ► The gastrointestinal effect elicited by neuronostatin was significantly reversed by melanocortin 3/4 receptor antagonist SHU9119 or classical opioid receptor antagonist naloxone. ► Intracerebroventricular administration of non-amidated neuronostatin had no influence on gastric emptying and gastrointestinal transit.Neuronostatin is a 13-amino acid amidated peptide widely distributed in various organs including gastrointestinal tract. However, the effect of neuronostatin on gastrointestinal motility has not been well characterized. In the present work, effects of central administration of neuronostatin on gastric emptying and gastrointestinal transit were investigated. The results indicated that intracerebroventricular (i.c.v.) administration of neuronostatin (1, 5, 10 or 20 nmol/mouse) delayed gastric emptying and gastrointestinal transit in a dose-related manner in mice. The effects were significantly reversed by melanocortin 3/4 receptor antagonist SHU9119 or classical opioid receptor antagonist naloxone, suggesting that the central melanocortin system and opioid system may be involved in the gastrointestinal effects elicited by i.c.v. administration of neuronostatin. In addition, we found that C-terminal amidation modification of neuronostatin is essential to exert its gastrointestinal effects. These results indicated that neuronostatin may play an important role in regulating gastrointestinal function.
Keywords: Neuronostatin; Gastric emptying; Gastrointestinal transit; Central melanocortin system; Opioid system;

Identification of orexins and cognate receptors in the lacrimal gland of sheep by Cecilia Dall’Aglio; Francesca Mercati; Margherita Maranesi; Cristiano Boiti (36-41).
► The orexinergic system is expressed in the lacrimal gland of sheep. ► OX1R transcript is two-fold higher than OX2R. ► Positive staining for OX1R is evidenced in the wall of small arteries. ► OX2R staining is localized in the secretory portion of the acinar gland cells. ► The orexinergic system might regulate the lacrimal gland function.The aim of the present work was to study, by means of immunohistochemical and RT-PCR techniques, the presence and distribution of immunopositivity for orexin A and B (OXA and OXB) and orexin type 1 and 2 receptors (OX1R and OX2R) in the lacrimal gland of sheep as well as the gene expressions for prepro-orexin (PPOX) and cognate receptors. In serial sections, positive staining for OXA and OXB were localized in the same nervous fibers within the connective tissue septa. Positive staining for OX1R was evidenced in the wall of small arteries while that for OX2R was observed in the secretory portion of the acinar gland cells with a characteristic localization in the apical cytoplasm. RT-PCR analysis showed the presence of transcripts for PPOX, OX1R and OX2R in the sheep lacrimal gland; the gene expression of OX1R was two-fold greater (p  < 0.01) than that of OX2R. Taken together the present findings raise intriguing questions on the potential role of the orexinergic system in the regulation of lacrimal gland functions that require further investigations.
Keywords: Orexin/hypocretin; OXA; OXB; PPOX; OX1R; OX2R;

Biosynthesis of proTRH-derived peptides in prohormone convertase 1 and 2 knockout mice by Nicole E. Cyr; Ronald C. Stuart; Xiaorong Zhu; Donald F. Steiner; Eduardo. A. Nillni (42-48).
► This is the first study where the fate of proTRH biosynthesis and processing is analyzed in PC1KO and PC2KO mice. ► The current in vivo study confirms our initial in vitro and ex vivo findings demonstrating that PC1 plays a primary role in proTRH processing. ► PC1 is necessary to maintain normal levels of the biologically active thyroid hormone T3. ► PC2 is required for the conversion of the proTRH-derived peptide pFE22 to pFQ7, and pSE14, which may function to regulate prolactin.Prohormone convertases (PCs) 1 and 2 are the primary endoproteases involved in the post-translational processing of proThyrotropin Releasing Hormone (proTRH) to give rise to TRH and other proposed biologically active non-TRH peptides. Previous evidence suggests that PC1 is responsible for most proTRH cleavage events. Here, we used the PC1 and PC2 knockout (KO) mouse models to examine the effects of PC1 or PC2 loss on proTRH processing. The PC1KO mouse presented a decrease in five proTRH-derived peptides, whereas the PC2KO mouse showed only lesser reduction in three TRH (Gln-His-Pro), TRH-Gly (Gln-His-Pro-Gly), and the short forms preproTRH178–184 (pFQ7) and preproTRH186–199 (pSE14) of pFE22 (preproTRH178–199). Also, PC1KO and not PC2KO showed a decrease in pEH24 indicating that PC1 is more important in generating this peptide in the mouse, which differs from previous studies using rat proTRH. Furthermore, downstream effects on thyroid hormone levels were evident in PC1KO mice, but not PC2KO mice suggesting that PC1 plays the more critical role in producing bioactive hypophysiotropic TRH. Yet loss of PC1 did not abolish TRH entirely indicating a complementary action for both enzymes in the normal processing of proTRH. We also show that PC2 alone is responsible for catalyzing the conversion of pFE22 to pFQ7 and pSE14, all peptides implicated in regulation of suckling-induced prolactin release. Collectively, results characterize the specific roles of PC1 and PC2 in proTRH processing in vivo.
Keywords: proTRH; TRH; Prohormone convertases; PC1; PC2, Neuropeptides; Prohormone processing;

► The rat Npy mRNA exists in two splice-variants. ► The two Npy variants share the same transcription start site. ► The “short”Npy mRNA does not seem to code for a protein. ► The “short”Npy mRNA is expressed in lower levels than the “long” one. ► Both Npy mRNAs are decreased in a genetic model of depression.Neuropeptide Y (NPY) is known to influence emotional processing and decreased NPY levels have been associated with mood and anxiety disorders. Alternative splicing of pre-messenger RNA is a cellular mechanism that allows for transcriptome diversity, yet there is limited knowledge in this respect with regard to Npy. Since the hippocampus and the prefrontal cortex play an important role in affective disorders, we investigated alternative splicing of Npy in these regions of a rat model of depression (Flinders Sensitive Line, FSL) and its controls (Flinders Resistant Line, FRL). The existence of different Npy messenger RNA (mRNA) variants was examined using 5′ and 3′ RACE. In addition to the Npy mRNA species annotated in GenBank and Ensembl, we identified a novel “short” mRNA splice variant. Immunoblotting results argued against a putative translation of this “short” mRNA into protein in brain tissue. Compared to the FRL, the FSL had reduced “short”Npy mRNA levels in the HIP (P  = 0.00014) and the PFC (P  = 0.016). Gene expression analyses in five brain regions of an outbred rat strain supported the presence of the “short”Npy transcript in all examined regions and showed that it is expressed in ∼2.4-fold lower levels than the “long”Npy mRNA. Finally, sequencing of the 5′ RACE products revealed a transcription start site of Npy that is different from the currently annotated position. These data add to the characterization of the rat Npy mRNA and demonstrate the presence of a novel transcript with a so far unknown function.
Keywords: NPY; Transcription start site (TSS); Alternative splicing; Psychiatry; Depression; Brain regions;

Orexin receptor type-1 antagonist SB-334867 inhibits the development of morphine analgesic tolerance in rats by Yadollah Ranjbar-Slamloo; Hossein Azizi; Yaghoub Fathollahi; Saeed Semnanian (56-59).
► The effect of orexin receptor type-1 antagonism on morphine tolerance was studied. ► Central injection of SB-334867 prior to morphine inhibited the development of analgesic tolerance. ► It is concluded that orexin might be involved in the development of tolerance to morphine.Herein the effect of orexin receptor type-1 antagonist SB-334867 on the development of tolerance to analgesic effects of morphine was studied in rats. To incite tolerance, morphine sulfate was injected intraperitoneally (i.p., 10 mg/kg) once a day for 7 days. The tail flick test was used to evaluate antinociceptive effects of the morphine. A selective OxR1 receptor antagonist, SB-334867, was microinjected (i.c.v.) into the right cerebral ventricle (10 μg/10 μl) immediately before each morphine injection. Repeated morphine application resulted in tolerance to morphine analgesic effects as a decreasing trend during 7 days. Also, repeated administration of SB-334867 (i.c.v.) alone was without significant effect on the nociception as compared to control. Microinjection of SB-334867 prior to each morphine injection inhibited the development of tolerance, so that the analgesic effects of morphine were significantly higher in SB-334867 plus morphine treated rats than that of vehicle plus morphine treated ones on days 4–7. It is concluded that orexin receptor type-1 might be involved in the development of tolerance to morphine analgesic effects.
Keywords: Orexin; Orexin receptor type-1; SB-334867; Morphine; Morphine tolerance; Rat;

Substance P stimulates Growth Hormone (GH) and GH-Releasing Hormone (GHRH) secretions through tachykinin NK2 receptors in sheep by Guy-Joseph Lemamy; Viviane Guillaume; Bénédicte Ndéboko; Justine Mouecoucou; Charles Oliver (60-64).
► Substance P stimulates GH and GHRH secretion in sheep. ► NK2 receptor antagonist blocks substance P-induced GH and GHRH release. ► NK1 receptor antagonist does not inhibit substance P-induced GH and GHRH release.Substance P is ubiquitous undecapeptide belonging to the tachykinins family. It has been found in the hypothalamus and is involved in the hypothalamo-hypophysial axis in several mammals, including human. Previous studies have shown that substance P increases GH secretions in rats and human. In this study, we have shown that intravenously infused substance P in sheep caused an increased level of Growth Hormone (GH) and GH-Releasing Hormone (GHRH), and decreased Somatotropin Release Inhibiting Hormone (SRIH) secretions. GH was obtained from peripheral blood. GHRH and SRIH were directly collected from hypophysial portal blood, using a trans-nasal surgery technique in a vigil sheep that allowed accessing to hypothalamo-hypophysial portal vessels. Hormones assays were performed by radioimmunoassay (RIA). Moreover, we showed that substance P-induced GH and GHRH secretion appears to be mediated by NK2 tachykinin receptors, since it is specifically blocked by a non peptidic tachykinin NK2 receptor antagonist (SR48968, Sanofi, Montpellier, France) whereas a non peptidic tachykinin NK1 antagonist (SR140333, Sanofi, Montpellier, France) failed to modify GH and GHRH hormones secretions.
Keywords: Substance P; GH; GHRH; SRIH; NK2 receptor;

Ghrelin signaling in heart remodeling of adult obese mice by Glauciane Lacerda-Miranda; Vivian M. Soares; Anatalia K.G. Vieira; Juliana G. Lessa; Alessandra C.S. Rodrigues-Cunha; Erika Cortez; Erica P. Garcia-Souza; Anibal S. Moura (65-73).
► Hypernutrition in early life leads to obesity with myocardial remodeling in adulthood. ► SL presented increased heart hypertrophy. Obesity reduced plasma acylated ghrelin level, increased GHS-R1a expression. ► Obesity changed ghrelin signaling proteins contents in heart.Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), has been suggested to be associated to obesity, insulin secretion, cardiovascular growth and homeostasis. GHS-R has been found in most of the tissues, and among the hormone action it is included the regulation of heart energy metabolism. Therefore, hypernutrition during early life leads to obesity, induces cardiac hypertrophy, compromises myocardial function, inducing heart failure in adulthood. We examined ghrelin signaling process in cardiac remodeling in these obese adult mice. The cardiomyocytes (cmy) of left ventricle were analyzed by light microscopy and stereology, content and phosphorilation of cardiac proteins: ghrelin receptor (growth hormone secretagogue receptor 1a, GHSR-1a), protein kinase B (AKT and pAKT), phosphatidil inositol 3 kinase (PI3K), AMP-activated protein kinase (AMPK and pAMPK) and actin were achieved by Western blotting. GHSR-1a gene expression was analyzed by Real Time-PCR. We observed hyperglycemia and higher liver and visceral fat weight in obese when compared to control group. Obese mice presented a marked increase in heart weight/tibia length, indicating an enlarged heart size or a remodeling process. Obese mice had increased GHSR-1a content and expression in the heart associated to PI3K content and increased AKT content and phosphorylation. In contrast, AMPK content and phosphorylation in heart was not different between experimental groups. Ghrelin plasma levels in obese group were decreased when compared to control group. Our data suggest that remodeled myocardial in adult obese mice overnourished in early life are associated with higher phosphorylation of GHSR-1a, PI3K and AKT but not with AMPK.
Keywords: Overnutrition; Heart; GHSR-1a; PI3K; AKT;

B-type natriuretic peptide predicts new-onset atrial fibrillation in patients with ST-segment elevation myocardial infarction treated by primary percutaneous coronary intervention by Milika Asanin; Sanja Stankovic; Igor Mrdovic; Dragan Matic; Lidija Savic; Nada Majkic-Singh; Miodrag Ostojic; Zorana Vasiljevic (74-77).
► BNP was measured in 180 patients with STEMI treated by primary PCI. ► ROC analysis identified the best BNP cut-off value for prediction AF (≥720 pg/mL). ► BNP predicts the occurrence of new-onset AF in STEMI patients treated by primary PCI.The predictive value of B-type natriuretic peptide (BNP) with respect to the occurrence of new-onset atrial fibrillation (AF) in patients with ST-segment elevation myocardial infarction (STEMI) treated by primary percutaneous coronary intervention (PCI) is unknown. The aim of this study was to evaluate whether BNP has a predictive value for the occurrence of new-onset AF in patients with STEMI treated by primary PCI. In 180 patients with STEMI treated by primary PCI, BNP concentrations were measured 24 h after chest pain onset. The Receiver Operating Characteristic analysis was performed to identify the most useful BNP cut-off level for the prediction of AF. The patients were divided into the two groups according to calculated cut-off level: high BNP group (BNP ≥ 720 pg/mL, n  = 33) and low BNP group (BNP < 720 pg/mL, n  = 147). The incidence of AF was 5.0%, and occurred more frequently in high BNP group (7/33, 21.2%) than in low BNP group (2/147, 1.4%), (p  < 0.001). Patients with high BNP were older (p  = 0.017), had more often anterior wall infarction (p  = 0.015), higher Killip class on admission (p  = 0.038), higher peak troponin I (p  = 0.002), lower left ventricular ejection fraction (p  = 0.029) than patients with low BNP. After multivariate adjustment, BNP was an independent predictor of AF (OR 3.70, 95% CI 1.40–9.77, p  = 0.008). BNP independently predicts the occurrence of new-onset AF in STEMI patients treated by primary PCI.
Keywords: B-type natriuretic peptide; Atrial fibrillation; ST-segment elevation myocardial infarction; Primary percutaneous coronary intervention;

► Peptide therapy attenuates hypoxia-induced pulmonary artery hypertension and vascular remodeling. ► The effect was found to be mediated by increased NO-release and inhibition of cGMP-specific PDE-5. ► Dual action of the peptide that enhance NO release and diminished PDE-5 activity resulted in sustained elevation of cGMP. ► This novel peptide has therapeutic potential for NO/cGMP-dependent modulation of hemodynamic and structural changes in the pulmonary circulation.Pulmonary vascular endothelial nitric oxide (NO) synthase (eNOS)-derived NO is the major stimulant of cyclic guanosine 5′-monophosphate (cGMP) production and NO/cGMP-dependent vasorelaxation in the pulmonary circulation. We recently synthesized multiple peptides and reported that an eleven amino acid (SSWRRKRKESS) peptide (P1) but not scrambled P1 stimulated the catalytic activity but not expression of eNOS and causes NO/cGMP-dependent sustained vasorelaxation in isolated pulmonary artery (PA) segments and in lung perfusion models. Since cGMP levels can also be elevated by inhibition of phosphodiesterase type 5 (PDE-5), this study was designed to test the hypothesis that P1-mediated vesorelaxation is due to its unique dual action as NO-releasing PDE-5 inhibitor in the pulmonary circulation. Treatment of porcine PA endothelial cells (PAEC) with P1 caused time-dependent increase in intracellular NO release and inhibition of the catalytic activity of cGMP-specific PDE-5 but not PDE-5 protein expression leading to increased levels of cGMP. Acute hypoxia-induced PA vasoconstriction ex vivo and continuous telemetry monitoring of hypoxia (10% oxygen)-induced elevated PA pressure in freely moving rats were significantly restored by administration of P1. Chronic hypoxia (10% oxygen for 4 weeks)-induced alterations in PA perfusion pressure, right ventricular hypertrophy, and vascular remodeling were attenuated by P1 treatment. These results demonstrate the potential therapeutic effects of P1 to prevent and/or arrest the progression of hypoxia-induced PAH via NO/cGMP-dependent modulation of hemodynamic and vascular remodeling in the pulmonary circulation.
Keywords: Pulmonary hypertension; Hypoxia; Nitric oxide; Phosphodiesterase-5; Synthetic peptide; Cyclic GMP; Telemetry; Pulmonary artery pressure;

Larazotide acetate regulates epithelial tight junctions in vitro and in vivo by Shobha Gopalakrishnan; Malarvizhi Durai; Kelly Kitchens; Amir P. Tamiz; Robert Somerville; Mark Ginski; Blake M. Paterson; Joseph A. Murray; Elena F. Verdu; Sefik S. Alkan; Niranjan B. Pandey (86-94).
► Larazotide acetate (LA) inhibits gliadin-induced actin and ZO-1 redistribution. ► LA inhibits cytokine-induced permeability. ► LA inhibits gliadin translocation across Caco-2 cell monolayers. ► LA preserves tight junction structure and function in gluten-sensitive mice. ► LA is a novel compound with therapeutic value in celiac disease management.Tight junctions (TJs) control paracellular permeability and apical-basolateral polarity of epithelial cells, and can be regulated by exogenous and endogenous stimuli. Dysregulated permeability is associated with pathological conditions, such as celiac disease and inflammatory bowel disease. Herein we studied the mechanism by which larazotide acetate, an 8-mer peptide and TJ regulator, inhibits the cellular changes elicited by gliadin fragments, AT-1002, and cytokines. Previously, we demonstrated that AT-1002, a 6-mer peptide derived from the Vibrio cholerae zonula occludens toxin ZOT, caused several biochemical changes in IEC6 and Caco-2 cells resulting in decreased transepithelial electrical resistance (TEER) and increased TJ permeability. In this study, larazotide acetate inhibited the redistribution and rearrangement of zonula occludens-1 (ZO-1) and actin caused by AT-1002 and gliadin fragments in Caco-2 and IEC6 cells. Functionally, larazotide acetate inhibited the AT-1002-induced TEER reduction and TJ opening in Caco-2 cells. Additionally, larazotide acetate inhibited the translocation of a gliadin 13-mer peptide, which has been implicated in celiac disease, across Caco-2 cell monolayers. Further, apically applied larazotide acetate inhibited the increase in TJ permeability elicited by basolaterally applied cytokines. Finally, when tested in vivo in gliadin-sensitized HLA-HCD4/DQ8 double transgenic mice, larazotide acetate inhibited gliadin-induced macrophage accumulation in the intestine and preserved normal TJ structure. Taken together, our data suggest that larazotide acetate inhibits changes elicited by AT-1002, gliadin, and cytokines in epithelial cells and preserves TJ structure and function in vitro and in vivo.
Keywords: Celiac disease; Gliadin; ZO-1; Actin; Tight junction; Permeability inducer;

Larazotide acetate promotes tight junction assembly in epithelial cells by Shobha Gopalakrishnan; Amit Tripathi; Amir P. Tamiz; Sefik S. Alkan; Niranjan B. Pandey (95-101).
► Larazotide acetate promotes tight junction (TJ) assembly in epithelial cells. ► Larazotide acetate promotes actin rearrangement during TJ assembly. ► Larazotide acetate promotes junctional distribution of TJ and AJ proteins. ► Larazotide acetate decreases TJ permeability in leaky Caco-2 cells.Tight junctions (TJ) control paracellular permeability and apical-basolateral polarity of epithelial cells. Dysregulated permeability is associated with pathological conditions, such as celiac disease and inflammatory bowel disease. TJ formation is dependent on E-cadherin-mediated cell-cell adhesion and actin rearrangement, and is regulated by the Rho family GTPase and aPKC signaling pathways. Larazotide acetate, an 8-mer peptide and TJ modulator, inhibits TJ disassembly and dysfunction caused by endogenous and exogenous stimuli in intestinal epithelial cells. Here, we examined the effect of larazotide acetate on de novo TJ assembly using 2 different model systems. In MDCK cells, larazotide acetate promoted TJ assembly in a calcium switch assay. Larazotide acetate also promoted actin rearrangement, and junctional distribution of zonula occludens-1 (ZO-1), occludin, claudins, and E-cadherin. Larazotide acetate promoted TJ maturation and decreased paracellular permeability in “leaky” Caco-2 cells. Taken together, our data indicate that larazotide acetate enhances TJ assembly and barrier function by promoting actin rearrangement and redistribution of TJ and AJ proteins.
Keywords: Epithelial tight junctions; Permeability; TJ assembly;

Chimeric relaxin peptides highlight the role of the A-chain in the function of H2 relaxin by Mohammed Akhter Hossain; John D. Wade; Ross A.D. Bathgate (102-106).
► Chimeric peptides combining H2 relaxin-B/INSL peptides-A have been synthesized. ► The peptides demonstrate reduced activity highlighting the importance of the A-chain. ► A-chain chimeras can be produced with receptor specificity. ► H2 relaxin chimeric peptides may be templates for specific RXFP1 agonist development.Human gene-2 (H2) relaxin is a member of the insulin–relaxin peptide superfamily. Because of the potential clinical applications of H2 relaxin, there is a need for novel analogs that have improved biological activity and receptor specificity. In this respect, we have chemically assembled chimeric peptides consisting of the B-chain of H2 relaxin in combination with A-chains from other insulin/relaxin family members. The peptides were prepared using solid phase peptide synthesis together with regioselective disulfide bond formation and characterized by RP-HPLC, MALDI-TOF MS and amino acid analysis. Their in vitro activity was assessed in RXFP1 or RXFP2 expressing cells. Replacement of the H2 relaxin A-chain resulted in parallel losses of binding affinity and activity on RXFP1. Not surprisingly H1A-H2B demonstrated the highest activity as the H1 A-chain shares high homology with H2 relaxin whereas INSLA-H2B, which shows low homology, had very poor activity. Importantly A-chain replacements had a dramatic effect on RXFP2 activity similar to previous results demonstrating different modes of activation of A-chain variants on RXFP1 and RXFP2. H3A-H2B is particularly interesting as it displays moderate activity at RXFP1 but poor activity at RXFP2 indicating that it may be a template for specific RXFP1 agonist development. Our study confirms that the activity of H2 relaxin at both RXFP1 and RXFP2 relies on interactions with both the B- and A-chains, and also provide new biochemical insights into the mechanism of relaxin action that the A-chain needs to be in native or near-native form for strong RXFP1 or RXFP2 agonist activity.
Keywords: Relaxin; RXFP1; GPCR; Peptide;

Isolation, modulatory functions on murine B cell development and antigen-specific immune responses of BP11, a novel peptide from the chicken bursa of Fabricius by Xiao-dong Liu; Xiu-li Feng; Bin Zhou; Rui-Bing Cao; Xin-feng Li; Zhi-Yong Ma; Pu-Yan Chen (107-113).
► We isolated and identified a new bursal peptide(BP11) from BF. ► BP11 could promoted CFU pre-B formation. ► BP11 has a role in regulate B cell development. ► BP11 exerted immunomodulatory function.The bursa of Fabricius (BF) is the central humoral immune organ unique to birds which plays important roles in B lymphocyte differentiation. Here, a new bursal peptide (BP11) with the amino acid sequence DVAGKLPDNRT was identified and characterized from BF. It was proved that BP11 promoted CFU pre-B formation, and regulated B cell differentiation, including increase the percentage of immature and mature B cells in BM cells co-cultured with IL-7. BP11 also exerted immunomodulatory function on antigen-specific immune responses in BALB/c mice immunized with inactivated influence virus (AIV, H9N2 subtype) vaccine, including enhancing AIV-specific antibody and cytokine production. Furthermore, it was noteworthy that BP11 stimulated antibody productions and potentiates the Th1 and Th2-type immune responses in dose-dependent manner in chicken. These results suggested that BP11 might be highly relevant for the development of avian immune system.
Keywords: Bursal peptide (BP11); B cell development; Immunomodulatory function; Immunization experiment;

Dipeptidyl-peptidase IV inhibitory activity of peptides derived from tuna cooking juice hydrolysates by Shih-Li Huang; Chia-Ling Jao; Kit-Pan Ho; Kuo-Chiang Hsu (114-121).
► Peptides with MW over 1422 Da showed great DPP-IV inhibitory activities. ► A peptide, PGVGGPLGPIGPCYE (1412.7 Da), showed DPP-IV inhibition with IC50 value of 116.1 μM. ► A peptide, CAYQWQRPVDRIR (1690.8 Da), showed DPP-IV inhibition with IC50 value of 78.0 μM. ► A peptide, PACGGFWISGRPG (1304.6 Da), showed DPP-IV inhibition with IC50 value of 96.4 μM.The in vitro DPP-IV inhibitory activity of isolated peptides from of tuna cooking juice hydrolyzed by Protease XXIII (PR) and orientase (OR) was determined. The results showed that the peptide fractions with the molecular weight over 1422 Da possessed the greatest DPP-IV inhibitory activity. The amino acid sequences of the three peptides isolated from PR and OR hydrolysates were identified by MALDI-TOF/TOF MS/MS, and they were Pro-Gly-Val-Gly-Gly-Pro-Leu-Gly-Pro-Ile-Gly-Pro-Cys-Tyr-Glu (1412.7 Da), Cys-Ala-Tyr-Gln-Trp-Gln-Arg-Pro-Val-Asp-Arg-Ile-Arg (1690.8 Da) and Pro-Ala-Cys-Gly-Gly-Phe-Try-Ile-Ser-Gly-Arg-Pro-Gly (1304.6 Da), while they showed the dose-dependent inhibition effect of DPP-IV with IC50 values of 116.1, 78.0 and 96.4 μM, respectively. In vitro simulated gastrointestinal digestion retained or even improved the DPP-IV inhibitory activities of the three peptides. The results suggest that tuna cooking juice would be a good precursor of DPP-IV inhibitor, and the DPP-IV inhibitory peptides can successfully passed through the digestive tract.
Keywords: Dipeptidyl-peptidase IV inhibitor; Tuna cooking juice; Type 2 diabetes; Bioactive peptide;

Allatotropin, leucokinin and AKH in honey bees and other Hymenoptera by Jan A. Veenstra; Léa Rodriguez; Robert J. Weaver (122-130).
► Hymenoptera have an allatotropin gene. ► In honeybees the allatotropin gene is only weakly expressed. ► Bees have a leucokinin gene. ► Acquisition of a second TATA box in the Apis AKH gene explains its weak expression.In the honey bee no allatotropin gene has been found, even though allatotropin stimulates the synthesis of juvenile hormone in this species. We report here that honey bees and other Hymenoptera do have a typical allatotropin gene, although the peptides predicted have a somewhat different structure from that of other insect allatotropins. Polyclonal antisera to honey bee allatotropin reacted with material in the neurohemal organs of the segmental nerves of abdominal ganglia. We were unable to find the allatotropin peptide using mass spectrometry in extracts from these tissues. Thus the expression of this gene in honey bees is less important than in other insect species. We also characterized the leucokinin gene which similarly appears to be very weakly expressed in worker honey bees. Unlike the allatotropin gene, which is conserved within Hymenoptera, the leucokinin gene is much more variable in structure and was not found in ants nor the parasitic wasp Nasonia vitripennis. The absence of significant expression of adipokinetic hormone (AKH) in the honey bee may be due to the existence of a second TATA box in the promotor region of the gene, which explains the production of an mRNA encoding a putative peptide precursor from which no AKH should be released. Such a second TATA box was not found in other Hymenoptera, and may therefore be specific for the two Apis species. It is suggested that functional disintegration of this important metabolic gene became possible in Apis because of the highly evolved social nature of the species.
Keywords: Insect neuropeptide; Juvenile hormone; Corpus allatum; Neuropeptide precursor; Ant; Mass spectrometry; Abdominal ganglia; Adipokinetic hormone; Eusociality;

Intracerebroventricular administration of urotensin II regulates food intake and sympathetic nerve activity in brown adipose tissue by Tohru Yasuda; Takayuki Masaki; Koro Gotoh; Seiichi Chiba; Tetsuya Kakuma; Hironobu Yoshimatsu (131-135).
► Central infusion of urotensin II suppressed food intake in rats. ► Central infusion of urotensin II increased uncoupling proteins in brown adipose tissue (BAT). ► Central infusion of urotensin II increased BAT temperature and sympathetic nerve activity.To clarify the functional roles of urotensin II in regulating energy balance, we investigated the effects of a central infusion of urotensin II on food intake, uncoupling protein (UCP) 1 mRNA expression, temperature, and sympathetic nervous system activity in brown adipose tissue (BAT), a site that regulates energy expenditure in rodents. A bolus central infusion of urotensin II at a dose of 1 nmol/rat into the third cerebral ventricle decreased food intake (p  < 0.05). Additionally, urotensin II induced c-Fos-like-immunoreactivity (c-FLI) in the paraventricular nucleus (PVN) as compared with that in the control (phosphate buffered saline [PBS]-treated) group. Furthermore, urotensin II increased BAT UCP 1 mRNA expression (p  < 0.05). Finally, central infusion of urotensin II significantly increased BAT sympathetic nerve activity, which was accompanied by a significant elevation in BAT temperature (p  < 0.05) in rats. Taken together, central infusion of urotensin II regulates food intake and BAT sympathetic nerve activity in rats.

Maternal hypertension induces tissue-specific modulations of the apelinergic system in the fetoplacental unit in rat by Joanna Ivars; Laura Butruille; Claude Knauf; Thomas Bouckenooghe; Sylvain Mayeur; Didier Vieau; Philippe Valet; Philippe Deruelle; Jean Lesage (136-138).
► The apelinergic (apelin/APJ) system is present in both fetal tissues and placenta in rat. ► Maternal hypertension modulates its expression in the feto-placental unit. ► Maternal and fetal apelin plasma levels are close at term. ► This system may control fetal growth and cardiovascular functions in utero. Apelin and its receptor APJ are expressed in fetal tissues but their function and regulation remain largely unknown. In rat, maternal treatment with a nitric oxide synthase inhibitor inducing hypertension was used to investigate apelin plasma levels in mother/fetus pairs and on the gene expression level of the apelin/APJ system in fetal tissues and placenta. At term, plasma levels of apelin were not modulated but APJ expression was increased in placenta and lung but reduced in heart. Apelin expression was increased only in the heart. We postulate that the apelinergic system may control fetal growth and cardiovascular functions in utero.

Suppressor of cytokine signaling 3 in the human hypothalamus by Anneke Alkemade; Unga A. Unmehopa; Ellen V.S. Hessel; Dick F. Swaab; Andries Kalsbeek; Eric Fliers (139-142).
► SOCS3 expression in the human hypothalamus is most prominent in the PVN. ► SOCS3 is expressed in the IFN, the human equivalent of the ARC. ► SOCS3 in the PVN is inversely correlated with serum leptin.In rodents, the mediobasal hypothalamus and the hypothalamic paraventricular nucleus (PVN) are implicated in leptin signaling. Surprisingly little data is available on the human hypothalamus. We set out to study the expression of suppressor-of-cytokine-signaling 3 (SOCS3), α-melanocyte stimulating hormone (αMSH) and agouti-related protein (AgRP) in the infundibular nucleus (IFN) and to investigate the relationship between these neuropeptide expressions and serum leptin concentrations in a blood sample taken within 24 h before death. We studied post-mortem human brain material by means of quantitative immunocytochemistry. We found that SOCS3 immunoreactivity was widely distributed throughout the hypothalamus, and most prominent in the PVN, whereas expression levels in the IFN were low. Surprisingly, SOCS3 expression in the PVN was inversely related to serum leptin. A significant positive correlation was observed between AgRP and NPY expression in the IFN. The inverse correlation between SOCS3 expression in the PVN and serum leptin was unexpected and may be related to the hypothalamic adaptation to fatal illness rather than to nutritional status, or may represent an interspecies difference.
Keywords: Hypothalamus; Suppressor of cytokine signaling 3; Leptin; Agouti related protein; Neuropeptide Y; α-Melanocyte stimulating hormone;