Peptides (v.31, #12)

Structure–activity studies of GmSubPep, a soybean peptide defense signal derived from an extracellular protease by Gregory Pearce; Gerhard Munske; Yube Yamaguchi; Clarence A. Ryan (2159-2164).
▶ Substitutions of alanine at each of the 12 amino acid positions revealed that the amino acids at positions 10 (arginine) and 12 (histidine) were essential for activity. ▶ Both analogs, [A10]GmSubPep and [A12]GmSubPep were able to reduce the physiological effects of GmSubPep associated with receptor binding ▶ Deletion of the C-terminal histidine [GmSubPep(1–11)] abolished the alkalinizing activity and this peptide was also a strong competitor for receptor binding. ▶ This study indicates that the extreme C-terminal of GmSubPep has important signal transduction properties while the C-terminal is essential for receptor interaction.GmSubPep, a 12-amino acid peptide isolated from soybean leaves, induces the expression of genes in soybean suspension-cultured cells that encode proteins involved in defense against pathogens. The peptide is derived from an extracellular subtilisin-like protease (subtilase) and binds a putative cell-surface receptor that initiates a defense signaling cascade. Interaction of the peptide with its receptor results in alkalinization of soybean suspension cell media which can be utilized to analyze the kinetics of receptor binding. Substitutions of alanine at each of the 12 amino acid positions revealed that the amino acids at positions 10 (arginine) and 12 (histidine) were essential for activity. Both analogs were able to reduce the physiological effects of GmSubPep associated with receptor binding. Deletion of the C-terminal histidine [GmSubPep(1–11)] abolished the alkalinizing activity and this peptide was also a strong competitor for receptor binding. Deletion of N-terminal amino acids from GmSubPep caused a sequential loss of activity with no alkalinizing activity for GmSubPep(4–12). However, the N-terminal deleted peptides did not compete with GmSubPep for receptor binding. Further modifications at the arginine-10 position indicated that an ionizable proton was not essential for activity as an attenuated response was found for a citrulline substitution. Substitution of the histidine-12 with methylated histidine at position N-1 of the imidazole group abolished activity, whereas substitution at N-3 was completely active, indicating that the N-3 analog retains important receptor binding properties. This study indicates that the extreme C-terminal of GmSubPep has important signal transduction properties while the C-terminal is essential for receptor interaction.
Keywords: GmSubPep; Subtilisin; Plant defense; Peptide signaling;

Conserved regions of the Plasmodium falciparum rhoptry-associated protein 3 mediate specific host-pathogen interactions during invasion of red blood cells by Jeison García; Hernando Curtidor; Magnolia Vanegas; Gabriela Arévalo-Pinzon; Manuel A. Patarroyo; Manuel E. Patarroyo (2165-2172).
▶ RAP-3 contains regions that specifically bind to RBCs. ▶ Both high and low molecular weight RBC surface receptors were found for RAP-3 HABPs. ▶ RAP-3 HABPs moderately inhibit merozoite invasion to RBCs in vitro.Invasion of red blood cells (RBCs) by the Plasmodium falciparum malaria merozoite is mediated by parasite surface molecules and proteins contained within apical organelles that are capable of recognizing receptors on the membrane of RBCs. The identification and characterization of these P. falciparum invasion-associated proteins is the first step for unveiling potential new drug and vaccine target molecules to eradicate this deadly disease. Among the exclusive set of malarial vaccine candidates, the members of the rhoptry-associated protein (RAP) family have been associated with the parasite's binding to and invasion of RBCs. Remarkably, the third member of this family (named RAP-3) has been recently detected on the surface of non-infected RBCs exposed to free merozoites, therefore suggesting the participation of this protein during RBC infection. In this study, the sequence of RAP-3 was finely mapped using synthetic peptides in order to identify which are the specific binding regions involved in RAP3–RBC interactions. Two high-activity binding peptides (HABPs) established high affinity interactions with RBC surface molecules of about 27–90 kDa, which were differentially affected by different enzymatic treatments. RAP-1 and RAP-2 HABPs inhibited binding of RAP-3 HABPs to different extents, thus suggesting the recognition of similar binding sites on RBC membrane, as well as ability of RAP-3 HABPs to inhibit P. falciparum infection in vitro. Altogether, these functional analyses of RAP-3 HABPs strongly suggest a potential role for this protein in RBC invasion, and highlight its HABPs as potential targets to develop a fully protective minimal subunit-based malarial vaccine.
Keywords: Malarial vaccine; High-activity binding peptides; HABPs; Plasmodium falciparum; Rhoptry-associated protein 3; RAP-3;

▶ BAR peptide inhibits adherence of Porphyromonas gingivalis to Streptococcus gordonii. ▶P. gingivalis-mediated proteolytic degradation of BAR occurs rapidly but BAR is protected from cleavage by its soluble receptor, the Mfa protein. ▶ Degradation of BAR peptide is primarily mediated by the lysine-specific gingipain of P. gingivalis. ▶ Substitution of d-Lys for l-Lys in BAR confers resistance to P. gingivalis-mediated proteolytic degradation. ▶ The d-Lys analog of BAR is a more potent inhibitor of P. gingivalis adherence to Streptococcus gordonii.The interaction of the periodontal pathogen, Porphyromonas gingivalis, with oral streptococci such as Streptococcus gordonii precedes colonization of the subgingival pocket and represents a target for limiting P. gingivalis colonization of the oral cavity. Previous studies showed that a synthetic peptide (designated BAR) derived from the antigen I/II protein of S. gordonii was a potent competitive inhibitor of P. gingivalis adherence to S. gordonii and subsequent biofilm formation. Here we show that despite its inhibitory activity, BAR is rapidly degraded by intact P. gingivalis cells in vitro. However, in the presence of soluble Mfa protein, the P. gingivalis receptor for BAR, the peptide is protected from proteolytic degradation suggesting that the affinity of BAR for Mfa is higher than for P. gingivalis proteases. The rate of BAR degradation was reduced when the P. gingivalis lysine-specific gingipain was inhibited using the specific protease inhibitor, z-FKcK, or when the gene encoding the Lys-gingipain was inactivated. In addition, substituting d-Lys for l-Lys residues in BAR prevented degradation of the peptide when incubated with the Lys-gingipain and increased its specific adherence inhibitory activity in a S. gordoniiP. gingivalis dual species biofilm model. These results suggest that Lys-gingipain accounts in large part for P. gingivalis-mediated degradation of BAR and that more effective peptide inhibitors of P. gingivalis adherence to streptococci can be produced by introducing modifications that limit the susceptibility of BAR to the Lys-gingipain and other P. gingivalis associated proteases.
Keywords: Porphyromonas gingivalis; Streptococcus gordonii; Biofilm; Gingipain;

▶ The blockade of CGRP signaling prevents the development of morphine tolerance. ▶ BIBN4096BS prevents the right-shift of the dose–response curve of acute morphine. ▶ Chronic morphine time-dependently increases microglial p38 phosphorylation. ▶ The number and morphology of microglia are subject to modulation by CGRP signaling.Calcitonin gene-related peptide (CGRP) exerts an effect on the development of morphine antinociceptive tolerance, which may in part involve the activation of p38 kinase. In the present study, we investigated the temporal expression and spatial distribution of p38 phosphorylation as well as their possible regulation by CGRP receptor signaling following chronic morphine treatment. A 7-day intrathecal treatment with morphine (15 μg/day) produced tolerance to its analgesic effects as well as a rightward shift in the dose–response curve to its antinociceptive effects. This treatment time-dependently increased p38 phosphorylation in the spinal dorsal horn, as shown by phosphorylated p38-immunoreactive (p-p38-ir) cell counts. The increased phosphorylation occurred first in superficial layers of the spinal dorsal horn and then extended to deeper laminae. Interestingly, accompanying the development of morphine tolerance, p-p38-ir cells, identified as microglia, displayed hypertrophy and increased number of branched processes, suggesting their activation. These various behavioral and morphological changes were blocked by the intrathecal treatment with BIBN4096BS, a non-peptide CGRP receptor antagonist. These data provide additional morphological evidence in support of a role for CGRP in the development of morphine antinociceptive tolerance, possibly by regulating the expression and distribution of p38 phosphorylation in microglia.
Keywords: Calcitonin gene-related peptide; Phosphorylation; Glia; Morphine;

Regulation of gastric hormones by systemic rapamycin by Geyang Xu; Yin Li; Wenjiao An; Jing Zhao; Xinxin Xiang; Li Ding; Ziru Li; Youfei Guan; Xian Wang; Chaoshu Tang; Yi Zhu; Nanping Wang; Xiaoying Li; Michael Mulholland; Weizhen Zhang (2185-2192).
▶ mTOR signaling molecules are expressed in gastric endocrine cells. ▶ Ghrelin-positive cells express pmTOR, rapamycin stimulates ghrelin production. ▶ 1/3 gastrin-positive cells express pS6K1, rapamycin inhibits gastrin synthesis. ▶ Somatostatin-immunoreactive cells do not express pS6K1.The mammalian target of rapamycin (mTOR), an evolutionarily conserved serine–threonine kinase, is an intracellular fuel sensor critical for cellular energy homeostasis. Gastrointestinal endocrine cells play a vital role in the regulation of energy balance by secreting hormones that inform the brain about energy supply. Here we showed the localization of mTOR signaling molecules in more than 90% of gastric ghrelin cells and 36 ± 3% of gastrin cells, while no somatostatin-positive cell showed phospho-S6K1 immunoreactivity. Inhibition of mTOR significantly stimulated expression of gastric ghrelin mRNA and protein, and the concentration of plasma ghrelin (2.06 ± 0.34 ng/ml vs. 12.53 ± 3.9 ng/ml, p  < 0.05), inhibited gastrin synthesis and secretion (75.01 ± 6.71 pg/ml vs. 54.04 ± 3.65 pg/ml, p  < 0.05), but had no effect on somatostatin production (165.2 ± 25.07 pg/ml vs. 178.9 ± 29.14 pg/ml, p  = 0.73). Gastric mTOR is a gastric sensor whose activity is linked to the differential regulation of gastric hormone production and release.
Keywords: Gastric mTOR; Gastric endocrine cells; Gastric hormones;

▶ Saporin, when conjugated to NPY (NPY–SAP), kills cells expression NPY receptors. ▶ Injecting NPY–SAP into the central amygdala (CeA) reduces local Y1 receptor levels. ▶ Injecting NPY–SAP into the CeA increases anxiety-like behavior. ▶ NPY–SAP may be a useful tool to study the central NPY circuitry modulating anxiety.Neuropeptide Y (NPY) is a 36-amino-acid neuromodulator that is distributed throughout the central nervous system and has been implicated in a wide range of neurobiological responses including the integration of emotional behavior. The anxiolytic properties of NPY are modulated by NPY signaling in the hippocampus and in the central (CeA) and basolateral (BLA) nuclei of the amygdala. Recently, the neurotoxin saporin, when conjugated to NPY (NPY–SAP), was shown to selectively kill NPY receptor-expressing neurons and has been used as a tool to study the central NPY neurocircuitry involved with feeding behaviors. Here we determined if NPY–SAP can be used as a tool to study the central NPY neurocircuitry that modulates anxiety-like behaviors. BALB/cJ mice were given injection of either NPY–SAP or a control blank saporin (B-SAP) into the CeA or the basomedial hypothalamus (BMH) as a control injection site. The elevated zero maze test was used to assess anxiety-like behavior and NPY–SAP-induced lesions were verified using NPY Y1 receptor (Y1R) immunoreactivity (IR). Results showed that injection of NPY–SAP into the CeA site-specifically blunted Y1R IR in the CeA which was associated with a significant increase in anxiety-like behavior. Injection of NPY–SAP into the BMH, while locally blunting Y1R IR, promoted a compensatory increase of Y1R IR in the BLA and the CA3 region of the hippocampus which was associated with a significant reduction of anxiety-like behavior. The present set of experiments suggest that the NPY–SAP neurotoxin may be a useful tool for studying the NPY neurocircuitry that modulates anxiety-like behaviors.
Keywords: Neuropeptide Y; Anxiety-like behavior; Amygdala; Hippocampus; Saporin; BALB/cJ;

mRNA expression of corticotropin-releasing factor and urocortin 1 after restraint and foot shock together with alprazolam administration by Isabel C. Cespedes; Amanda R. de Oliveira; Joelcimar M. da Silva; André V. da Silva; Luciane V. Sita; Jackson C. Bittencourt (2200-2208).
fos-ir was increased in the PVN after restraint comparing to foot shock but significantly lower in the restraint plus alprazolam and foot shock plus alprazolam. ▶ CRF mRNA expression in the PVN was significantly higher in the restraint than in the foot shock, but was even lower in the restraint plus alprazolam and foot shock plus alprazolam. However, only the difference between restraint plus alprazolam and restraint-only groups was significant. ▶fos-ir in the EW was significantly increased in the restraint and foot shock groups than in the control, but higher in the foot shock than in the restraint group. ▶ Ucn 1 mRNA expression was higher in the restraint than in the foot shock group. ▶ Alprazolam administration significantly increased fos-ir and upregulated Ucn 1 mRNA expression in the EW within the restraint and foot shock groups.Corticotropin-releasing factor (CRF) is expressed in the paraventricular nucleus of the hypothalamus (PVN), and act centrally to provoke stress-like autonomic and behavioral responses. Urocortins 1–3 are additional ligands to the CRF receptors 1 and 2. Ucn 1 neurons are primarily concentrated in the Edinger–Westphal (EW) nucleus and also have been associated with stress responses. It is also known that UCN 1 respond in different ways depending on the stressor presented. Benzodiazepines can act via the CRF peptidergic system and chronic administration of alprazolam does not interfere with CRF mRNA expression in the PVN, but significantly increase Ucn 1 mRNA expression in the EW. The aim of our study was to investigate the relationship between different stressor stimuli, foot shock (FS) and restraint (R), and the mRNA expression of CRF and Ucn 1 in the PVN and EW using alprazolam (A). We employed fos activation and in situ hybridization. Restraint group presented increased fos-ir and CRF mRNA expression in the PVN compared to FS group. The stress responses of R group were prevented by A. In the EW, fos-ir was higher in the FS group than in the R group, whereas Ucn 1 mRNA expression was higher in the R group than in the FS group. Alprazolam significantly increased fos-ir and Ucn 1 mRNA expression in both groups. Our results show that PVN and EW respond in different ways to the same stressors. Furthermore, EW of stressed animals replies in a complementary way comparing to PVN with the use of Alprazolam.
Keywords: Stress; CRF receptors; Benzodiazepines; Anxiety; Urocortins; Paraventricular nucleus of the hypothalamus; Edinger–Westphal nucleus;

Intrahippocampal scopolamine impairs spatial win-shift performance in the RAM. ▶ Intrahippocampal Nle1-AngIV mitigates scopolamine impairment. ▶ Nle1-AngIV improvement of delay-independent memories is immediate. ▶ Nle1-AngIV improvement of delay-dependent memories is not immediate.Depletion of cholinergic neurons in the hippocampus has been implicated in memory impairment and Alzheimer's Disease (AD). The brain angiotensin AT4 receptor is co-localized with cholinergic neurons, and the AT4 receptor has also been implicated in cognitive processing. The current investigation used the spatial win-shift version of the radial arm maze to determine the involvement of AT4 receptors in spatial working memory formation. We initially established that intrahippocampal scopolamine significantly impaired the spatial working memory performance of Sprague-Dawley rats in the radial arm maze. We also demonstrated that subsequent intrahippocampal infusions of Norleucine1-Angiotensin IV (Nle1-AngIV) significantly prevented the scopolamine-induced deficit. Consistent with previously published data on long-term spatial memory, our findings suggest that activation of AT4 receptors can compensate for impaired spatial working memory resulting from compromised muscarinic acetylcholine receptor function. We further demonstrate that the hippocampus is a site of action for Nle1-AngIV-mediated cognitive improvement.
Keywords: Angiotensin IV; AT4 receptor; Hippocampus; RAM; Scopolamine; Spatial working memory;

Involvement of Src family kinase activation in angiotensin II-induced hyperresponsiveness of rat bronchial smooth muscle by Hiroyasu Sakai; Ayako Nishimura; Yu Watanabe; Yuko Nishizawa; Yuki Hashimoto; Yoshihiko Chiba; Miwa Misawa (2216-2221).
Display Omitted▶ Angiotensin II causes SFK activation in bronchial smooth muscle (BSM). ▶ All of the SFK genes were expressed in the lungs and main bronchi. ▶ Angiotensin II causes BSM hyperresponsiveness. ▶ Angiotensin II induces a BSM hyperresponsiveness through activation of SFK.Angiotensin II (Ang II) might be an important mediator in pathogenesis of airway hyperresponsiveness (AHR) that is the asthmatic characteristic feature of asthma, although the mechanisms of AHR caused by Ang II are not yet clear. Presently, the RT-PCR analyses revealed that all the Src family kinases (SFKs), such as Fyn, Lck, Lyn, Hck, Src, Yes, Blk, Fgr and Frk, were expressed in the lungs and main bronchi of rats. The phosphorylation (activation) of SFK (Tyr416) was increased in bronchial smooth muscle (BSM) by Ang II. The Ang II-induced SFK phosphorylation was inhibited by pretreatment with SU6656, an SFK inhibitor. The concentration–contraction curves to carbachol (CCh) were shifted to the left in the presence of Ang II. The maximal contraction of CCh was also significantly increased by pretreatment with Ang II. These results indicate that Ang II causes BSM hyperresponsiveness. The Ang II-induced BSM hyperresponsiveness was significantly inhibited by SU6656, although the carbachol (CCh)-induced contraction itself was not changed by SU6656. In conclusion, Ang II induced a BSM hyperresponsiveness through activation of SFK, and might play an important role in pathophysiology of bronchial asthma.
Keywords: Airway hyperresponsiveness; Angiotensin II; Src family kinase; Bronchial smooth muscle;

Altered myocardial expression of ghrelin and its receptor (GHSR-1a) in patients with severe heart failure by Andres Beiras-Fernandez; Simone Kreth; Florian Weis; Carola Ledderose; Thomas Pöttinger; Carlos Dieguez; Andres Beiras; Bruno Reichart (2222-2228).
▶ Failing hearts express decreased levels of ghrelin and increased levels of the GHS-R. ▶ The expression of these proteins does not differ between the cardiac anatomical areas. ▶ The ghrelin-system appears as part of the network regulating cardiac performance.Ghrelin is a peptide hormone mainly produced by the stomach, which strongly stimulates the release of growth hormone (GH) via the GH secretagogue receptor 1a (GHSR-1a) located in the hypothalamus. It has been reported to exert performance-enhancing effects on myocardial function, and as both ghrelin and GHSR-1a are expressed in myocardial tissues, the ghrelin system may have a direct GH-independent impact on cardiac function. We intended to investigate the expression of ghrelin and its receptor GHSR-1a in different myocardial areas of patients with chronic heart failure (CHF) as compared to heart-healthy subjects to better define the role of the ghrelin signaling system in the networks regulating cardiac function and its potential as a target for diagnosis and/or treatment of CHF. Myocardium biopsies of 12 patients undergoing heart transplantation and suffering from CHF were obtained. Expression of both ghrelin and GHSR-1a was assessed by means of immunohistochemistry and real-time PCR. Expression of ghrelin was significantly decreased in CHF hearts both in atrium and ventricles in comparison to the control hearts (p  < 0.05). The expression of the GHS-1a receptor was significantly increased in the CHF biopsies as compared to controls (p  < 0.05). No significant differences were found between the anatomical areas studied. Expression of myocardial ghrelin and GHSR-1a is directly associated with myocardial function: CHF hearts exhibit an impaired ghrelin production which might reflect maladaptive processes and an – probably compensatory – increase in GHSR-1a expression. These findings may open up new perspectives regarding the potential of ghrelin signaling as a target for pharmacological modulation.
Keywords: Ghrelin; GHSR-1a; Immunohistochemistry; Heart failure; Myocardium;

Cold ambient temperature reverses abdominal surgery-induced delayed gastric emptying and decreased plasma ghrelin levels in rats by Andreas Stengel; Miriam Goebel; Andrew Luckey; Pu-Qing Yuan; Lixin Wang; Yvette Taché (2229-2235).
▶ Abdominal surgery-induced decreased gastric emptying is prevented by cold exposure. ▶ TRH analog intracisternally also prevents postoperative gastric ileus. ▶ Exposure to cold increases plasma acyl (AG) and desacyl ghrelin (DAG) levels. ▶ Cold exposure reverses the abdominal surgery-induced decrease of plasma AG and DAG. ▶ TRH activation by cold prevents surgery-induced ileus likely via releasing AG/DAG.We investigated whether acute cold-induced vagal activation through brainstem thyrotropin-releasing hormone (TRH) signaling influences abdominal surgery-induced delayed gastric emptying (GE) in fasted rats. Laparotomy and cecal palpation or sham (short anesthesia alone) was performed 10 min before or 30 min after cold exposure (4–6 °C) lasting 90 min. Non-nutrient GE was assessed during 70–90 min of cold exposure. Control groups remained at room temperature (RT). The stable TRH analog, RX-77368 (50 ng/rat) was injected intracisternally immediately before surgery and GE monitored 30–50 min postsurgery in rats maintained at RT. Plasma acyl (AG) and total ghrelin levels were assessed using the new RAPID blood processing method and radioimmunoassays. Desacyl ghrelin (DAG) was derived from total minus AG. In rats maintained at RT, abdominal surgery decreased GE by 60% compared to sham. Cold before or after surgery or RX-77368 normalized the delayed GE. In non-fasted rats, cold exposure increased plasma AG and DAG levels at 2 h (2.4- and 2.7-times, respectively) and 4 h (2.2- and 2.0-times, respectively) compared to values in rats maintained at RT. In fasted rats, abdominal surgery decreased AG and DAG levels by 2.4- and 2.1-times, respectively, at 90 min. Cold for 90 min after surgery normalized AG and DAG levels to those observed in sham-treated animals kept at RT. These data indicate that endogenous (cold exposure) and exogenous (TRH analog) activation of medullary TRH vagal signaling prevent abdominal surgery-induced delayed GE. The restoration of circulating AG levels inhibited by abdominal surgery may contribute to alleviate postoperative gastric ileus.
Keywords: Abdominal surgery; Acyl ghrelin; Cold; Desacyl ghrelin; Gastric emptying; RAPID method; Rat; TRH analog;

▶ The peptides apelin, ghrelin and nesfatin-1 are present in the human breast milk. ▶ Their concentration is lowered by gestational diabetes mellitus. ▶ These peptides could be important for growth, energy regulation and maturation of the gastrointestinal system in neonates.Numerous bioactive peptides (such as ghrelins) have been identified in breast milk but there is no information concerning apelin and nesfatin-1. Therefore, present study was designated to explore whether breast milk contains apelin and nesfatin-1, to determine the concentrations and to compare these with serum levels. In addition, the concentrations of these peptides were compared in patients with gestational diabetes and normal lactating samples. Furthermore, this study explored the effectivity of various commercial diagnostic kits for determining ghrelin concentrations in breast milk. Ten gestational diabetic lactating women (29.1 ± 2.2 years old and BMI: 33.2 ± 4.8) and 10 control lactating women (28.2 ± 1.8 years old and BMI: 39.48 ± 1.7) were enrolled in the study. An ELISA was used to determine concentrations of apelin-36 and -12, nesfatin-1, and acylated and desacylated ghrelin in serum, colostrum and mature milk. Serum apelin-36 and -12 concentrations were correlated with colostrum and mature milk, and the same trends were observed for nesfatin-1. Apelins and nesfatin-1 concentrations were higher in mature milk than in colostrum (P  < 0.05). The concentration of apelins, ghrelins and nesfatin-1 in serum and milk in gestational diabetic lactating women was lower than in control samples. The majority of ghrelin circulating and in milk was the free form (desacylated) in both groups of women. This is the first report to describe the presence of apelins and nesfatin-1 in breast milk. It is suggested that the source of ghrelins, apelins and nesfatin-1 in breast milk is likely to be breast tissue (autonomous production). These bioactive peptides found in breast milk could be important for growth, energy regulation and maturation of the gastrointestinal system in neonates.
Keywords: Apelin; Nesfatin-1; Ghrelin; Breast milk; Gestational diabetes mellitus;

Intracerebroventricular administration of apelin-13 inhibits distal colonic transit in mice by Yan-Jie Yang; Shuang-Yu Lv; Ming-Hui Xiu; Ning Xu; Qiang Chen (2241-2246).
▶ Apelin-13 dose-dependently inhibits fecal pellet output and bead expulsion in mice. ▶ Apelin-13 inhibits distal colonic transit through the activation of APJ receptors. ▶ Opioid receptors are involved in apelin-13-induced suppression of distal colon transit. ▶ In vitro, apelin-13 has no effect on distal colonic contractions.Apelin is a novel bioactive peptide as the endogenous ligand for the orphan G-protein-coupled receptor (GPCR), APJ, a receptor distributed in various tissues such as the hypothalamus and the gastrointestinal tract. Recent reports showed that apelin regulated many biological functions, including blood pressure, neuroendocrine, drinking behavior and food intake. However, the role of apelin in regulating gastrointestinal motility remains unknown. The present study aimed to investigate the actions of intracerebroventricularly administered apelin-13 on colonic transit as well as the actions of apelin-13 on the contraction of isolated distal colon in vitro. Intracerebroventricular (i.c.v.) injection of apelin-13 (0.3, 0.5, 1 and 3 μg/mouse) dose-dependently inhibited fecal pellet output and bead expulsion. This effect was significantly antagonized by the APJ receptor antagonist apelin-13(F13A), indicating an APJ receptor-mediated mechanism. Furthermore, naloxone could also reverse the inhibitory effect of apelin-13 on fecal pellet output and bead expulsion, suggesting the involvement of opioid receptors in the suppressive effect of apelin-13 on distal colon transit. However, apelin-13 (10−8–10−6  M) did not affect distal colonic contractions in vitro.
Keywords: Apelin-13; APJ receptor; Opioid receptor; Fecal pellet output; Bead expulsion; Distal colonic transit;

Identification of a peptide sequence targeting mammary vasculature via RPLP0 during lactation by Nam Kyung Lee; Min kook Kim; Jin Huk Choi; Eun Bae Kim; Hong Gu Lee; Sang Kee Kang; Yun Jaie Choi (2247-2254).
▶ Lactating mammary tissue-specific peptides were identified by in vivo phage display. ▶ CLHQHNQMC peptide (MG1) specifically homes to mammary vasculature during lactation. ▶ MG1 directly binds to RPLP0 expressed dominantly on lactating mammary tissue. ▶ MG1 internalizes into the endothelial cell HUVECs by specific interaction with RPLP0.To find novel targeting moieties to lactating mammary gland, in vivo phage display screening was conducted with lactating rats and a peptide ligand, CLHQHNQMC (designated as MG1), which specifically homes to the mammary tissue during lactation, was identified through the consecutive in vivo biopannings. MG1 peptide ligand showed specific binding affinity to lactating mammary tissue without any preference to other organs tested in ex vivo and in vivo validation, and microscopy analysis revealed that systemically introduced MG1 could be specifically localized in the lactating mammary gland associated with mammary epithelia and alveolar vasculature. Based on the observation that binding of MG1-encoding phage to lactating mammary gland was competitively inhibited by synthetic MG1 peptide ligand, we attempted to identify a counterpart molecule corresponding to specific recognition of the MG1 and the acidic Ribosomal Protein Large P0 (RPLP0) was selected as a candidate receptor for MG1 by peptide affinity pull-down assay with protein extracts from lactating mammary tissue. We demonstrated specific expression of RPLP0 in mammary tissue, especially during lactation, by immunoblotting assays and also demonstrated that MG1 peptide ligand could be bound to, and internalized into, the cells effectively via specific interaction with RPLP0 by analysis using an in vitro endothelial cell model. The overall results suggest that the MG1 has a specific affinity with RPLP0 which are dominantly expressed on the mammary vasculature during lactation and this specific affinity enables the MG1 would be served as an effective homing ligand to deliver functional molecules to the lactating mammary gland.
Keywords: Lactation; Mammary vasculature; Phage display; RPLP0; Targeting moiety;

Plasma levels of kisspeptins in postmenopausal Chinese women do not show substantial elevation by Jing Peng; Hong Xu; Bei Yang; Jia Hu; Bao-Ping Zhang; Lin Zou; Hai-Bin Kuang (2255-2258).
▶ Plasma levels of kisspeptin in postmenopausal women. ▶ There was no significant difference in post- and premenopausal plasma kisspeptin levels. ▶ Plasma kisspeptin levels were not significantly correlated to FSH and LH. ▶ There was no any correlation between plasma kisspeptin and E2 in postmenopausal women.The menopause, defined as the permanent cessation of menstruation resulting from ovarian failure, is characterized by elevated levels of serum gonadotropins. Recent studies have demonstrated that the gonadotropin hypersecretion in postmenopausal women is secondary to increase of KiSS-1 mRNA from the hypothalamus neurons, which encoded kisspeptin peptides. The present study was designed to determine whether plasma kisspeptins levels are altered in postmenopausal women. Blood samples were taken from 145 postmenopausal women, 35 young women and 30 pregnant women control in the first trimester. The plasma concentration of kisspeptins, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2) was measured using immunoassay kits. Results indicated that plasma kisspeptins levels in postmenopausal women had higher than those in young women (5.25 ± 0.36; 4.48 ± 0.34 pmol/L), but no significant difference was found between the two groups (p  = 0.179). Plasma FSH and LH levels were significantly higher in postmenopausal women (124.67 ± 12.78, 57.14 ± 3.57 mIu/mL) than those in young women (9.23 ± 2.78, 7.56 ± 2.71 mIu/mL, p  < 0.001). However, Plasma kisspeptins levels were not significantly correlated to FSH and LH in postmenopausal women (r  = −0.23, 0.324; p  = 0.927, 0.176, respectively), and also there was no any correlation between plasma kisspeptins and E2 in postmenopausal women (r  = −0.065; p  = 0.792). Collectively, there was no significant difference in plasma kisspeptins levels between postmenopausal and young women. Our result suggested that kisspeptins’ role during menopause might mainly act in central rather than peripheral system and it could not be currently used as a clinical marker for menopause.
Keywords: Kisspeptin; Menopause; Gonadotropins; Estradiol; G protein-coupled receptor 54;

Central Neuropeptide S inhibits food intake in mice through activation of Neuropeptide S receptor by Ya-Li Peng; Ren-Wen Han; Min Chang; Lei Zhang; Rui-San Zhang; Wei Li; Yi-Fan Han; Rui Wang (2259-2263).
▶ NPS dose-related inhibited food intake in fasted mice. ▶ NPSR antagonist blocked the inhibitory effect of NPS on food intake. ▶ CRF1 receptor antagonist inhibited the hyperlocomotor action of NPS. ▶ CRF1 receptor antagonist did not affect the role of NPS on food intake.Neuropeptide S (NPS), the endogenous ligand of NPS receptor (NPSR), can regulate a variety of biological functions, including arousal, anxiety, locomotion, memory and drug abuse. Previous studies have shown that central NPS inhibited food intake in rats and chicks. In the present study, we investigated the role of central NPS on food intake in fasted mice, and detected the underlying mechanism(s) by using NPSR antagonist [D-Val5]NPS and Corticotropin-Releasing Factor 1 (CRF1) Receptor antagonist NBI-27914. The present results indicated that intracerebroventricular injection of NPS (0.001–0.1 nmol) dose-dependently inhibited food intake in fasted mice. The anorectic effect of NPS reached the maximum at the dose of 0.1 nmol, which could be antagonized by co-injection of 10 nmol NPSR antagonist [D-Val5]NPS. Furthermore, CRF1 receptor antagonist NBI-27914 at the dose of 2 μg antagonized the hyperlocomotor action of NPS, but did not affect the role of NPS on food intake. In conclusion, our results demonstrated central NPS inhibited food intake in fasted mice, mediated by its cognate NPSR, but not by CRF1 receptor.
Keywords: Neuropeptide S (NPS); Neuropeptide S receptor (NPSR); CRF; Food intake; Mice;

Locus coeruleus galanin expression is enhanced after exercise in rats selectively bred for high capacity for aerobic activity by Patrick S. Murray; Jessica L. Groves; Brett J. Pettett; Steven L. Britton; Lauren G. Koch; Rod K. Dishman; Philip V. Holmes (2264-2268).
▶ Selection for running capacity dramatically influences running behavior. ▶ Greater overall running distance is related to more robust galanin mRNA upregulation in the locus coeruleus.The neuropeptide galanin extensively coexists with norepinephrine in locus coeruleus (LC) neurons. Previous research in this laboratory has demonstrated that unlimited access to activity wheels in the home cage increases mRNA for galanin (GAL) in the LC, and that GAL mediates some of the beneficial effects of exercise on brain function. To assess whether capacity for aerobic exercise modulates this upregulation in galanin mRNA, three heterogeneous rat models were tested: rats selectively bred for (1) high intrinsic (untrained) aerobic capacity (High Capacity Runners, HCR) and (2) low intrinsic aerobic capacity (Low Capacity Runners, LCR) and (3) unselected Sprague–Dawley (SD) rats with and without free access to running wheels for 3 weeks. Following this exercise protocol, mRNA for tyrosine hydroxylase (TH) and GAL was measured in the LC. The wheel running distances between the three models were significantly different, and age contributed as a significant covariate. Both selection and wheel access condition significantly affected GAL mRNA expression, but not TH mRNA expression. GAL was elevated in exercising HCR and SD rats compared to sedentary rats while LCR rats did not differ between conditions. Overall running distance significantly correlated with GAL mRNA expression, but not with TH mRNA expression. No strain differences in GAL or TH gene expression were observed in sedentary rats. Thus, intrinsic aerobic running capacity influences GAL gene expression in the LC only insofar as actual running behavior is concerned; aerobic capacity does not influence GAL expression in addition to changes associated with running.
Keywords: Galanin; Tyrosine hydroxylase; Aerobic capacity; Running-wheel;

Glial response to lipopolysaccharide: Possible role of endothelins by Talia Filipovich-Rimon; Sigal Fleisher-Berkovich (2269-2275).
▶ Endothelins inhibited the LPS-induced glial synthesis of prostaglandin E2 and nitric oxide. ▶ By contrast, endothelins enhanced unstimulated synthesis of prostaglandin E2 and nitric oxide. ▶ COX-2 and iNOS mediate endothelins effects on glial inflammation.Glial inflammation plays a major role in the development of neurodegenerative diseases. Although endothelins (ETs) are known as modulators of inflammation in the periphery, little is known about their possible role in brain inflammation. Previously, we demonstrated that all three endothelins (ET-1, ET-2 and ET-3) enhanced unstimulated synthesis of the glial pro-inflammatory mediators, prostaglandin E2 (PGE2) and nitric oxide (NO). In the present study, glial cells were stimulated in an in vitro model of inflammation by incubation with the bacterial endotoxin lipopolysaccharide (LPS). Indeed, the present study shows that ETs regulate basal and LPS-induced glial inflammation in an opposite fashion. Here we demonstrate that ETs significantly inhibited the LPS-induced glial synthesis of PGE2 and NO, and each of the selective antagonists for ETA and ETB receptors (BQ123 and BQ788 respectively), significantly inhibited the ETs effects in LPS-treated cells. Similar results were observed when expression of key enzymes namely, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in PG and NO synthesis respectively, was measured. ET-1 significantly enhanced the expression of both COX-2 and iNOS. Whereas, it inhibited the LPS-induced expression of both enzymes. These observations suggest a novel neuro-immune feedback pathway through which inflammatory mediators’ synthesis is initially enhanced by ETs and are eventually blocked by the same neuropeptide when excessive production of inflammatory mediators occurs following an inflammatory insult.
Keywords: Endothelins; Prostaglandin E2; Nitric oxide; Lipopolysaccharide; Glial cells; Brain inflammation;

Effects of PACAP and VIP on hyperglycemia-induced proliferation in murine microvascular endothelial cells by Alessandro Castorina; Salvatore Giunta; Venera Mazzone; Venera Cardile; Velia D’Agata (2276-2283).
▶ Prolonged exposure to high glucose conditions increase endothelial cell growth. ▶ Hyperglycemia-induced endothelial cell growth increases PAC1 and VPAC2 receptor expression. ▶ Exogenous PACAP or VIP stimulation inhibits cell proliferation.Hyperglycemia is implicated both in micro- and macro-vascular complications in diabetes mellitus. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are two known nonclassic regulators of angiogenesis, although their biological role on endothelial cell proliferation remains poorly defined. In the present study we hypothesized that either peptides might play an inhibitory role on hyperglycemia-induced cell growth. To this end, we investigated the effect of both PACAP and VIP on cell proliferation in murine microvascular endothelial cells (H5V) cultured both under euglycemic and hyperglycemic conditions (5 and 25 mM glucose, respectively) for 24, 48 h, 7 and 15 days. Results demonstrated that high glucose treatment induced a time-dependent increase in cell viability after 48 h (p  < 0.05), which was much more evident after 7 and 15 days (p  < 0.001). Similar effects were observed in cell proliferation, although significant changes were obtained after prolonged exposures to high glucose (7 and 15 days; p  < 0.001). The proliferative response to the glucose-enriched environment was correlated to changes in the expression of PAC1 and, to a minor extent, to VPAC2, but not VPAC1 receptors, as measured by quantitative real-time PCR. These results were further confirmed by Western blot and immunofluorescence analyses. Interestingly, 10−7  M PACAP or VIP treatment significantly attenuated hyperglycemia-induced increase in cell viability and proliferation after 7 and 15 days. Taken together, our findings demonstrate that both PACAP and VIP peptides exert an inhibitory activity on hyperglycemia-induced endothelial cell proliferation, thus suggesting that the effect might be mediated by PAC1 and VPAC2 receptors.
Keywords: PACAP; VIP; Endothelial cells; Proliferation; Hyperglycemia;

Insulin detemir is not transported across the blood–brain barrier by William A. Banks; John E. Morley; Jessica L. Lynch; Kristin M. Lynch; Arshag D. Mooradian (2284-2288).
▶ Insulin detemir does not cross the blood–brain barrier. ▶ Insulin detemir inhibits the transport of insulin across the blood–brain barrier. ▶ The effects of insulin detemir are likely mediated through indirect mechanisms.Insulin detemir has a different profile of action on the central nervous system (CNS) than human insulin. It has been hypothesized that this is caused by an altered ability of insulin detemir to cross the blood–brain barrier (BBB). Here, we measured the permeability of the BBB to insulin detemir. We labeled insulin detemir with radioactive iodine (I-Det) and examined its ability to cross the BBB of the mouse. Permeation was assessed after intravenous injection and by brain perfusion in the presence or absence of excess insulin detemir. The ability of insulin detemir to inhibit human insulin transport across the BBB was also assessed. I-Det did not cross the BBB either after intravenous injection or when studied by brain perfusion, a method which removes or reduces the influence of circulating proteins. Unlabeled detemir was about 10 times less potent than human insulin at inhibiting the transport of radioactive human insulin across the BBB. The altered CNS profile of insulin detemir may be caused by its poor access to CNS receptors and by a block of human insulin from crossing the BBB.
Keywords: Blood–brain barrier; Insulin; Peptide; Brain;

Functional coupling of Cys-226 and Cys-296 in the glucagon-like peptide-1 (GLP-1) receptor indicates a disulfide bond that is close to the activation pocket by Rosalind J. Mann; Suleiman Al-Sabah; Rakel López de Maturana; John K. Sinfield; Dan Donnelly (2289-2293).
▶ A phenotypic change caused by mutating Cys-226 was reversed by the additional mutation of Cys-296. ▶ This functional coupling suggests the presence of a disulfide bond between these cysteines. ▶ A side chain larger than a methyl group at residue 226 leads to reduced agonist potency. ▶ ECL2 lies close to the activation pocket in the receptor core domain.G protein-coupled receptors (GPCRs) are seven transmembrane α-helical (7TM) integral membrane proteins that play a central role in both cell signaling and in the action of many pharmaceuticals. The crystal structures of several Family A GPCRs have shown the presence of a disulfide bond linking transmembrane helix 3 (TM3) to the second extracellular loop (ECL2), enabling ECL2 to stabilize and contribute to the ligand binding pocket. Family B GPCRs share no significant sequence identity with those in Family A but nevertheless share two conserved cysteines in topologically equivalent positions. Since there are no available crystal structures for the 7TM domain of any Family B GPCR, we used mutagenesis alongside pharmacological analysis to investigate the role of ECL2 and the conserved cysteine residues. We mutated Cys-226, at the extracellular end of TM3 of the glucagon-like peptide-1 (GLP-1) receptor, to alanine and observed a 38-fold reduction in GLP-1 potency. Interestingly, this potency loss was restored by the additional substitution of Cys-296 in ECL2 to alanine. Alongside the complete conservation of these cysteine residues in Family B GPCRs, this functional coupling suggested the presence of a disulfide bond. Further mutagenesis demonstrated that the low potency observed at the C226A mutant, compared with the C226A-C296A double mutant, was the result of the bulky nature of the released Cys-296 side chain. Since this suggested that ECL2 was in close proximity to the agonist activation pocket, an alanine scan of ECL2 was carried out which confirmed the important role of this loop in agonist-induced receptor activation.
Keywords: GPCR; GLP-1; Mutagenesis; Agonist; Receptor;

Bombesin and neurotensin exert antiproliferative effects on oval cells and augment the regenerative response of the cholestatic rat liver by Stelios F. Assimakopoulos; Athanassios C. Tsamandas; Christos D. Georgiou; Constantine E. Vagianos; Chrisoula D. Scopa (2294-2303).
▶ BBS and NT attenuate oval cell proliferation and accumulation in the cholestatic rat liver. ▶ BBS and NT regulate hepatocyte proliferation/apoptosis improving cholestatic liver's regenerative response. ▶ BBS and NT regulate cholangiocyte proliferation/apoptosis improving obstructive cholangiopathy.The regenerative capacity of the cholestatic liver is significantly attenuated. Oval cells are hepatic stem cells involved in liver's regeneration following diverse types of injury. The present study investigated the effect of the neuropeptides bombesin (BBS) and neurotensin (NT) on oval cell proliferation as well as on hepatocyte and cholangiocyte proliferation and apoptosis in the cholestatic rat liver. Seventy male Wistar rats were randomly divided into five groups: controls, sham operated, bile duct ligated (BDL), BDL + BBS (30 μg/kg/d), BDL + NT (300 μg/kg/d). Ten days later, alpha-fetoprotein (AFP) mRNA (in situ hybridization), cytokeratin-19 and Ki67 antigen expression (immunohistochemistry) and apoptosis (TUNEL) were evaluated on liver tissue samples. Cells with morphologic features of oval cells that were cytokeratin-19(+) and AFP mRNA(+) were scored in morphometric analysis and their proliferation was recorded. In addition, the proliferation and apoptotic rates of hepatocytes and cholangiocytes were determined. Alanine aminotransferase (ALT) levels and hepatic oxidative stress (lipid peroxidation and glutathione redox state) were also estimated. The neuropeptides BBS and NT significantly reduced ALT levels and hepatic oxidative stress. Both agents exerted similar and cell type-specific effects on oval cells, hepatocytes and cholangiocytes: (a) oval cell proliferation and accumulation in the cholestatic liver was attenuated, (b) hepatocyte proliferation was increased along with a decreased rate of their apoptosis and (c) cholangiocyte proliferation was attenuated and their apoptosis was increased. These observations might be of potential value in patients with extrahepatic cholestasis.
Keywords: Bile duct ligation; Hepatic progenitor cells; Oval cells; Apoptosis; Proliferation; Oxidative stress;

γ2-Melanocyte stimulation hormone (γ2-MSH) truncation studies results in the cautionary note that γ2-MSH is not selective for the mouse MC3R over the mouse MC5R by Christine G. Joseph; Hua Yao; Joseph W. Scott; Nicholas B. Sorensen; Rebecca N. Marnane; Kathleen G. Mountjoy; Carrie Haskell-Luevano (2304-2313).
Display Omitted▶ Identification of γ2-MSH peptide amino acids important for melanocortin receptor potency. ▶ The γ2-MSH ligands have different mouse versus human species specific potencies. ▶ Intraperitoneal (i.p.) injection of NDP-MSH into mice reduces hypothalamic MC3R and MC5R mRNA gene expression levels.The melanocortin system has been implicated in a multitude of physiological pathways including obesity, satiety, energy homeostasis, sexual behavior, pigmentation, sodium regulation, hypertension, and many others. Based upon studies of the endogenous melanocortin receptor agonists at the cloned human melanocortin receptor proteins, it was concluded that the γ-MSH related agonist ligands are selective for the MC3 versus the MC4 and MC5 receptors. In attempts to understand and identify the specific amino acids of γ2-MSH important for MC3R selectivity, we have performed N- and C-terminal truncation studies and pharmacologically characterized twenty-eight ligands at the mouse MC1 and MC3-5 melanocortin receptors. The C-terminal Trp-Asp9-Arg10-Phe11 residues are important for nM potency at the mMC3R and the Arg7-Trp8 residues are important for mMC5R nM potency. We observed the unanticipated results that several of the C-terminal truncated analogs possessed nM agonist potency at the mMC3 and mMC5Rs which lead us to perform a comparative side-by-side study of the mouse and human MC5R. These data resulted in μM γ2-MSH analog potency at the hMC5R, consistent with previous reports, however at the mMC5R, nM γ2-MSH analog potency was observed. Thus, these data support the hypothesis of important species specific differences in γ-MSH related ligand potency at the rodent versus human MC5R subtype that is critical for the interpretation of in vivo rodent physiological studies. These results prompted us to examine the affects of a peripherally administered melanocortin agonist on hypothalamic gene expression levels of the MC3R, MC4R, and MC5R. The super potent non-selective NDP-MSH agonist was administered i.p. and resulted in significantly decreased levels of mMC3R and mMC5R hypothalamic mRNA versus saline control. These data provide for the first time data demonstrating peripherally administered NDP-MSH can modify hypothalamic melanocortin receptor expression levels.
Keywords: Melanotropin; Heart; Blood pressure; Obesity; Receptor brain expression; GPCR;

Effect of MTII on food intake and brain c-Fos in melanocortin-3, melanocortin-4, and double MC3 and MC4 receptor knockout mice by Neil E. Rowland; Jay W. Schaub; Kimberly L. Robertson; Amy Andreasen; Carrie Haskell-Luevano (2314-2317).
▶ Both MC3R and MC4R knockout mice show partial anorectic responses to MTII. ▶ Fos induced by MTII reduced in PVN of MC3R knockout and in AP of MC4R knockout. ▶ Mice with knockout of both receptors show neither anorexia nor induced Fos. ▶ Suggests dissociation of brain region and MC receptor subtype in feeding behavior.Mice with genomic knockout of either melanocortin type 3 receptors (MC3R−/−), type 4 receptors (MC4R−/−) or knockout of both (double knockout, DKO) were tested for their anorectic response to the mixed MC3/4R agonist, MTII, injected into the anterior cerebral ventricle. Wild type (WT) mice showed a strong anorexia and, as expected, DKO were completely unresponsive to MTII. In contrast, both MC3R−/− and MC4R−/− showed a partial anorectic response. Induction of c-Fos immunoreactivity by MTII was examined in brain regions including paraventricular hypothalamus (PVN) and area postrema (AP). Compared with WT, MC4R−/− showed no activation in AP but showed normal activation in PVN, whereas MC3R−/− showed reduced activation in PVN but not in AP. RT-PCR analysis showed that hypothalamic mRNA for MC3R in MC4R−/− and for MC4R in MC3R−/− was unaltered from WT levels. These data suggest that both receptor subtypes are involved in the behavioral action of MTII, and that the critical receptors are in different brain regions.

▶ Subtle changes in the linear peptide part drastically affect the activity. ▶ Substituting Thr for Dab at position 8 decreases direct but not sensitizing activity. ▶ Lengthening the fatty acyl tail to dodecanoyl decreases the activity.Polymyxin B and colistin are pentacationic lipopeptides that possess a cyclic heptapeptide portion, a linear tripeptide portion, and a fatty acyl tail. They are used, in spite of nephrotoxicity, to treat infections caused by extremely multiresistant Gram-negative bacteria. We have recently developed novel derivatives, that carry three cationic charges only. Some of them, including NAB739, are directly antibacterial whereas others, including NAB7061, lack the direct activity but sensitize bacteria to other antibiotics. NAB739 and NAB7061 differ from the old polymyxins in their renal handling and have reduced affinity to kidney brush border membrane. To further study the structure–activity relationships, we here synthesized eight additional derivatives and tested their antibacterial activity. NAB751 carries methylheptanoyl as the fatty acyl instead of octanoyl in NAB739 and was as active as NAB739, whereas NAB750 with dodecanoyl was less active. NAB781 and NAB782 with the linear peptide portion Ser-DSer and Ser-Ser-DSer, respectively, were less active than NAB739 that carries Thr-DSer. NAB771 with Thr at position 8 in the cyclic portion (instead of Dab in NAB7061) and Thr-Dab as the linear peptide portion (instead of Thr-Abu in NAB7061), resembled NAB7061 in its activity. However, replacement of two Dab residues in the cyclic portion with Thr greatly decreased the activity, even though the loss of the cationic charges was compensated by introducing two Dab residues in the linear portion. These findings reveal that subtle structural modifications have a major effect on the antibacterial activity and that it is possible to design numerous tricationic polymyxin derivatives that are antibacterial.

▶ Cell volume regulation. ▶ The role of the renin–angiotensin system. ▶ Influence of angiotensin (1–7) on cell volume and ionic currents. ▶ Implications for cardiac arrhythmias.The influence of angiotensin II and angiotensin (1–17) on cell volume and on the activation of ionic channels including the swelling-dependent chloride channel was reviewed. Particular emphasis was given to the influence of the balance between the ACE–angiotensin II and of the ACE2–angiotensin (1–7)–Mas receptor axis on heart cell volume regulation and on the swelling-dependent chloride current. The implications for myocardial ischemia and cardiac arrhythmias are discussed.
Keywords: Cell swelling; Angiotensin II; Angiotensin (1–7); Swelling-dependent chloride current; Cardiac arrhythmias; Myocardial ischemia;

Endogenous opiates and behavior: 2009 by Richard J. Bodnar (2325-2359).
▶ This review summarizes articles about the endogenous opiates published in 2009. ▶ Molecular-biochemical and neuroanatomical substrates of opiates. ▶ Opiates and stress, tolerance/dependence, learning/memory and ingestion. ▶ Opiates and addiction, mental illness, neurollogical disorders and neurophysiology. ▶ Opiates and locomotor visceral, cardiovascular, respiratory and immune functions.This paper is the 32nd consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2009 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular–biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (Section ), and the roles of these opioid peptides and receptors in pain and analgesia (Section ); stress and social status (Section ); tolerance and dependence (Section ); learning and memory (Section ); eating and drinking (Section ); alcohol and drugs of abuse (Section ); sexual activity and hormones, pregnancy, development and endocrinology (Section ); mental illness and mood (Section ); seizures and neurologic disorders (Section ); electrical-related activity and neurophysiology (Section ); general activity and locomotion (Section ); gastrointestinal, renal and hepatic functions (Section ); cardiovascular responses (Section ); respiration and thermoregulation (Section ); and immunological responses (Section ).
Keywords: Opioid peptides; Opioid receptors; Pain; Analgesia; Tolerance; Dependence; Learning and memory; Locomotor behavior; Immunology;