Neuropeptides - Articles in Press

Influence of tachykinin NK2 receptors on intestinal sensitivity and motility in newborn rats - Corrected Proof

2010-02-08

Abstract: The effect of tachykinin neurokinin NK2 receptors activation on intestinal propulsion and colorectal sensitivity was studied in 7–15days old newborn rats.In a first set of experiments investigating the intestinal transit, the selective NK2 receptor agonist, [βAla8]NKA-(4-10) was used. It produced an increase of the small intestinal transit measured by charcoal test of 54%, that was inhibited in a dose-dependent manner by nepadutant ([N4-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-l-asparaginyl-l-aspartyl-l-tryptophyl-l-phenylalanyl-l-2,3-diaminopropionyl-l-leucyl]-C-4.2-N-3.5-lactam-C-1.6-N-2.1-lactam), a known selective NK2 receptor antagonist, orally administered 2–48h before the challenge with the NK2 receptor agonist. Nepadutant did not affect the basal intestinal propulsion and showed a good oral bioavailability and long duration of action.In another set of experiments investigating visceral sensitivity, a fixed distension volume of a balloon inserted intrarectally in 14–15days old newborns rats produced abdominal contractions (AC) that were increased after colonic application of acetic acid (50μl, 0.5%). In this latter condition nepadutant, at 0.5 and 2.5mg/kg p.o., significantly reduced the resulting AC. In control rats, untreated with acetic acid, nepadutant did not affect AC evoked by colorectal distension.These findings show for the first time two models to assess intestinal motility and visceral sensitivity in newborn rats and indicate nepadutant as a valuable tool to assess the role of NK2 receptors in the intestinal propulsive and nociceptive activity in infants.

Wake promoting effects of cocaine and amphetamine-regulated transcript (CART) - Corrected Proof

Glenda L. Keating, Michael J. Kuhar, Donald L. Bliwise, David B. Rye — 2010-02-01

Abstract: Cocaine- and amphetamine-regulated transcript (CART) peptides modulate anxiety, food intake, endocrine function, and mesolimbic dopamine related reward and reinforcement. Each of these disparate behaviors takes place during the state of wakefulness. Here, we identify a potential wake promoting role of CART by characterizing its effects upon sleep/wake architecture in rats. Dose-dependent increases in wake were documented following intracerebroventricular CART 55–102 administered at the beginning of the rat’s major sleep period. Sustained wake was observed for up to 4h following delivery of 2.0μg of CART peptide. The wake promoting effect was specific to active CART 55–102 because no effect on sleep/wake was observed with the inactive form of the peptide. Increased wake was followed by robust rebound in NREM and REM sleep that extended well into the subsequent lights-off, or typical wake period, of the rat. These findings point to a potential novel role for CART in regulating wakefulness.

Central litorin injection is associated with primary anorexigenic effects that coincide with activation of the magnocellular division of the paraventricular nucleus - Corrected Proof

Mark A. Cline, Stuart A. Cofield, Tetsuya Tachibana — 2010-02-01

Abstract: The central mechanism that mediates litorin-induced satiety is poorly understood, and has not been studied in a non-mammalian species. Therefore, the aim of this study was to determine if litorin-induced satiety in an alternative vertebrate model, the chick, and to elucidate some of the central mechanisms that are associated with this response. In Experiment 1, chicks responded to intracerebroventricular (ICV) injection of litorin with reduced food intake at all doses tested (0.1, 1.0, and 10nmol), while concurrently, an anti-dipsogenic effect was observed in the two higher doses tested. Whole blood glucose concentrations were not affected. In Experiment 2, chicks that were food-withheld did not reduce their water intake after ICV litorin injection. To determine if litorin affected behaviors unrelated to ingestion, a comprehensive behavior analysis was conducted as Experiment 3. Of the behaviors observed, only the number of feeding pecks was reduced. Other behaviors such as movement, defecation, escape, posture, or deep rest were not affected. Lastly, in Experiment 4, litorin-treated chicks had an increased number of c-Fos immunoreactive cells in the magnocellular division of the paraventricular nucleus. The arcuate nucleus, dorsomedial nucleus, lateral hypothalamus, parvicellular division of the paraventricular nucleus, suprachiasmatic nucleus, periventricular nucleus and the ventromedial hypothalamus were not affected. Therefore, we conclude that ICV litorin causes anorexigenic effects in chicks associated with changes in hypothalamic chemistry that appear to be behavior specific.

Y4 receptors and pancreatic polypeptide regulate food intake via hypothalamic orexin and brain-derived neurotropic factor dependent pathways - Corrected Proof

2010-02-01

Abstract: Gut-derived peptides are known to regulate food intake by activating specific receptors in the brain, but the target nuclei and neurons influenced are largely unknown. Here we show that peripherally administered pancreatic polypeptide (PP) stimulates neurons in key nuclei of the hypothalamus critical for appetite and satiety regulation. In the lateral hypothalamic area (LHA), also known as the feeding center, neurons expressing the orexigenic neuropeptide orexin co-localize with the early neuronal activation marker c-Fos upon i.p. injection of PP into mice. In the ventromedial hypothalamus (VMH), also known as the satiety center, neurons activated by PP, as indicated by induction of c-Fos immunoreactivity, express the anorexigenic brain-derived neurotrophic factor (BDNF). Activation of neurons in the LHA and VMH in response to PP occurs via a Y4 receptor-dependent process as it is not seen in Y4 receptor knockout mice. We further demonstrate that in response to i.p. PP, orexin mRNA expression in the LHA is down-regulated, with Y4 receptors being critical for this effect as it is not seen in Y4 receptor knockout mice, whereas BDNF mRNA expression is up-regulated in the VMH in response to i.p. PP in the fasted, but not in the non-fasted state. Taken together these data suggest that PP can regulate food intake by suppressing orexigenic pathways by down-regulation of orexin and simultaneously increasing anorexigenic pathways by up-regulating BDNF.

Acoustic startle response and sensorimotor gating in a genetic mouse model for the Y1 receptor - Corrected Proof

T. Karl, R. Chesworth, L. Duffy, H. Herzog — 2010-01-25

Abstract: Recent research has highlighted a potential role for neuropeptide Y (NPY) and its Y1 receptor in the development of schizophrenia. Genetic as well as molecular biological studies have demonstrated reduced levels of NPY in schizophrenia patients. Importantly, Y1 receptors may mediate some of the potential effects of NPY on schizophrenia, as decreased Y1 receptor expression has been found in the lymphocytes of schizophrenia patients. To clarify NPY’s role in schizophrenia, we investigated a genetic animal model for Y1 deficiency in regard to (i) acoustic startle response (ASR), (ii) habituation to ASR and (iii) sensorimotor gating [i.e. prepulse inhibition (PPI)] using two different PPI protocols. Mutant and wild type-like mice were screened for baseline behaviours and after pharmacological challenge with the psychotropic drugs dexamphetamine (DEX) and MK-801. Y1 knockout mice (Y1−/−) showed a moderate reduction of the ASR and an impaired ASR habituation at baseline and after DEX treatment. The baseline PPI performance of Y1 mutant mice was unaltered their response to DEX and MK-801 challenge was moderately different compared to control mice, which was dependent on the PPI protocol used. MK-801 challenge had a protocol-dependent differential effect in Y1−/− mice and DEX a more pronounced impact at the highest prepulse intensities. In conclusion, it appears that the Y1 receptor influences the acoustic startle response and its habituation but does not play a major role in sensorimotor gating. Further explorations into the effects of Y1 deficiency seem valid.

Pathogenic involvement of neuropeptides in anxiety and depression - Corrected Proof

Brett Alldredge — 2010-01-25

Abstract: Anxiety and depression are highly prevalent disorders of mood posing significant challenges to individuals and society. Current evidence indicates no single neurobiological determinant underpins these conditions and an integrated approach in both research and treatment is expedient. Basic, behavioral, and clinical science indicates various stress-responsive neuropeptides in the neuroendocrine, autonomic, and behavioral pathophysiology of stress-related disorders including anxiety and depression. This review draws on recent research to capture the consensus and implications of neuropeptide research concerning the pathogenesis of anxiety and depression.

Correlations between angiotensinase activity asymmetries in the brain and paw preference in rats - Corrected Proof

2010-01-25

Abstract: The function of angiotensin peptides is dependent upon the action of several aminopeptidases (APs) termed angiotensinases. Soluble (SOL) and membrane (MEM)-bound alanyl-AP (AlaAP) and cystinyl-AP (CysAP) are involved in the metabolism of angiotensins and related to the modulation of behavior and memory. To study the interactions between angiotensinase activity in the hippocampus and behavioral lateralization, Wistar rats were selected on the basis of their performance in the paw preference test (left-handed, ambidextrous and right-handed) and the activities of SOL-AlaAP/CysAP and MEM-AlaAP/CysAP were measured in the both hippocampuses. We observed that: (1) the left hippocampus had higher activities of SOL-AlaAP/CysAP and MEM-AlaAP/CysAP than the right hippocampus; (2) rats showed significant differences in the activities of SOL-AlaAP/CysAP and MEM-AlaAP/CysAP in the hippocampus depending on the behavioral lateralization detecting by paw preference; (3) in three groups of rats, hemispheric dominance – %R/T [%R/T=right hemisphere/(right hemisphere+left hemisphere)×100] activities of MEM-AlaAP, SOL-CysAP and MEM-CysAP was significantly different whereby %RT was lower in left-handed, higher in ambidextrous and intermediate in right-handed rats; (4) individual %R/T activities of SOL-CysAP and MEM-CysAP in the hippocampus were positively correlated with paw preference scores. Finally, we used the passive avoidance behavior test to demonstrate the differences of long-term memory among the three groups. These results suggested that the asymmetric activity of angiotensinase in the rat hippocampus may be associated with both the direction and the intensity of behavioral lateralization as expressed by paw preference.

Antidiabetic efficacy of bradykinin antagonist R-954 on glucose tolerance test in diabetic type 1 mice - Corrected Proof

2010-01-22

Abstract: Insulin-dependent diabetes mellitus (type 1 diabetes) is an inflammatory autoimmune disease associated with many complications including nephropathy, retinopathy, neuropathy and hyperalgesia. Experimental evidence has shown that the bradykinin B1 receptor (BKB1-R) is involved in the development of type 1 diabetes and found to be upregulated alongside the disease. In the present study the effects of the selective BKB1-R antagonist the R-954 (Ac-Orn-[Oic2, α-MePhe5, D-β Nal7, Ile8 ]des-Arg9-BK and the BKB1-R agonist des Arg9-BK (DBK) were studied on diabetic hyperglycemia. Diabetic type 1 was induced in C57 BL/KsJ mdb male mice by five consecutives doses of STZ (45mg/kg i.p.). A glucose tolerance test (GTT) was performed by an intraperitoneal administration of glucose, 8, 12 and 18days after the diabetes induction. The induction of type 1 diabetes provoked a significant hyperglycemia levels in diabetic mice at 12 and 18days after STZ. The administration of R-954 (400μg/kg i.p.) at 12 and 18days after STZ returned the glycemia levels of this animals to normal values. In addition the administration of DKB (300μg/kg i.p.) significantly potentiated the diabetes-induced hyperglycemia; this effect that was totally reversed by R-954. These results provide further evidence for the implication of BKB1-R in the type 1 diabetes mellitus (insulitis).

Compensatory mechanisms to maintain blood pressure in paraplegic rats: Implication of central tachykinin NK-1 and NK-3 receptors? - Corrected Proof

Frank Cloutier, Jenny L. Lauschke, Pascal Carrive — 2010-01-22

Abstract: People with high level spinal cord injury (SCI) suffer from both hypotension and spontaneous hypertension due to loss of supraspinal control of spinal sympathetic outflow. Few reports have addressed whether any changes occur in central regulation of blood pressure (BP) and heart rat (HR) at the supraspinal level. Central tachykinin NK-1 and NK-3 receptors are located in many cardiovascular areas in the brain and are known to modulate BP and HR. This study examined the intracerebroventricular (i.c.v.) effects of the selective NK-1 receptor agonist [Sar9, Met(O2)11]SP (65pmol, n=6) and NK-3 receptor agonist senktide (650pmol, n=6) on mean arterial pressure (MAP) and HR before and after complete spinal cord transection at thoracic level 4 (T4). [Sar9, Met(O2)11]SP evoked increases in MAP and HR which were still present 4days after the T4 SCI. Further analysis using the β1-adrenoceptor antagonist atenolol (10mgkg−1) revealed an increased contribution of HR in the MAP increase after SCI. For senktide, 2 and 5weeks after T4 SCI, the rise in MAP induced by senktide was significantly increased in magnitude and was similar to a normal response at 8weeks. These effects were accompanied by a bradycardia, which was still present and amplified at 8weeks. Our results reveal a transient potentiation of the senktide-mediated MAP effect and a greater contribution of the HR in MAP increase by [Sar9, Met(O2)11]SP in T4 transected rats. Although the significance of these changes remains to be established. This suggest a reorganization of supraspinal mechanisms regulating BP and HR after a high level SCI. Central NK-1 and NK-3 receptors might therefore contribute to the maintenance of MAP following high thoracic SCI.

Selective tumor blood–brain barrier opening with the kinin B2 receptor agonist [Phe8ψ(CH2NH)Arg9]-BK in a F98 glioma rat model: An MRI study - Corrected Proof

2010-01-18

Abstract: Treatment of malignant glioma with chemotherapy is limited mostly because of delivery impediment related to the blood–brain barrier (BBB). One approach for transporting drugs across the BBB involves the activation of bradykinin-B2 receptors (BK-B2R). Our objective was to pharmacologically characterize the BBB permeability induced by the synthetic biostable BK-B2R analogue [Phe8ψ(CH2NH)Arg9]-BK (R523) in F98 glioma-implanted Fischer rats. On day 10 post-inoculation, we detected the presence of B2R in the tumor cells and the peritumoral microvasculature (RT-PCR and immunohistochemistry). We assessed BBB permeability before and after the intracarotid (i.c.) infusion of R523 (0.1ml/min for 5min; 2.5, 10, and 50nmol/kg/min) using non-invasive dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) with the different sized-contrast agents Gd-DTPA (0.5kDa) and Gadomer (17kDa) (0.25mmol/kg via the caudal vein). T1-weighted images were analyzed for the presence or absence of contrast enhancement within and surrounding the tumor area and mathematically processed to yield a contrast agent distribution volume (CADV), which was used as an indicator of vascular permeability. Our results showed that the agonist R523 increased, in a dose-dependent manner, the CADV indexes of Gd-DTPA and Gadomer, with a maximum 2-fold increase in brain uptake of both CA. The increase in CADV induced by R523 (10nmol/kg/min) was prevented by the B2R antagonist HOE140 (20nmol/kg/min, i.c.) and the nitric oxide synthase inhibitor L-NA (5mg/kg, i.v.) but not by the B1R antagonist R892 (20nmol/kg/min, i.c.) or the cyclooxygenase inhibitor Meclofenamate (5mg/kg, i.v.). The BBB permeabilizing effect of R523 (10nmol/kg/min) lasted for <1h and was accompanied by a dose-related fall in arterial blood pressure. We concluded that R523 allows the extravasation of hydrophilic macromolecular agents (⩽17kDa) into tumor tissues by inducing selective tumor BBB permeability via B2R- and NO-dependent mechanisms.

Contribution of the central dopaminergic system in the anti-hypertensive effect of kinin B1 receptor antagonists in two rat models of hypertension - Corrected Proof

H. De Brito Gariepy, P. Carayon, B. Ferrari, R. Couture — 2010-01-14

Abstract: Kinins are neuroactive peptides that could play a role in central autonomic control of blood pressure. Whereas kinin B1R binding sites were increased in specific brain areas of spontaneously hypertensive rats (SHR) and Angiotensin II (AngII)-hypertensive rats, the contribution of kinin B1R in hypertension remains controversial. The aims of the study were to determine: (a) the effects on mean arterial blood pressure (MAP) of centrally and peripherally administered B1R antagonists in SHR (16weeks) and AngII-hypertensive rats (200ng/kg/min×2weeks, s.c.); (b) the contribution of central dopamine in the effects of SSR240612. The rationale is based on the overactivity of the dopaminergic system in hypertension. In both models, SSR240612 (1, 5 and 10mg/kg, gavage) reduced dose-dependently MAP (⩾−75mm Hg at least up to 6–8h) and this therapeutic effect was resolved after 24h. At the dose of 5mg/kg, SSR240612-induced anti-hypertension was prevented by two dopamine receptor blockers, namely raclopride (0.16mg/kg, i.v.) and haloperidol (10mg/kg, s.c.). I.c.v. SSR240612 (1μg) decreased rapidly MAP in both models (1–6h) via a raclopride sensitive mechanism. In comparison, peripherally acting B1R antagonists (R-715 and R-954, 2mg/kg, s.c.) caused shorter and very modest decreases of MAP (from −20 to −30mm Hg). Centrally or peripherally administered B1R antagonists had no effect on MAP in control Wistar–Kyoto rats. Data provide the first pharmacological evidence that the up-regulated brain kinin B1R contributes through a central dopaminergic mechanism (DA-D2R) to the maintenance of arterial hypertension in genetic and experimental animal models of hypertension.

Plasma Kallikrein and Angiotensin I-converting enzyme N- and C-terminal domain activities are modulated by the insertion/deletion polymorphism - Corrected Proof

2010-01-11

Abstract: Angiotensin I-converting enzyme (ACE) is recognized as one of the main effector molecules involved in blood pressure regulation. In the last few years some polymorphisms of ACE such as the insertion/deletion (I/D) polymorphism have been described, but their physiologic relevance is poorly understood. In addition, few studies investigated if the specific activity of ACE domain is related to the I/D polymorphism and if it can affect other systems. The aim of this study was to establish a biochemical and functional characterization of the I/D polymorphism and correlate this with the corresponding ACE activity. For this purpose, 119 male brazilian army recruits were genotyped and their ACE plasma activities evaluated from the C- and N-terminal catalytic domains using fluorescence resonance energy transfer (FRET) peptides, specific for the C-domain (Abz-LFK(Dnp)OH), N-domain (Abz-SDK(Dnp)P-OH) and both C- and N-domains (Abz-FRK(Dnp)P-OH). Plasma kallikrein activity was measured using Z-Phe-Arg-AMC as substrate and inhibited by selective plasma kallikrein inhibitor (PKSI). Some physiological parameters previously described related to the I/D polymorphism such as handgrip strength, blood pressure, heart rate and BMI were also evaluated. The genotype distribution was II n=27, ID n=64 and DD n=28. Total plasma ACE activity of both domains in II individuals was significantly lower in comparison to ID and DD. This pattern was also observed for C- and N-domain activities. Difference between ID and DD subjects was observed only with the N-domain specific substrate. Blood pressure, heart rate, handgrip strength and BMI were similar among the genotypes. This polymorphism also affected the plasma kallikrein activity and DD group presents high activity level. Thus, our data demonstrate that the I/D ACE polymorphism affects differently both ACE domains without effects on handgrip strength. Moreover, this polymorphism influences the kallikrein–kinin system of normotensive individuals.

Role of kinin B1 and B2 receptors in memory consolidation during the aging process of mice - Corrected Proof

2010-01-11

Abstract: Under physiological conditions, elderly people present memory deficit associated with neuronal loss. This pattern is also associated with Alzheimer’s disease but, in this case, in a dramatically intensified level. Kinin receptors have been involved in neurodegeneration and increase of amyloid-β concentration, associated with Alzheimer’s disease (AD). Considering these findings, this work evaluated the role of kinin receptors in memory consolidation during the aging process. Male C57Bl/6 (wt), knock-out B1 (koB1) or B2 (koB2) mice (3, 6, 12 and 18-month-old – mo; n=10 per group) were submitted to an acquisition session, reinforcement to learning (24h later: test 1) and final test (7days later: test 2), in an active avoidance apparatus, to evaluate memory. Conditioned avoidance responses (CAR, % of 50 trials) were registered. In acquisition sessions, similar CAR were obtained among age matched animals from all strains. However, a significant decrease in CAR was observed throughout the aging process (3mo: 8.8±2.3%; 6mo: 4.1±0.6%; 12mo: 2.2±0.6%, 18mo: 3.6±0.6%, P<0.01), indicating a reduction in the learning process. In test 1, as expected, memory retention increased significantly (P<0.05) in all 3- and 6-month-old animals as well as in 12-month-old-wt and 12-month-old-koB1 (P<0.01), compared to the training session. However, 12-month-old-koB2 and all 18-month-old animals did not show an increase in memory retention. In test 2, 3- and 6-month-old wt and koB1 mice of all ages showed a significant improvement in memory (P<0.05) compared to test 1. However, 12-month-old wt and koB2 mice of all ages showed no difference in memory retention. We suggest that, during the aging process, the B1 receptor could be involved in neurodegeneration and memory loss. Nevertheless, the B2 receptor is apparently acting as a neuroprotective factor.

Akt pathway activation and increased neuropeptide Y mRNA expression in the rat hippocampus: Implications for seizure blockade - Corrected Proof

2010-01-11

Abstract: The aim of this study was to analyze the expression of survival-related molecules such Akt and integrin-linked kinase (ILK) to evaluate Akt pathway activation in epileptogenesis process. Furthermore, was also investigated the mRNA expression of neuropeptide Y, a considered antiepileptic neuropeptide, in the pilocarpine-induced epilepsy. Male Wistar rats were submitted to the pilocarpine model of epilepsy. Hippocampi were removed 6h (acute phase), 12h (late acute), 5d (silent) and 60d (chronic) after status epilepticus (SE) onset, and from animals that received pilocarpine but did not develop SE (partial group). Hippocampi collected were used to specify mRNA expression using Real-Time PCR. Immunohistochemistry assay was employed to place ILK distribution in the hippocampus and Western blot technique was used to determine Akt activation level. A decrease in ILK mRNA content was found during acute (0.39±0.03) and chronic (0.48±0.06) periods when compared to control group (0.87±0.10). Protein levels of ILK were also diminished during both periods. Partial group showed increased ILK mRNA expression (0.80±0.06) when compared with animals in the acute stage. Silent group had ILK mRNA and immunoreactivity similar to control group. Western blot assay showed an augmentation in Akt activation in silent period (0.52±0.03) in comparison with control group (0.44±0.01). Neuropeptide Y mRNA expression increased in the partial group (1.67±0.22) and in the silent phase (1.45±0.29) when compared to control group (0.36±0.12). Results suggest that neuropeptide Y (as anticonvulsant) might act in protective mechanisms occurred during epileptic phenomena. Together with ILK expression and Akt activation, these molecules could be involved in hippocampal neuroprotection in epilepsy.

Effect of kinin B2 receptor ablation on skeletal muscle development and myostatin gene expression - Corrected Proof

2010-01-04

Abstract: Bradykinin (BK) is an active peptide that binds to the kinin B2 receptor and induces biological events during the development and adult life. In this study we aimed to investigate the effect of kinin B2 receptor ablation in the postnatal skeletal muscle development and body composition in adult life. For studies of skeletal muscle development, control (C57Bl6 – WT) and B2 receptor knockout mice () were sacrificed at 15, 30 and 90days after birth, the gastrocnemius skeletal muscle was weighed and myostatin gene expression evaluated by real time PCR. For energy balance determination, data from control and at 90 and 120days were collected by calorimetric method. Body composition at 120days was determined by chloroform–methanol (total body fat) and Lowry-modified method (total body protein). The results show that have significantly increased total body weight at 15, 30 and 90days of life, when compared to WT. The weight of the gastrocnemius skeletal muscle was also significantly increased at 30 and 90days of life. Body composition analyses revealed that mice exhibit more total corporal protein and less total corporal fat. Energy balance revealed that have increased metabolizable energy intake and energy expenditure when compared to control mice, resulting in a lower energy gain. Interestingly, myostatin mRNA expression was significantly decreased in 15 and 30days old mice and after icatibant treatment of WT adult mice for 5days. In conclusion, together our results show that kinin B2 receptor deletion increases lean mass, reduces fat mass and improves metabolism efficiency in mice. The mechanism involved in this phenotype could be related to the reduction of myostatin gene expression during postnatal life.

Differential regulation of inducible and endothelial nitric oxide synthase by kinin B1 and B2 receptors - Corrected Proof

F. Kuhr, J. Lowry, Y. Zhang, V. Brovkovych, R.A. Skidgel — 2010-01-04

Abstract: Kinins are vasoactive peptides that play important roles in cardiovascular homeostasis, pain and inflammation. After release from their precursor kininogens, kinins or their C-terminal des-Arg metabolites activate two distinct G protein-coupled receptors (GPCR), called B2 (B2R) or B1 (B1R). The B2R is expressed constitutively with a wide tissue distribution. In contrast, the B1R is not expressed under normal conditions but is upregulated by tissue insult or inflammatory mediators. The B2R is considered to mediate many of the acute effects of kinins while the B1R is more responsible for chronic responses in inflammation. Both receptors can couple to Gαi and Gαq families of G proteins to release mediators such as nitric oxide (NO), arachidonic acid, prostaglandins, leukotrienes and endothelium-derived hyperpolarizing factor and can induce the release of other inflammatory agents. The focus of this review is on the different transduction events that take place upon B2R and B1R activation in human endothelial cells that leads to generation of NO via activation of different NOS isoforms. Importantly, B2R-mediated eNOS activation leads to a transient (∼5min) output of NO in control endothelial cells whereas in cytokine-treated endothelial cells, B1R activation leads to very high and prolonged (∼90min) NO production that is mediated by a novel signal transduction pathway leading to post-translational activation of iNOS.

Distribution and expression of B2-kinin receptor on human leukocyte subsets in young adults and elderly using flow cytometry - Corrected Proof

2010-01-04

Abstract: The kallikrein-kinin system has been investigated in many experimental models. Dysregulations of the KKS are likely to be involved in pathologies such as inflammation, cancer and cardiovascular diseases. Previous works on the human KKS mostly rely on gene polymorphism and mRNA expression. In order to assess the KKS in human at the protein level, we have developed an approach based on flow cytometric analysis of leukocytes. Whole blood samples were collected and erythrocytes were lysed. Permeabilised leukocytes were incubated with anti-B2R (IgG2b), anti-IgG2b-PE, anti-CD3-PerCP (lymphocytes) and anti-CD14-APC (monocytes) antibodies. FACScalibur analyzed fluorescence intensities. Results were expressed as per cent of B2R-positive cells in each leukocyte subset and as B2R fluorescence intensity per positive cell. Detection of the B2R protein by this methodology was validated by (i) correlation with Western blotting using two different B2R antibodies, (ii) BK-induced Erk activation, (iii) B2R mRNA expression. The methodology was then applied to evaluate variations of B2R expression in a population including young healthy, elderly healthy, and elderly treated hypertensive men and women. In the young healthy subjects, B2R distribution was: monocytes>polymorphonuclear neutrophils (PMN)>lymphocytes and no difference with gender was observed. Moreover, no difference was observed on PMN B2R expression. B2R expression remained unchanged in the elderly healthy or hypertensive men. By contrast, monocytes and lymphocytes B2R expressions were decreased in the elderly healthy women. Finally, FACS analysis of B2R expression on leukocytes subsets provides single cell quantification of B2R expression allowing comparison of cellular sub-populations. This approach provides a new efficient tool to investigate B2R profiling of immune system in pathological states.

Modulation of feeding and locomotion through mu and delta opioid receptor signaling in the nucleus accumbens - Corrected Proof

Yoshihiro Katsuura, Sharif A. Taha — 2009-12-31

Abstract: Opioid signaling has been strongly implicated in driving palatable food consumption. The nucleus accumbens (NAcc) is one important site of this effect; hyperphagia elicited by administration of exogenous mu opioid receptor (MOR) ligands in this brain region has been well documented. However, the role that endogenous opioid ligands in the NAcc play in controlling food intake remains poorly understood. Enkephalins, which signal through both the MOR and delta opioid receptor (DOR), are highly expressed within a subset of NAcc neurons, and have been shown to be sensitive to manipulations of diet and motivation. To investigate a potential role for these signaling molecules in regulating palatability-driven consumption, we measured high fat chow intake in rats following a series of pharmacological manipulations of NAcc opioid signaling. NAcc infusion of the MOR agonist [d-Ala2, N-MePHe4, Gly-ol]-enkephalin (DAMGO) robustly increased palatable food intake, as has previously been demonstrated. In contrast, neither infusion of Met-enkephalin, its synthetic analogue [d-Ala2] Met-enkephalin (DALA) nor the DOR-specific ligand [d-Pen2, Pen5]-enkephalin (DPDPE) had significant effects on food intake. However, when administered in combination with DAMGO, DPDPE significantly suppressed the magnitude of DAMGO-evoked feeding. Further analysis of DPDPE effects revealed that the drug strongly increased locomotor activity. Suppressive effects on feeding, then, may have occurred through competition between feeding and locomotion for behavioral expression.

Kinins in cardiac inflammation and regeneration: Insights from ischemic and diabetic cardiomyopathy - Corrected Proof

Konstantinos Savvatis, Dirk Westermann, Heinz-Peter Schultheiss, Carsten Tschöpe — 2009-12-25

Abstract: The kallikrein–kinin system (KKS) is a system of vasoactive peptides, the kinins, involved in different aspects of remodeling, inflammation and angiogenesis. Kinins mediate their actions through two receptors, B1R and B2R. It is increasingly recognized that the KKS is involved in the inflammatory processes of the heart. Evidence shows that the B2R is beneficial in myocardial diseases, protecting from inflammation, fibrosis and apoptosis, while B1R shows a proinflammatory character contributing to the disease progression by increasing the production of cytokines and stimulating the migration of immune cells. Furthermore, novel important actions of the KKS and its receptors contribute to neovascularization and recruitment of endothelial progenitor cells in ischemic areas and endothelial dysfunction. The kinin receptors could therefore constitute potential therapeutic targets in the treatment of myocardial ischemia and diabetic cardiomyopathy.

VPAC1 receptor binding site: Contribution of photoaffinity labeling approach - Corrected Proof

A. Couvineau, E. Ceraudo, Y.-V. Tan, M. Laburthe — 2009-12-25

Abstract: The vasoactive intestinal peptide (VIP) is a prominent 28 aminoacid neuropeptide with wide distribution in both central and peripheral nervous systems, where it plays important regulatory role in many physiological processes. VIP has a large spectrum of biological functions including exocrine secretions, hormone release, foetal development, immune response and also exerts beneficial effect in neuro-degenerative and inflammatory diseases. Few years ago, it has been shown that VIP can be a promising anti-inflammatory agent. VIP mechanisms of action implicate two sub-types of receptors (VPAC1 and VPAC2) which are members of class B receptors belonging to the super-family of G protein-coupled receptor (GPCR). Because, VPAC1 receptor plays an important role in the modulation of the ant-inflammatory response and represent an archetype of class B GPCR, we have extensively studied the structure–function relationship of this receptor, which allowed us to define the molecular basis of that receptor in term of affinity, specificity, desensitization and coupling to adenylyl cyclase. Those studies showed the crucial role of the N-terminal ectodomain (N-ted) of VPAC1 receptor in VIP binding. Using different techniques including photoaffinity labeling, NMR, molecular modeling and molecular dynamic simulation, it has been possible to define how VIP interacts with its receptor. We have shown that most of the VIP molecule, 1–28 (α-helix) sequence, tightly binds the N-ted part of the receptor which is himself structured as a «Sushi» domain. In contrast, the N-terminal part of the specific antagonist PG97–269 is in physical contact with the N-ted but in different region. These studies define the molecular mechanism implicated in the activation of class B VPAC1 receptor and should allow the development of new VIP pharmacology using rational synthesis of agonist molecules.

Neuronal circuits involving ghrelin in the hypothalamus-mediated regulation of feeding - Corrected Proof

Haruaki Kageyama, Fumiko Takenoya, Kanako Shiba, Seiji Shioda — 2009-12-25

Abstract: Ghrelin, an n-octanoylated 28-amino acid brain–gut peptide, was first isolated from extracts of porcine stomach. Ghrelin is an endogenous ligand for the growth hormone secretagogue type 1a receptor (GHS-R1a), the functionally active form of GHS-R, and stimulates feeding and growth hormone secretion. Ghrelin is mainly produced in the A/X-like cells of the oxyntic glands of the stomach and is the main orexigenic circulating hormone that acts on the hypothalamus to affect feeding behavior and energy metabolism.Ghrelin-containing neuronal cell bodies are localized in the hypothalamic arcuate nucleus, a center that integrates signals for energy homeostasis. Ghrelin-containing nerve fibers are widely distributed in the brain. Accumulated evidence shows that hypothalamic neuropeptides such as neuropeptide Y (NPY), orexin and proopiomelanocortin (POMC) are involved in the regulation of feeding behavior and energy homeostasis via neuronal circuits in the hypothalamus. Ghrelin also forms part of the feeding-regulating neuronal circuitry in conjunction with other feeding-regulating peptide-containing neurons within the hypothalamus. In view of the fact that one decade has now passed since ghrelin was first discovered, we review advances that have been made in ghrelin research during that time and how this has impacted on our knowledge of feeding regulation in the hypothalamus. We also summarize our current understanding of the neuronal interactions between ghrelin and the different kinds of feeding-regulating peptide-containing neurons in the hypothalamus based on evidence at the ultrastructural level.

Structural analysis of three peptides related to the transmambranic helix VI of AT1 receptor - Corrected Proof

2009-12-14

Abstract: Introduction: Angiotensin II (AII) is the main active product of the renin angiotensin system. Better known effects of AII are via AT1 receptor (AT1R). Expression of AT1R mutants (L265D and L262D) in CHO cells increased cAMP formation when compared to CHO cells expressing the wild type (WT) AT1R. Morphological transformation of CHO cells transfected with mutants correlated with their increased cAMP formation. DNA synthesis was inhibited in these cells too, indicating that cAMP promotes inhibitory effects on transfected CHO cells growth and causes their morphological change from a tumorigenic phenotype to a non-tumorigenic one.Objectives: To assess the importance of leucine 262 and 265 in determining AT1R structure by means of a comparative structural analysis of two mutant peptides and of a wild-type fragment.Methodology: Three peptides had their conformation compared by circular dichroism (CD): L262D259–272, L265D259–272 (mutants) and WT260–277.Results: Secondary structures were: β-turn for WT and L262D and random coil for L265D.Conclusions: Strong correlation was found in the results of biochemical, cellular and structural approaches used to compare WT AT1R to mutant types. Random coil structure of the L265D mutant may be a key point to explain those changes observed in biochemical (binding and signal transduction) and proliferation assays (). β-Turn formation is an important step during early protein folding and this secondary simple structure is present in L262D and WT, but not in L265D. Therefore, leucine 265 seems to play a crucial role in determining an entirely functional AT1R.

Bradykinin B1 receptor antagonist R954 inhibits eosinophil activation/proliferation/migration and increases TGF-β and VEGF in a murine model of asthma - Corrected Proof

Luciana M.C. Vasquez-Pinto, François Nantel, Pierre Sirois, Sonia Jancar — 2009-12-04

Abstract: In the present study the effects of bradykinin receptor antagonists were investigated in a murine model of asthma using BALB/c mice immunized with ovalbumin/alum and challenged twice with aerosolized ovalbumin. Twenty four hours later eosinophil proliferation in the bone marrow, activation (lipid bodies formation), migration to lung parenchyma and airways and the contents of the pro-angiogenic and pro-fibrotic cytokines TGF-β and VEGF were determined. The antagonists of the constitutive B2 (HOE 140) and inducible B1 (R954) receptors were administered intraperitoneally 30min before each challenge. In sensitized mice, the antigen challenge induced eosinophil proliferation in the bone marrow, their migration into the lungs and increased the number of lipid bodies in these cells. These events were reduced by treatment of the mice with the B1 receptor antagonist. The B2 antagonist increased the number of eosinophils and lipid bodies in the airways without affecting eosinophil counts in the other compartments. After challenge the airway levels of VEGF and TGF-β significantly increased and the B1 receptor antagonist caused a further increase. By immunohistochemistry techniques TGF-β was found to be expressed in the muscular layer of small blood vessels and VEGF in bronchial epithelial cells. The B1 receptors were expressed in the endothelial cells. These results showed that in a murine model of asthma the B1 receptor antagonist has an inhibitory effect on eosinophils in selected compartments and increases the production of cytokines involved in tissue repair. It remains to be determined whether this effects of the B1 antagonist would modify the progression of the allergic inflammation towards resolution or rather towards fibrosis.

Distribution of neuropeptide W in the rat brain - Corrected Proof

2009-11-30

Abstract: Neuropeptide W (NPW), which was recently isolated from the porcine hypothalamus, has been identified as the endogenous ligand of the orphan G protein-coupled receptors GPR7 (NPBWR1) and GPR8 (NPBWR2). Infusion of NPW increases food intake in the light phase, whereas in the dark phase, it has the opposite effect. In this study, we used RT-PCR analysis to examine the gene expression of NPW mRNA in the rat brain, and performed a detailed analysis of the distribution of NPW-positive neurons by use of immunohistochemistry at both the light and electron microscopic levels. NPW mRNA expression was demonstrated in the hypothalamic paraventricular nucleus (PVN), arcuate nucleus (ARC), ventromedial nucleus (VMH) and lateral hypothalamus (LH). At the light microscopic level, NPW-like immunoreactive (NPW-LI) cell bodies were found in the preoptic area (POA), PVN, ARC, VMH, LH, PMD (dorsal premammillary nucleus), periaqueductal gray (PAG), lateral parabrachial nucleus (LPB), and prepositus nucleus (Pr). NPW-LI axon terminals were shown in the POA, bed nucleus of the stria terminalis (BST), amygdala, PVN, ARC, VMH, LH, and PAG, LPB. In addition, at the electron microscopic level, NPW-LI cell bodies and dendritic processes were often seen to receive inputs from other unknown neurons in the ARC, PVN, VMH and amygdala. Our observations indicate that NPW-LI neurons widely distributed in the rat brain region. These finding suggest that NPW may have important roles in feeding behavior, energy homeostasis, emotional response and regulation of saliva secretion.

Participation of kinin receptors on memory impairment after chronic infusion of human amyloid-β 1-40 peptide in mice - Corrected Proof

2009-11-19

Abstract: Chronic infusion of human amyloid-β 1-40 (Aβ) in the lateral ventricle (LV) of rats is associated with memory impairment and increase of kinin receptors in cortical and hippocampal areas. Deletion of kinin B1 or B2 receptors abolished memory impairment caused by an acute single injection of Aβ in the LV. As brain tissue and kinin receptors could unlikely react to acute or chronic administration of a similar quantity of Aβ, we evaluated the participation of B1 or B2 receptors in memory impairment after chronic infusion of Aβ. Male C57Bl/6J (wt), knock-out B1 (koB1) or B2 (koB2) mice (12weeks of age) previously trained in a two-way shuttle-box and achieving conditioned avoidance responses (CAR, % of 50 trials) were infused with AB (550pmol, 0.12μL/h, 28days) or vehicle in the LV using a mini-osmotic pump. They were tested before the surgery (T0), 7 and 35days after the infusion started (T7; T35). In T0, no difference was observed between CAR of the control (Cwt=59.7±6.7%; CkoB1=46.7±4.0%; CkoB2=64.4±5.8%) and Aβ (Aβwt=66.0±3.0%; AβkoB1=66.8±8.2%; AβkoB2=58.7±5.9%) groups. In T7, AβkoB2 showed a significant decrease in CAR (41.0±8.6%) compared to the control-koB2 (72.8±2.2%, P<0.05). In T35, a significant decrease (P<0.05) was observed in Aβwt (40.7±3.3%) and AβkoB2 (41.2±10.7%) but not in the AβkoB1 (64.0±14.0%) compared to their control groups. No changes were observed in the controls at T35. We suggest that in chronic infusion of BA, B1 receptors could play an important role in the neurodegenerative process. Conversely, the premature memory impairment of koB2 suggests that it may be a protective factor.

Kinin B1 and B2 receptors contribute to orofacial heat hyperalgesia induced by infraorbital nerve constriction injury in mice and rats - Corrected Proof

2009-11-16

Abstract: Mechanisms coupled to kinin B1 and B2 receptors have been implicated in sensory changes associated to various models of neuropathy. The current study aimed to investigate if kinins also participate in orofacial thermal hyperalgesia induced by constriction of the infraorbital nerve (CION), a model of trigeminal neuropathic pain which displays persistent hypersensitivity to orofacial sensory stimulation, in rats and mice. Male Swiss mice (30–35g) or Wistar rats (200–250g; n=6–10 per group in both cases) underwent CION or sham surgery and were submitted repeatedly to application of heat (∼50°C) to the ipsilateral or contralateral snout, delivered by a heat source placed 1cm from the vibrissal pad. Decreases in latency to display head withdrawal or vigorous snout flicking were considered indicative of heat hyperalgesia. CION caused long-lasting heat hyperalgesia which started on Day 2 after surgery in both species and lasted up to Day 17 in mice and Day 10 in rats. Administration of DALBK or HOE-140 (peptidic B1 and B2 receptor antagonists, respectively; each at 3nmol in 10μl) onto the exposed infraorbital nerve of mice at the moment of surgery delayed the development of the thermal hyperalgesia. Systemic treatment on Day 5 (mice) or Day 4 (rats) with Des-Arg9, Leu8-Bradykinin (DALBK, B1 receptor antagonist, 0.1–1μmol/kg, i.p.) or HOE-140 (B2 receptor antagonist, 0.001–1μmol/kg, i.p.) transiently reduced heat hyperalgesia in both species. Due to the peptidic nature of DALBK and HOE-140, it is likely that their effects reported herein resulted from blockade of peripheral kinin receptors. Thus, mechanisms operated by kinin B1 and B2 receptors, contribute to orofacial heat hyperalgesia induced by CION in both mice and rats. Perhaps kinin B1 and B2 receptor antagonists might constitute effective preventive and curative treatments for orofacial thermal hyperalgesia induced by nerve injury.

Neuropeptide Y receptor subtypes in the dorsal vagal complex under acute feeding adaptation in the adult rat - Corrected Proof

Stéphanie Mahaut, Yvan Dumont, Alain Fournier, Rémi Quirion, Emmanuel Moyse — 2009-11-02

Abstract: Neuropeptide Y (NPY), Peptide YY (PYY) and pancreatic polypeptides (PPs) belong to the same peptide family called the Y or NPY family. Central and peripheral injections of these peptides are implicated in the regulation of food intake at the level of the hypothalamus (central effects; increased food intake) and dorsal vagal complex (DVC) (peripheral effects; decreased food intake). The DVC of the brainstem is a satiety reflex key region, which includes the nucleus tractus solitarius (NTS), area postrema (AP) and dorso motor nucleus of the vagus (DMX). NPY binding sites were quantified on serial DVC sections using in vitro receptor autoradiography in two feeding adaptation models: fasting and inflammatory anorexia. Receptor autoradiography revealed that Y1, Y2, Y4 and Y5 receptor subtypes are present in all nuclei of the DVC. Additionally, we also observed significant amount of specific labelling remaining even after having blocked all known NPY receptor subtypes targeted by radioligands such as [125I][Leu31, Pro34]PYY, [125I]PYY3-36 and [125I]hPP. This binding is referred as an atypical NPY site. Lipopolysaccharide (LPS) injection and food deprivation (24–48h) did not induce any change in the expression of NPY Y1, Y2, Y4 and Y5 receptors at the level of the NTS and DMX. However, a significant decrease in [125I]PYY3-36/Y2 and [125I]hPP/Y4- and Y5-insensitive binding sites (residual or atypical site) was observed in the AP. Together, these data could suggest that residual or atypical NPY binding site in the AP is modulated by food deprivation and may be physiologically relevant and implicated in feeding behaviors.

Guest Editorial for the Special Issue Peptide Receptors: Focus on Neuropeptides and Kinins - Corrected Proof

François Marceau, Rémi Quirion — 2009-10-21

The Joint International Symposium Peptide Receptors – Kinin 2009 was successfully held in Quebec City (June 26–30, 2009), and the Editor-in-Chief of Neuropeptides, Dr. Jacqueline N. Crawley, graciously accepted to host a Special Issue open to the 150 participants of this event. This was the 5th Symposium of the Peptide Receptors series and the preceding editions (1994, 1996, 2001, 2004) were all held in Montreal, Canada. The 2009 symposium benefited from the experience of a pretty stable core of members of the successive local organizing committees. The 2009 edition of Peptide Receptors was recognized as the First meeting of the Canadian Branch of the International Neuropeptide Society (www.inps.org). Thus, the usual interest in the neurosciences was strongly represented with cutting edge basic and clinical investigations of pain and CNS disorders. Bradykinin-related peptides and their receptors have always been highlighted at successive Peptide Receptors symposia, not surprisingly if one considers the co-chair function of Prof. Regoli in the symposium series. The present edition was held jointly with Kinin 2009, sponsored by the Frey–Werle Foundation (www.frey-werle-foundation.org) and part of a meeting series held every other year in the recent past (2007 Berlin/Germany; 2005 Lund/ Sweden). This naturally led to sessions devoted to inflammation, hypertension and diabetes. The joint meeting was a perfect occasion to follow up on topics of common interest to all peptide scientists, the medicinal chemistry and molecular pharmacology of peptide receptors. Two poster sessions illustrated in a creative way many novel approaches and results in the peptide hormone field and six awards were presented to training investigators for excellent posters presentations: Jérôme Côté (Université de Sherbrooke), Hajer Jegham (Centre Hospitalier Universitaire de Québec), Frank Kuhr (University of Illinois Chicago), Marilia G. Pereira (Universidade de São Paulo), Mylène Pouliot (Université de Montréal) and Matt Rowan (University of Texas San Antonio).

Altered cardiac bradykinin metabolism in experimental diabetes caused by the variations of angiotensin-converting enzyme and other peptidases - Corrected Proof

2009-10-19

Abstract: The peptidases angiotensin-converting enzyme (ACE) and neutral endopeptidase 24.11 (NEP) mediate most of the kinin catabolism in normal cardiac tissue and are the molecular targets of inhibitory drugs that favorably influence diabetic complications. We studied the variations of those kininases in the myocardium of rats in experimental diabetes. ACE and NEP activities were significantly decreased in heart membranes 4–8weeks post-streptozotocin (STZ) injection. However, insulin-dependent diabetes did not modify significantly bradykinin (BK) half-life (t1/2) while the effect of both ACE (enalaprilat) and ACE and NEP (omapatrilat) inhibitors on BK degradation progressively decreased, which may be explained by the upregulation of other unidentified metallopeptidase(s). In vivo insulin treatment restored the activities of both ACE and NEP. ACE and NEP activities were significantly higher in hearts of young Zucker rats than in those of Sprague–Dawley rats. BK t1/2 and the effects of peptidase inhibitors on t1/2 varied accordingly. It is concluded that kininase activities are subjected to large and opposite variations in rat cardiac tissue in type I and II diabetes models. A number of tissue or molecular factors may determine these variations, such as remodeling of cardiac tissue, ectoenzyme shedding to the extracellular fluid and the pathologic regulation of peptidase gene expression.