Experimental study on renoprotective effect of intermedin on diabetic nephropathy.

Intermedin(IMD) is a novel member of the calcitonin/calcitonin gene-related peptide (CT/CGRP) family that has anti-inflammatory, antioxidant and anti-apoptosis properties. This study aimed to evaluate the renoprotective effects of IMD on podocyte apoptotic loss and slit diaphragm protein deficiency the kidneys of rats with in streptozotocin (STZ) induced diabetes in high glucose-exposed podocytes. Our results showed that IMD significantly attenuated proteinuria, and alleviated the abnormal alterations in glomerular ultrastructure in vivo. IMD also improved the induction of slit diaphragm proteins, and restored the decreased Bcl-2 expression and suppressed Bax and caspase-3 induction in the diabetic glomeruli. In addition, IMD attenuated podocyte apoptosis and filamentous actin (F-actin) rearrangement in high glucose-exposed podocytes. Exposure to high glucose elevated the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress in renal podocytes, and IMD treatment blocked such ER stress responses pertinent to podocyte apoptosis and reduced synthesis of slit diaphragm proteins in vivo and in vitro. These observations demonstrate that targeting ER stress is an underlying mechanism of IMD-mediated amelioration of diabetes-associated podocyte injury and dysfunction.

Intermedin Alleviates Renal Ischemia-Reperfusion Injury and Enhances Neovascularization in Wistar Rats.

BACKGROUND: Ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) and increases the risk of subsequently developing chronic kidney disease. Angiogenesis has been shown to play an important role in reducing renal injury after ischemia reperfusion. In this study, we investigated whether IMD could reduce renal IRI by promoting angiogenesis. METHODS: The kidneys of Wistar rats were subjected to 45 min of warm ischemia followed by 24 h of reperfusion. IMD was overexpressed in vivo using the vector pcDNA3.1-IMD transfected by an ultrasound-mediated system. The renal injury after ischemia reperfusion was assessed by detection of the serum creatinine concentration and histologic examinations of renal tissues stained by PAS and H&E. Real-time PCR and Western blotting were used to determine the mRNA and protein levels, respectively. Histological examinations were used to assess the expression of CD31, MMP2, MMP9, ET-1, VEGF and VEGFR2 in tissues. RESULTS: Renal function and renal histological damage were significantly ameliorated in IMD-transfected rats after ischemia reperfusion. Compared to the IRI, IMD significantly promoted angiogenesis. IMD also upregulated the protein and mRNA expression levels of VEGF and VEGFR2 and downregulated the expression level of MMP2, MMP9 and ET-1. CONCLUSION: IMD could protect the kidney after renal ischemia-reperfusion injury by promoting angiogenesis and reducing the destruction of the perivascular matrix.

Adrenomedullin has a role in angiogenic effects of resveratrol in adipose tissues of obese female rats.

Obesity is a complex, chronic disease that arises according to the interaction between genetic and environmental factors. The expansion and growth of white adipose tissue (WAT) could be related to angiogenesis. Resveratrol and adrenomedullin (AdM) were used for the inhibition of angiogenesis in metabolically passive WAT for inhibiting the expansion of this tissue, and the activation of angiogenesis in metabolically active brown adipose tissue (BAT) for increasing daily energy consumption as a way of reducing obesity. Rats were divided into eight groups. Four obese groups were fed with a high-fat diet containing 60% fat as energy for three months. After obtaining obesity, 2.5 nmol/kg AdM and 10 mg/kg resveratrol were treated to experiment groups intraperitoneally (i.p.) every other day for four weeks. AdM and vascular endothelial growth factor A (VEGF-A) mRNA levels were detected with semi-quantitative PCR; protein levels were detected with Western Blotting. AdM and resveratrol are multifactorial molecules, thus, this study has revealed a few novel evidence. The results were distinct in the group and treatment levels. The results showed that resveratrol has a role in angiogenesis in obesity and contributed to AdM production. It is observed that AdM has regulated its expression and increased the effect of resveratrol in WAT. AdM and VEGF-A gene expressions could not be detected in BAT; however, it is suggested that resveratrol may have a pro-angiogenic effect in BAT of obese rats according to the protein levels. AdM also has regulated VEGF-A level according to the metabolic situation of the organism.

Subcutaneously administered adrenomedullin exerts a potent therapeutic effect in a murine model of ulcerative colitis.

Adrenomedullin (AM) exerts a potent anti-inflammatory effect. Intrarectal or consecutive intravenous administrations of AM reduce pathological manifestations in rodent colitis models. However, in clinical applications, a safer administration route that provides stronger alleviation of patient burden is preferred. We investigated whether subcutaneously administered AM is effective against dextran sulfate sodium (DSS)-induced colitis. C57BL/6J mice were administered 1% DSS in drinking water and received AM at 8, 40 or 80 nmol/kg subcutaneously once a day for 7 consecutive days. Subcutaneously administered AM significantly and dose-dependently ameliorated body weight loss, diarrhea, and histological severity of colonic inflammation in DSS-treated mice. The AM therapeutic effect was associated with the upregulation of the production of autocrine AM, and expression of cAMP, c-fos, KLF4, and downregulation of STAT3 and NF-κB p65 phosphorylation, as well as a decrease in proinflammatory cytokine expression in the colon. Subcutaneous AM treatment potently attenuated DSS-induced colitis, which suggests that AM administered subcutaneously in ulcerative colitis (UC) patients may decrease diseases burden and improve quality of life.

Small molecules related to adrenomedullin reduce tumor burden in a mouse model of colitis-associated colon cancer.

To investigate the contribution of adrenomedullin (AM) and its gene-related peptide, proadrenomedullin N-terminal 20 peptide (PAMP), to the progression and potential treatment of colon cancer we studied the effects of four small molecules (SM) related to AM and PAMP on a mouse model of colon cancer. For each SM, four experimental groups of male mice were used: (i) Control group; (ii) SM group; (iii) DSS group (injected with azoxymethane [AOM] and drank dextran sulfate sodium [DSS]); and (iv) DSS + SM group (treated with AOM, DSS, and the SM). None of the mice in groups i and ii developed tumors, whereas all mice in groups iii and iv developed colon neoplasias. No significant differences were found among mice treated with PAMP modulators (87877 and 106221). Mice that received the AM negative modulator, 16311, had worse colitis symptoms than their control counterparts, whereas mice injected with the AM positive modulator, 145425, had a lower number of tumors than their controls. SM 145425 regulated the expression of proliferation marker Lgr5 and had an impact on microbiota, preventing the DSS-elicited increase of the Bacteroides/Prevotella ratio. These results suggest that treatment with AM or with positive modulator SMs may represent a novel strategy for colon cancer.

Anti-Inflammatory Effects of PEGylated Human Adrenomedullin in a Mouse DSS-Induced Colitis Model.

Preclinical Research Human adrenomedullin (hAM), a hypotensive peptide, also has anti-inflammatory effects. hAM can reduce the severity of the dextran sulphate sodium (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in animal models. Furthermore, in a clinical study, hAM treatment reduced the Disease Activity Index in ulcerative colitis. However, these therapeutic effects required continuous administration of hAM as the half-life of native hAM is quite short in blood. To resolve this problem, hAM N-terminal was conjugated with two kinds of polyethylene glycol (PEG); 5 kDa PEG or 60 kDa PEG (5 kDa PEG-hAM and 60 kDa PEG-hAM respectively). In a previous study, 5 kDa PEG-hAM stimulated cAMP production and prolonged the plasma half-life compared with native hAM. Herein we examine the effect of PEG-hAM in the DSS colitis model. Treatment with both PEG-hAM preparations reduced the total inflammation score. In addition, the plasma half-life of 60 kDa PEG-hAM was much longer than 5 kDa PEG-hAM. In summary, a single subcutaneous administration of 60 kDa PEG-hAM reduced the total inflammation score in mice with DSS-induced colitis. Therefore, these results suggest that 60 kDa PEG-hAM is a possible therapeutic agent for the treatment of inflammatory bowel disease. Drug Dev Res 78 : 129-134, 2017. © 2017 Wiley Periodicals, Inc.

Adrenomedullin Suppresses Vascular Endothelial Growth Factor-Induced Vascular Hyperpermeability and Inflammation in Retinopathy.

Diabetic macular edema (DME) is caused by blood-retinal barrier breakdown associated with retinal vascular hyperpermeability and inflammation, and it is the major cause of visual dysfunction in diabetic retinopathy. Adrenomedullin (ADM) is an endogenous peptide first identified as a strong vasodilator. ADM is expressed in the eyes and is up-regulated in various eye diseases, although the pathophysiological significance is largely unknown. We investigated the effect of ADM on DME. In Kimba mice, which overexpress human vascular endothelial growth factor in their retinas, the capillary dropout, vascular leakage, and vascular fragility characteristic of diabetic retinopathy were observed. Intravitreal or systemic administration of ADM to Kimba mice ameliorated both the capillary dropout and vascular leakage. Evaluation of the transendothelial electrical resistance and fluorescein isothiocyanate-dextran permeability of an endothelial cell monolayer using TR-iBRB retinal capillary endothelial cells revealed that vascular endothelial growth factor enhanced vascular permeability but that co-administration of ADM suppressed the effect, in part by enhancing tight junction formation between endothelial cells. In addition, a comprehensive PCR array analysis showed that ADM administration suppressed various molecules related to inflammation and NF-κB signaling within retinas. From these results, we suggest that by exerting inhibitory effects on retinal inflammation, vascular permeability, and blood-retinal barrier breakdown, ADM could serve as a novel therapeutic agent for the treatment of DME.

[Effects of intrathecal administration of AM22-52 on mechanical allodynia and CCL2 expression in DRG in bone cancer rats].

The pain peptide adrenomedullin (AM) plays a pivotal role in pathological pain. The present study was designed to investigate the effect of blockade of AM receptor on bone cancer pain (BCP) and its mechanism. BCP was developed by inoculation of Walker 256 mammary gland carcinoma cells in the tibia medullary cavity of Sprague Dawley rats. The selective AM receptor antagonist AM22-52 was administered intrathecally on 15 d after the inoculation. Quantitative real-time PCR was used to detect mRNA level of CC chemokine ligand 2 (CCL2) in dorsal root ganglion (DRG). Double immunofluorescence staining was used to analyze the localizations of CCL2 and AM in DRG of normal rats. The results showed that, from 6 to15 d after the inoculation, the animals showed significant reduction in the mechanical pain threshold in the ipsilateral hindpaw, companied by the decline in bone density of tibia bone. The expression of CCL2 mRNA in DRG of BCP rats was increased by 3 folds (P < 0.001 vs saline group). Intrathecal administration of AM22-52 abolished bone cancer-induced mechanical allodynia and increase of CCL2 mRNA level (P < 0.001). In normal rats, CCL2 was co-localized with AM in DRG neurons. These results suggest that AM may play a role in the pathogenesis of BCP. The increased AM bioactivity up-regulates CCL2 expression in DRG, which may contribute to the induction of pain hypersensitivity in bone cancer.

Cardiovascular effects of exogenous adrenomedullin and CGRP in Ramp and Calcrl deficient mice.

Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) are potent vasodilator peptides and serve as ligands for the G-protein coupled receptor (GPCR) calcitonin receptor-like receptor (CLR/Calcrl). Three GPCR accessory proteins called receptor activity-modifying proteins (RAMPs) modify the ligand binding affinity of the receptor such that the CLR/RAMP1 heterodimer preferably binds CGRP, while CLR/RAMP2 and CLR/RAMP3 have a stronger affinity for AM. Here we determine the contribution of each of the three RAMPs to blood pressure control in response to exogenous AM and CGRP by measuring the blood pressure of mice with genetic reduction or deletion of the receptor components. Thus, the cardiovascular response of Ramp1-/-, Ramp2+/-, Ramp3-/-, Ramp1-/-/Ramp3-/- double-knockout (dKO), and Calcrl+/- mice to AM and CGRP were compared to wildtype mice. While under anesthesia, Ramp1-/- male mice had significantly higher basal blood pressure than wildtype males; a difference which was not present in female mice. Additionally, anesthetized Ramp1-/-, Ramp3-/-, and Calcrl+/- male mice exhibited significantly higher basal blood pressure than females of the same genotype. The hypotensive response to intravenously injected AM was greatly attenuated in Ramp1-/- mice, and to a lesser extent in Ramp3-/- and Calcrl+/- mice. However, Ramp1-/-/Ramp3-/- dKO mice retained some hypotensive response to AM. These results suggest that the hypotensive effect of AM is primarily mediated through the CLR/RAMP1 heterodimer, but that AM signaling via CLR/RAMP2 and CLR/RAMP3 also contributes to some hypotensive action. On the other hand, CGRP's hypotensive activity seems to be predominantly through the CLR/RAMP1 heterodimer. With this knowledge, therapeutic AM or CGRP peptides could be designed to cause less hypotension while maintaining canonical receptor-RAMP mediated signaling.

Cardiac sympathetic afferent reflex response to intermedin microinjection into paraventricular nucleus is mediated by nitric oxide and γ-amino butyric acid in hypertensive rats.

Intermedin (IMD) is a member of calcitonin/calcitonin gene-related peptide (CGRP) and involves in the regulation of cardiovascular function in both peripheral tissues and central nervous system (CNS). Paraventricular nucleus (PVN) of hypothalamus is an important site in the control of cardiac sympathetic afferent reflex (CSAR) which participates in sympathetic over-excitation of hypertension. The aim of this study is to investigate whether IMD in the PVN is involved in the inhibition of CSAR and its related mechanism in hypertension. Rats were subjected to two-kidney one-clip (2K1C) surgery to induce renovascular hypertension or sham-operation (Sham). Acute experiments were carried out four weeks later under anesthesia. The CSAR was evaluated with the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) responses to the epicardial application of capsaicin. The RSNA and MAP were recorded in sinoaortic-denervated, cervical-vagotomized and anesthetized rats. Bilateral PVN microinjection of IMD (25 pmol) caused greater decrease in the CSAR in 2K1C rats than in Sham rats, which was prevented by pretreatment with adrenomedullin (AM) receptor antagonist AM22-52, non-selective nitric oxide (NO) synthase (NOS) inhibitor L-NAME or γ-amino butyric acid (GABA)B receptor blocker CGP-35348. PVN pretreatment with CGRP receptor antagonist CGRP8-37 or GABA(A) receptor blocker gabazine had no significant effect on the CSAR response to IMD. AM22-52, L-NAME and CGP-35348 in the PVN could increase CSAR in Sham and 2K1C rats. These data indicate that IMD in the PVN inhibits CSAR via AM receptor, and both NO and GABA in the PVN involve in the effect of IMD on CSAR in Sham and renovascular hypertensive rats.

Biological properties of adrenomedullin conjugated with polyethylene glycol.

Adrenomedullin (AM) is a vasodilator peptide with pleiotropic effects, including cardiovascular protection and anti-inflammation. Because of these beneficial effects, AM appears to be a promising therapeutic tool for human diseases, while intravenous injection of AM stimulates sympathetic nerve activity due to short-acting potent vasodilation, resulting in increased heart rate and renin secretion. To lessen these acute reactions, we conjugated the N-terminal of human AM peptide with polyethylene glycol (PEG), and examined the biological properties of PEGylated AM in the present study. PEGylated AM stimulated cAMP production, an intracellular second messenger of AM, in cultured human embryonic kidney cells expressing a specific AM receptor in a dose-dependent manner, as did native human AM. The pEC50 value of PEGylated AM was lower than human AM, but no difference was noted in maximum response (Emax) between the PEGylated and native peptides. Intravenous bolus injection of 10nmol/kg PEGylated AM lowered blood pressure in anesthetized rats, but the acute reduction became significantly smaller by PEGylation as compared with native AM. Plasma half-life of PEGylated AM was significantly longer than native AM both in the first and second phases in rats. In summary, N-terminal PEGylated AM stimulated cAMP production in vitro, showing lessened acute hypotensive action and a prolonged plasma half-life in comparison with native AM peptide in vivo.

Involvement of adrenomedullin in spinal glial activation following chronic administration of morphine in rats.

BACKGROUND: Adrenomedullin (AM) belongs to the calcitonin gene-related peptide (CGRP) family. Our previous studies show that chronic exposure to morphine increases spinal AM bioactivity, contributing to the development and maintenance of morphine tolerance. This study investigated the possible involvement of AM in morphine-evoked gliosis. METHODS: Real-time polymerase chain reaction was performed to determine interleukin-1ß (IL-1ß), IL-6 and tumour necrosis factor-α (TNF-α) mRNAs in the spinal dorsal horn and cultured sensory ganglion explants. Immunohistochemistry was performed to identify spinal microglia and astrocytes. RESULTS: Repetitive intrathecal (i.t.) injection of morphine (20 µg) increased the expression of IL-1ß, IL-6 and TNF-α mRNAs in the spinal dorsal horn. The co-administration of the selective AM receptor antagonist AM22-52 (36 µg) markedly attenuated chronic morphine-evoked increase in IL-1ß and IL-6, but not TNF-α, mRNA levels. Exposure of cultured dorsal root ganglion (DRG) explants to morphine (3.3 µmol/L) for 6 days up-regulated IL-1ß and IL-6 mRNA expressions. The depletion of AM gene using small interfering RNA (siRNA) approach abolished morphine-evoked increase in IL-1ß and IL-6 syntheses in the cultured DRG. The blockade of AM receptors by i.t. AM22-52 also inhibited chronic morphine-evoked cell hypertrophy of microglia and astrocytes as well as an increase in OX-42 and GFAP (glial fibrillary acidic protein) immunoreactivities. Furthermore, the 6-day treatment with AM (10 µg, i.t.) induced morphological changes of microglia and astrocytes as well as an increase in IL-1ß, IL-6 and TNF-α mRNA levels in the spinal dorsal horn. CONCLUSION: The present study supports the idea that up-regulation of the pronociceptive mediator AM can recruit spinal glial cells, resulting in an increase in cytokines during chronic use of morphine.

Adrenomedullin as a potential therapeutic agent for inflammatory bowel disease.

Adrenomedullin (AM) was originally isolated from human pheochromocytoma as a biologically active peptide with potent vasodilating action but is now known to exert a wide range of physiological effects, including cardiovascular protection, neovascularization, and apoptosis suppression. A variety of tissues, including the gastrointestinal tract, have been shown to constitutively produce AM. Pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin-1, and lipopolysaccharides, induce the production and secretion of AM. Conversely, AM induces the downregulation of inflammatory cytokines in cultured cells. Furthermore, AM downregulates inflammatory processes in a variety of different colitis models, including acetic acid-induced colitis and dextran sulfate sodium-induced colitis. AM exerts antiinflammatory and antibacterial effects and stimulates mucosal regeneration for the maintenance of the colonic epithelial barrier. Here, we describe the first use of AM to treat patients with refractory ulcerative colitis. The results strongly suggest that AM has potential as a new therapeutic agent for the treatment of refractory ulcerative colitis.

The effects of adrenomedullin in traumatic brain injury.

Traumatic brain injury (TBI) is a common cause of death and disability throughout the world. A multifunctional peptide adrenomedullin (AM) has protective effects in the central nervous system. We evaluated AM in an animal model as a therapeutic agent that reduces brain damage after traumatic brain injury. A total of 36 rats was divided into 3 groups as sham, head trauma plus intraperitoneal (ip) saline, and head trauma plus adrenomedullin ip. The diffuse brain injury model of Marmarou et al. was used. Blood samples were taken from all groups at the 1st, 6th and 24th hours for analysis of TNF-α (tumor necrosis factor-α), IL-1ß (interleukin-1ß) and IL-6 (interleukin-6) levels. At the end of the study (at the 24th hour) a neurological examination was performed and half of the rats were decapitated to obtain blood and tissue samples, the other half were perfused transcardiacally for studying the histopathology of the brain tissue. There were no statistically significant changes in plasma levels of IL-1ß, IL-6 and TNF-α relative to the sham group. Also, changes in tissue levels of malonedialdehyde, myeloperoxidase and glutathione were not statistically significant. However, neurological scores and histopathological examinations revealed healing. AM individually exerts neuroprotective effects in animal models of acute brain injury. But the mechanisms of action remain to be assessed.

Papel de la adrenomedulina cerebelosa en la regulación de la presión arterial / Role of cerebelar adrenomedullin in blood pressure regulation

La adrenomedulina (AM) es un péptido ubicuo de 52 residuos de aminoácidos que cumple funciones importantes en la regulación de la función cardiovascular (CDV). La AM ejerce sus acciones a través de su unión con tres subtipos de receptores, el receptor del péptido relacionado al gen de la calcitonina tipo 1 (CGRP1), el receptor de AM tipo 1 (AM1) y tipo 2 (AM2). El CGRP1 está formado por el receptor similar al receptor de calcitonina (CRLR) y la proteína que modifica la actividad del receptor tipo 1 (RAMP1). El AM1 por el CRLR+RAMP2 y el AM2 por el CRLR+RAMP3. A nivel del sistema nervioso central, la AM y sus receptores se localizan en diversas regiones, incluyendo el cerebelo. Se ha demostrado marcados incrementos en la densidad de los sitios de unión para la AM en el cerebelo durante la hipertensión, lo que sugiere un papel del sistema adrenomedulinérgico cerebeloso en la regulación de la presión arterial (PA). En el presente estudio se evaluó la participación de la AM cerebelosa en la regulación de la PA. Nuestros hallazgos muestran la existencia de desregulación de los componentes del sistema AM cerebeloso durante la hipertensión, ya que se encontró una reducida expresión de CRLR, RAMP1 y RAMP3 y una incrementada expresión de la AM y RAMP2 en el vermis de cerebelo de ratas hipertensas (SHR), cuando se comparó con las ratas controles, Wistar Kyoto (WKY), de 8 y 16 semanas de edad. La reducción de la PA mediante el tratamiento crónico con valsartán (60mg/Kg/día,p.o.) revirtió las desregulación de la AM y los componentes de su receptor, observados en las ratas SHR. El papel de las especies reactivas de oxígeno (EROS) en la acción de la AM cerebelosa quedó evidenciado, ya que la AM fue capaz de reducir la actividad de las tres enzimas antioxidantes, superóxiodo dismutasa (SOD), catalasa (CAT) y glutatión peroxidasa (GPx), en las ratas WKY y Sprague - Dawley (SD). Aún mas, nuestros hallazgos mostraron claramente el efecto antagónico entre la AM y la ANG II sobre la actividad de las enzimas antioxidantes inducida por la ANG II. El efecto de la AM sobre las enzimas antioxidantes no se manifestó en la ratas hipertensas, sin embargo el mismo fue restaurado mediante la disminución de la presión arterial con la administración crónica de dos antihipertensivos de mecanismo de acción distintos como la amlodipina (5mg/Kg/día,p.o) o el valsartán (60mg/Kg/día,p.o), lo que sugiere una relación entre la hipertensión y ausencia del efecto de AM en la ratas SHR. Al evaluar la posible vía de señalización que media la acción de la AM y el antagonismo con la ANG II sobre la actividad de las enzimas antioxidantes, demostramos que no existe una vía final común para dicho antagonismo, siendo la proteína quinasa A (PKA) y los 3 subtipos de receptores CGRP1, AM1 y AM2 los que median la acción de la AM, mientras que la acción de la ANG II se encuentra mediada a través de una vía que involucra la PKC/NAD(P)H oxidasa. Los hallazgos demuestran el antagonismo entre la AM y la ANG II en la regulación del estrés oxidativo en el cerebelo y ratifican la desregulación de la señalización de la AM mediada por EROs durante la hipertensión. Al evaluar las vías de señalización intracelular que median la acción de la AM en el cerebelo, demostramos que la AM es capaz de activar a las ERK, la producción de GMPc y NO a través de la estimulación del receptor AM1, y del AMPc a través de los tres subtipos de receptores de AM, lo que apoya que en el cerebelo la AM ejerce acciones a través de diversas vías de señalización como lo son NO/GMPc, AC/AMPc/PKA y/o ERK. El posible papel funcional de la AM in vivo fue inequívoco, ya que se demostró que la microinyección de AM en el vermis cerebeloso produjo una respuesta hipotensora profunda en las ratas SHR pero no en las normotensas. El hecho que la microinyección de AM en el vermis cerebelar en las ratas SD, WKY y SHR disminuyó significativamente la respuesta presora frente al estrés simpatoadrenal inducido por el estímulo eléctrico plantar, sugiere que la acción hipotensora está mediada a través de la regulación del eflujo simpático e indica un posible papel de la AM en la regulación de la respuesta CDV frente al estrés. En conjunto, nuestros resultados demuestran la existencia de un sistema adrenomedulinérgico funcional en el cerebelo, e indican por primera vez, que la AM cumple un papel importante en la regulación de la PA durante la hipertensión y el estrés.

Cardiovascular effects of the intracerebroventricular injection of adrenomedullin: roles of the peripheral vasopressin and central cholinergic systems

Our objective was to investigate in conscious Sprague-Dawley (6-8 weeks, 250-300 g) female rats (N = 7 in each group) the effects of intracerebroventricularly (icv) injected adrenomedullin (ADM) on blood pressure and heart rate (HR), and to determine if ADM and calcitonin gene-related peptide (CGRP) receptors, peripheral V1 receptors or the central cholinergic system play roles in these cardiovascular effects. Blood pressure and HR were observed before and for 30 min following drug injections. The following results were obtained: 1) icv ADM (750 ng/10 µL) caused an increase in both blood pressure and HR (DMAP = 11.8 ± 2.3 mmHg and ΔHR = 39.7 ± 4.8 bpm). 2) Pretreatment with a CGRP receptor antagonist (CGRP8-37) and ADM receptor antagonist (ADM22-52) blocked the effect of central ADM on blood pressure and HR. 3) The nicotinic receptor antagonist mecamylamine (25 µg/10 µL, icv) and the muscarinic receptor antagonist atropine (5 µg/10 µL, icv) prevented the stimulating effect of ADM on blood pressure. The effect of ADM on HR was blocked only by atropine (5 µg/10 µL, icv). 4) The V1 receptor antagonist [β-mercapto-β-β-cyclopentamethylenepropionyl¹, O-me-Tyr²,Arg8]-vasopressin (V2255; 10 µg/kg), that was applied intravenously, prevented the effect of ADM on blood pressure and HR. This is the first study reporting the role of specific ADM and CGRP receptors, especially the role of nicotinic and muscarinic central cholinergic receptors and the role of peripheral V1 receptors in the increasing effects of icv ADM on blood pressure and HR.

Attenuation of hyperoxia-induced lung injury in rats by adrenomedullin.

Oxidative stress and inflammation are involved in the pathogenesis of acute lung injury (ALI). Adrenomedullin (AM) is an endogenous peptide with anti-inflammatory and antioxidant properties. This study investigated that whether AM treatment may ameliorate hyperoxia-induced ALI in rats via inhibition of oxidative stress and inflammation. Rats were randomized to receive continuous intravenous infusion of AM or saline through a microosmotic pump, and then ALI was induced by exposing the animals in sealed cages >95% oxygen for 72 h. Exposure to hyperoxia caused lung injury as increased infiltration of inflammatory cells and disruption of lung architecture. AM administration markedly improved these changes. Additionally, AM administration significantly increased glutathione peroxidase and superoxide dismutase activities. Meanwhile, hyperoxia-induced increase of lipid hydroperoxide level was markedly reduced by AM treatment. Moreover, nuclear factor-kappa B-DNA-binding activity, and production of the inflammatory mediators interleukin-6, keratinocyte-derived chemokine, and matrix metalloproteinase 9, were significantly inhibited by AM treatment. AM ameliorates hyperoxia-induced ALI in rats by suppression of oxidative stress and inflammation.

Haemodynamic, endocrine and renal actions of adrenomedullin 5 in an ovine model of heart failure.

AM5 (adrenomedullin 5), a newly described member of the CGRP (calcitonin gene-related peptide) family, is reported to play a role in normal cardiovascular physiology. The effects of AM5 in HF (heart failure), however, have not been investigated. In the present study, we intravenously infused two incremental doses of AM5 (10 and 100 ng/min per kg of body weight each for 90 min) into eight sheep with pacing-induced HF. Compared with time-matched vehicle control infusions, AM5 produced progressive and dose-dependent increases in left ventricular dP/dt(max) [LD (low dose), +56 mmHg/s and HD (high dose), +152 mmHg/s] and cardiac output (+0.83 l/min and +1.81 l/min), together with decrements in calculated total peripheral resistance (-9.4 mmHg/min per litre and -14.7 mmHg/min per litre), mean arterial pressure (-2.8 mmHg and -8.4 mmHg) and LAP (left atrial pressure; -2.6 mmHg and -5.6 mmHg) (all P<0.001). HD AM5 significantly raised PRA (plasma renin activity) (3.5-fold increment, P<0.001), whereas plasma aldosterone levels were unchanged over the intra-infusion period and actually fell in the post-infusion period (70% decrement, P<0.01), resulting in a marked decrease in the aldosterone/PRA ratio (P<0.01). Despite falls in LAP, plasma atrial natriuretic peptide and B-type natriuretic peptide concentrations were maintained relative to controls. AM5 infusion also induced significant increases in urine volume (HD 2-fold increment, P<0.05) and urine sodium (2.7-fold increment, P<0.01), potassium (1.7-fold increment, P<0.05) and creatinine (1.4-fold increment, P<0.05) excretion and creatinine clearance (60% increment, P<0.05). In conclusion, AM5 has significant haemodynamic, endocrine and renal actions in experimental HF likely to be protective and compensatory in this setting. These results suggest that AM5 may have potential as a therapeutic agent in human HF.

Adrenomedullin(22-52) combats inflammation and prevents systemic bone loss in murine collagen-induced arthritis.

OBJECTIVE: Adrenomedullin(22-52) is a truncated peptide derived from adrenomedullin, a growth factor with antiapoptotic and immunoregulatory properties. It can act as an agonist or an antagonist depending on cell type. Its in vivo effects are unknown, but adrenomedullin(22-52) could possess immunomodulatory properties. This study was undertaken to evaluate the effect of adrenomedullin(22-52) in a mouse model of arthritis. METHODS: DBA/1 mice with collagen-induced arthritis (CIA) were treated with 1.2 µg/gm adrenomedullin(22-52) , adrenomedullin, or saline at arthritis onset. Bone mineral density was measured at the beginning of the experiment and when mice were killed. Mouse joints were processed for histologic analysis and protein studies, and spleens were examined for Treg cell expression. Cytokine expression was studied in mouse joint tissue and serum. RESULTS: In mice with CIA, adrenomedullin and adrenomedullin(22-52) reduced clinical and histologic arthritis scores and shifted the pattern of articular and systemic cytokine expression from Th1 to Th2, as compared to untreated mice with CIA (controls). Tumor necrosis factor α, interleukin-6 (IL-6), and IL-17A levels were significantly decreased in the joints of mice with CIA treated with adrenomedullin or adrenomedullin(22-52) as compared to controls, whereas IL-4 and IL-10 levels were increased. Adrenomedullin(22-52) was more effective than adrenomedullin in modulating cytokine content and enhanced Treg cell function without changing Treg cell expression compared to controls. Adrenomedullin receptor binding and transcriptional adrenomedullin receptor expression were markedly increased in joints from controls, whereas adrenomedullin receptor binding was considerably decreased in treated animals. Mice with CIA treated with adrenomedullin or adrenomedullin(22-52) had considerably fewer apoptotic chondrocytes and diminished cartilage degradation. Adrenomedullin(22-52) completely prevented systemic bone loss by preserving osteoblastic activity, but without changes in osteoclastic activity. CONCLUSION: Our findings indicate that adrenomedullin(22-52) , which has no vasoactive or tumor-inducing effects, is a potent antiinflammatory and bone-protective agent in this arthritis model.

[Effect and mechanism of intermedin in acute rat cardiac ischemic injury].

OBJECTIVE: To investigate the effect and potential mechanism of intermedin (IMD) in acute cardiac ischemic injury and to provide a new approach for exploring mechanism of sudden cardiac death. METHODS: Seventy-two healthy male rats were randomly divided into 3 groups: control, ischemic and the IMD-treated group. The activity of lactate dehydrogenase (LDH), malondialdehyde (MDA) and superoxide dismutase (SOD) in heart blood were tested by enzyme chemistry method. The mRNA changes of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMPs) in cardiac were measured by real-time PCR analysis. Myocardial cyclic adenosine monophosphate (cAMP) content was determined by enzyme linked immunosorbent assay (ELISA). Apoptosis related factors Bcl-2 and Bax were detected by immunohistochemistry. RESULTS: Comparing with the control group, LDH and MDA activity of ischemic group in heart blood increased and SOD activity decreased. The concentration of cAMP increased in ventricular muscle, Bcl-2 and Bax proteins expression ratio level decreased. The intravenation of IMD decreased the level of increased activity of LDH and MDA, and lessened the level of decreased activity of SOD. The mRNA expression of CRLR and RAMPs obviously increased in ventricular muscle. CONCLUSION: The protective effect of IMD against myocardial ischemic injury could be caused by decreasing the oxidative stress of ischemia and inhibiting the myocardial apoptosis.