Rat Adrenomedullin Research Articles

Plasma Clearance of Intravenously Infused Adrenomedullin in Rats with Acute Renal Failure.

Plasma adrenomedullin concentrations are reportedly elevated in patients with renal failure; however, the underlying mechanism is unclear. In this study, we investigated the plasma clearance of synthetic human adrenomedullin (AM) in two models of rats with renal dysfunction; one was induced by subcutaneous injection of mercury chloride (RD-Ag) and the other by completely blocking bilateral renal blood flow (RD-Bl). Sixty minutes after starting intravenous AM infusion, AM levels in RD-Ag, RD-Bl, and rats with normal renal function (NF) were still increased slightly; however, plasma AM levels in RD-Ag rats were approximately three times as high as in RD-Bl and NF rats. Plasma AM disappearance after the end of treatment was similar among the three groups. Pharmacokinetic analysis revealed that elevated plasma AM in RD-Ag rats may be caused by a reduced volume of distribution. The adrenomedullin functional receptor is composed of heterodimers, including GPCR, CLR (calcitonin receptor-like receptor, CALCRL), and the single transmembrane proteins, RAMP2 or RAMP3 (receptor activity modifying protein). Calcrl expression was downregulated in the lungs and kidneys of RD-Ag rats. Furthermore, the plasma concentration of exogenous AM was elevated in mice deficient in vascular endothelium-specific Ramp2. These results suggest that decreased plasma AM clearance in RD-Ag is not due to impaired renal excretion but to a decreased volume of distribution caused by a reduction in adrenomedullin receptors.

Plasma clearance of intravenously infused human adrenomedullin in rats with renal dysfunction

Plasma clearance of intravenously infused human adrenomedullin in rats with renal dysfunction

Modulation of osteoclastogenesis through adrenomedullin receptors on osteoclast precursors: initiation of differentiation by asymmetric cell division

Adrenomedullin (ADM), a member of the calcitonin family of peptides, is a potent vasodilator and was shown to have the ability to modulate bone metabolism. We have previously found a unique cell surface antigen (Kat1 antigen) expressed in rat osteoclasts, which is involved in the functional regulation of the calcitonin receptor (CTR). Cross-linking of cell surface Kat1 antigen with anti-Kat1 antigen monoclonal antibody (mAbKat1) stimulated osteoclast formation only under conditions suppressed by calcitonin. Here, we found that ADM provoked a significant stimulation in osteoclastogenesis only in the presence of calcitonin; a similar biological effect was seen with mAbKat1 in the bone marrow culture system. This stimulatory effect on osteoclastogenesis mediated by ADM was abolished by the addition of mAbKat1. 125I-labeled rat ADM (125I-ADM)-binding experiments involving micro-autoradiographic studies demonstrated that mononuclear precursors of osteoclasts abundantly expressed ADM receptors, and the specific binding of 125I-ADM was markedly inhibited by the addition of mAbKat1, suggesting a close relationship between the Kat1 antigen and the functional ADM receptors expressed on cells in the osteoclast lineage. ADM receptors were also detected in the osteoclast progenitor cells in the late mitotic phase, in which only one daughter cell of the dividing cell express ADM receptors, suggesting the semiconservative cell division of the osteoclast progenitors in the initiation of osteoclastogenesis. Messenger RNAs for the receptor activity-modifying-protein 1 (RAMP1) and calcitonin receptor-like receptor (CRLR) were expressed in cells in the osteoclast lineage; however, the expression of RAMP2 or RAMP3 was not detected in these cells. It is suggested that the Kat1 antigen is involved in the functional ADM receptor distinct from the general ADM receptor, consisting of CRLR and RAMP2 or RAMP3. Modulation of osteoclastogenesis through functional ADM receptors abundantly expressed on mononuclear osteoclast precursors is supposed to be important in the fine regulation of osteoclast differentiation in a specific osteotrophic hormonal condition with a high level of calcitonin in blood.Adrenomedullin (ADM) significantly stimulates osteoclastogenesis only in the presence of calcitonin. Functional ADM receptor partly composed of osteoclast-specific cell surface Kat1 antigen was detected in cells in the osteoclast-lineage. Expression of ADM receptor was firstly detected in proliferating osteoclast progenitors performing asymmetric cell division and is then expressed in mononuclear osteoclast precursors. Specific regulation of ADM receptors expressed on mononuclear osteoclast precursors could provide an alternative way to modulate bone remodeling therapeutically.

Modulatory effect of resveratrol and melatonin on natural killer cell activity and adrenomedullin in diabetic rats.

Epidemiological evidence suggests that diabetes poses a high risk for many chronic diseases, especially cardiovascular diseases, and cancer by stimulating many inflammatory and immunological pathogenic mediators and affecting natural killer (NK)-cell activity. In this study, the effects of melatonin and resveratrol on IL-6, TNF-alpha, oxidant/antioxidant capacity, NK-cell activity, and mid-regional proadrenomedullin (MR-proADM) levels of diabetic rats were investigated. In the study, 28 Sprague Dawley rats were randomly divided into the control group (group I) and 3 streptozotocininduced diabetes mellitus (DM) groups (group II, III, and IV), each group consisting of 7 rats. Five mg/kg/day melatonin to group III and 5 mg/kg/day resveratrol (intraperitoneal) to group IV was given. At the end of 3 weeks, NK-cell activity, total antioxidant/oxidant capacity, MR-proADM, IL-6, and TNF-alpha levels were measured in intracardiac blood taken under anesthesia. NK-cell activity of group II was found lower than group I, group III, and group IV (7.4 ± 2.0 vs. 22.5 ± 11.9, 30.6 ± 22.5 and 20.4 ± 9.1 pg/mL; p = 0.0018, respectively). The difference was more prominent in diabetic rats receiving melatonin (p < 0.01). TNF-alpha levels of group II were higher than the group I (p < 0.05). The MR-proADM levels of group II were found to be lower than the group I and group III (6.4 ± 3.6 vs. 14.4 ± 3.2 and 14.0 ± 4.2 ng/L; p < 0.05, respectively). In addition, NK-cell activity was moderately correlated with MR-proADM (r = 0.5618, p = 0.0019).

Expression of adrenomedullin in rats after spinal cord injury and intervention effect of recombinant human erythropoietin.

The expression of adrenomedullin (ADM) in injured tissue of rat spinal cord was observed and the effect of recombinant human erythropoietin was analyzed. A total of 45 Sprague-Dawley rats were selected and divided into 3 equal groups including, a sham-operation group in which rats received an excision of vertebral plate; a spinal cord injury model group and a recombinant human erythropoietin group in which rats with spinal cord injury received a caudal vein injection of 300 units recombinant human erythropoietin after injury. Hematoxylin and eosin staining was performed to observe the spinal cord injury conditions. Immunohistochemical staining was performed to observe the expression of ADM. Pathologic changes in the group of recombinant human erythropoietin at various times were significantly less severe than those in the group of spinal cord injury model. The expression of ADM was increased particularly in the group of recombinant human erythropoietin (P<0.01). The improved Tarlov scores of the group of spinal cord injury model and the group of recombinant human erythropoietin were lower than those of the sham-operation group at 3, 6 and 9 days (P<0.01). Thus, the recombinant human erythropoietin is capable of alleviating the secondary injury of spinal cord. One of the mechanisms may be achieved by promoting the increase of ADM expression.

Bone Marrow Stromal Cells Inhibit the Activation of Liver Cirrhotic Fat-Storing Cells via Adrenomedullin Secretion.

Cirrhosis, or liver fibrosis, which is mainly triggered by cirrhosis fat-storing cells (CFSCs) activation, has traditionally been considered an irreversible disease. However, recent observations indicate that even advanced fibrosis is still reversible by removing the causative agents. Anti-fibrotic effects of bone marrow-derived stromal cells (BMSCs) have been demonstrated by inhibiting CFSCs via cytokines secretion; however, the mechanisms are still unclear. The purpose of this study was to explore the underlying mechanisms by which BMSCs modulate the function of activated CFSCs. After the co-culture of CFSCs with BMSCs supernatants with or without the addition of recombinant rat adrenomedullin (AM)/AM-specific siRNA, western blot analysis was mainly used to detect the differences of relative protein expression on CFSCs. BMSC-secreted adrenomedullin (AM) effectively inhibited the proliferation and activation of CFSCs by suppressing the expression of Ang II and its binding receptor, AT1, which resulted in a reduction of p47-phox formation. Our data suggested that BMSCs inhibited CFSC activation in vitro via the AM-Ang II-p47-phox signaling pathway, and since CFSC activation is an essential part of hepatic fibrosis process, this inhibition by BMSCs implies us new insights into the potential treatment of hepatic fibrosis via BMSCs.

Expression of hypoxia-inducible factor-1α, endothelin-1 and adrenomedullin in newborn rats with hypoxia-induced pulmonary hypertension.

Hypoxia-inducible factor (HIF)-1α is associated with hypoxia-induced pulmonary hypertension (HPH) in adults. In the present study, the expression levels of HIF-1α, endothelin (ET)-1 and adrenomedullin (ADM) were analyzed during HPH in neonates. In total, 96 newborn rats were subjected to hypoxia or normoxia for 3, 5, 7, 10, 14 or 21 days (n=8 per subgroup). HIF-1α, ET-1 and ADM expression levels were measured by quantitative polymerase chain reaction. In addition, the intima-media thickness/external diameter ratio (MT%) and medial wall cross-sectional area/vessel total cross-sectional area ratio (MA%) were calculated to evaluate pulmonary vascular remodeling. The mean pulmonary arterial pressure (mPAP) increased with exposure to hypoxia. Furthermore, the expression levels of HIF-1α, ET-1 and ADM in the lungs were shown to increase after three and five days of hypoxia, while the MT% and MA% increased after seven days of hypoxia, as compared with the controls (P<0.05). Therefore, the expression of HIF-1α, ET-1 and ADM is upregulated in the lungs of newborn rats during early HPH. At later stages, the mPAP increases, vascular remodeling occurs and HIF-1α, ET-1 and ADM expression levels restore to normal levels.

Changes of hypoxia-inducible factor-1α, endothelin-1 and adrenomedullin in newborn rats with hypoxia-induced pulmonary hypertension

Objective To provide the foundation of theory on prevention of hypoxia-induced pulmonary hypertension(HPH) by investigating the concentrations in the serum and expression levels in the lung tissue, and by studying the roles of hypoxia-inducible factor-1α(HIF-1α), endothelin-1(ET-1) and adrenomedullin (ADM) in newborn rats with HPH. Methods Seventy-two newborn rats were randomly divided into hypoxic group(hypoxia exposure) and normoxic group.The mean pulmonary arteria pressure(mPAP), concentrations in the serum and expression levels in the lung tissue of the above-mentioned factors were measured in these neonatal rats after 3, 5, 7, 10, 14 and 21 days, including hypoxic group and normoxic group, respectively.The morphology of pulmonary arterioles was observed and the vascular remodeling indexes were measured. Results 1.Compared with the normoxic group, the mPAP increased with the prolonged hypoxic time in the hypoxic group(P<0.05).2.Concentrations in the serum and expression levels in the lung tissue of HIF-1α were higher than those of the normoxic group on 3, 5, 7, 10, 14 days(all P<0.05). The serum concentration of ET-1 elevated significantly in the hypoxic group at each time point, and ET-1 mRNA increased only on 3 and 5 days of the hypoxia group.The serum concentration of ADM level in the hypoxic group was higher than that of the normoxic group on 3 and 5 days, but lower than the normoxic group after 14 and 21 days of hypoxia, and the expression of ADM mRNA in the lung tissue was higher on 3 and 5 days in the hypoxic group.3.Morphology and ultrastructure of the pulmonary arteries change were found after 7 days of hypoxia exposure. Conclusions HIF-1α may regulate ET-1 and ADM in the pathogenesis of HPH.ET-1 plays a powerful role by pulmonary vasoconstriction and vascular remodeling, whereas ADM displays the protective effects on blood vessels in the early stages of HPH.Pulmonary vascular remodeling occurs in the hypoxic group after 7 days of hypoxia. Key words: Hypoxia-inducible factor-1α; Endothelin-1; Adrenomedullin; Hypertension; Rat

Pharmacological characterization of rat amylin receptors: implications for the identification of amylin receptor subtypes

Amylin (Amy) is an important glucoregulatory peptide and AMY receptors are clinical targets for diabetes and obesity. Human (h) AMY receptor subtypes are complexes of the calcitonin (CT) receptor with receptor activity-modifying proteins (RAMPs); their rodent counterparts have not been characterized. To allow identification of the most clinically relevant receptor subtype, the elucidation of rat (r) AMY receptor pharmacology is necessary. Receptors were transiently transfected into COS-7 cells and cAMP responses measured in response to different agonists, with or without antagonists. Competition binding experiments were performed to determine rAmy affinity. rCT was the most potent agonist of rCT((a)) receptors, whereas rAmy was most potent at rAMY(1(a)) and rAMY(3(a)) receptors. rAmy bound to these receptors with high affinity. Rat α-calcitonin gene-related peptide (CGRP) was equipotent to rAmy at both AMY receptors. Rat adrenomedullin (AM) and rAM2/intermedin activated all three receptors but were most effective at rAMY(3(a)) . AC187, AC413 and sCT(8-32) were potent antagonists at all three receptors. rαCGRP(8-37) displayed selectivity for rAMY receptors over rCT((a)) receptors. rAMY(8-37) was a weak antagonist but was more effective at rAMY(1(a)) than rAMY(3(a)) . AMY receptors were generated by co-expression of rCT((a)) with rRAMP1 or 3, forming rAMY(1(a)) and rAMY(3(a)) receptors, respectively. CGRP was more potent at rAMY than at hAMY receptors. No antagonist tested was able to differentiate the rAMY receptor subtypes. The data emphasize the need for and provide a useful resource for developing new CT or AMY receptor ligands as pharmacological tools or potential clinical candidates.

1000 INTRINSIC PEPTIDES MEDIATES ADIPOSE TISSUE-DERIVED STEM CELL-INDUCED RESTORATION OF ERECTILE FUNCTION IN DIABETES RATS

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research II1 Apr 20121000 INTRINSIC PEPTIDES MEDIATES ADIPOSE TISSUE-DERIVED STEM CELL-INDUCED RESTORATION OF ERECTILE FUNCTION IN DIABETES RATS Hiroaki Nishimatsu, Etsu Suzuki, Shintaro Kumano, Akira Nomiya, Motofumi Suzuki, Tetsuya Fujimura, Hiroshi Fukuhara, Yutaka Enomoto, Akira Ishikawa, Haruki Kume, Yasuhiko Igawa, and Yukio Homma Hiroaki NishimatsuHiroaki Nishimatsu Tokyo, Japan More articles by this author , Etsu SuzukiEtsu Suzuki Kawasaki, Japan More articles by this author , Shintaro KumanoShintaro Kumano Tokyo, Japan More articles by this author , Akira NomiyaAkira Nomiya Tokyo, Japan More articles by this author , Motofumi SuzukiMotofumi Suzuki Tokyo, Japan More articles by this author , Tetsuya FujimuraTetsuya Fujimura Tokyo, Japan More articles by this author , Hiroshi FukuharaHiroshi Fukuhara Tokyo, Japan More articles by this author , Yutaka EnomotoYutaka Enomoto Tokyo, Japan More articles by this author , Akira IshikawaAkira Ishikawa Tokyo, Japan More articles by this author , Haruki KumeHaruki Kume Tokyo, Japan More articles by this author , Yasuhiko IgawaYasuhiko Igawa Tokyo, Japan More articles by this author , and Yukio HommaYukio Homma Tokyo, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.1102AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Diabetic patients are more refractory to the treatment with phosphodiesterase type 5 inhibitors (PDE-5I) because of severe damage in their cavernous body. We have previously reported that acute administration of Adrenomedullin (AM) into the cavernous body enhances erectile function in rats. During the screening of cytokines that adipose tissue-derived stem cells (ASC) produce, we found that ASC produced AM especially when ASC were cultured in medium containing growth factors for vascular endothelial cells (VEC). In this study, we examined whether AM would be implicated in ASC-induced restoration of erectile function. METHODS ASC isolated from the subcutaneous adipose tissue of a Wister rat was cultured for one week in either EBM or EGM medium which contains vascular endothelial cell growth factor such as VEGF-A, IGF with 5% FBS. Streptozotocin (STZ) was injected into a male Wister rat. Stem cells cultured using the two methods above were injected into the corpus spongiosum penis of the model rat and a normal rat in the ninth week after STZ administration. The mean arterial blood pressure (MAP) and the intracavernosal pressure (ICP) were measured. We used lentivirus(LV) expressing rat AM siRNA and AdAM, and examined whether AM produced by ASC played pivotal roles in ASC-induced restoration of erectile function. RESULTS 1. ASC administration restores ICP; Administration of ASC significantly increased ICP/MAP and AUC. 2. ASC administration restores the structure of cavernous body; When ASC were injected, the trabeculae of cavernous body became larger and were distributed more densely. 3. ASC produce AM; The ASC secreted AM in the culture medium in a time-dependent fashion. When ASC were infected with LV that expresses AM siRNA and cultured for 1 week, AM secretion by ASC was significantly suppressed. 4. Effects of AM knockdown and forced expression of AM; ASC were infected with LV expressing AM siRNA (LV_AM siRNA) or its negtive control NC siRNA (LV_NC siRNA), cultured for 1 week, and injected in the cavernous body. When ASC were infected with LV_NC siRNA, ASC significantly restored erectile function, as assessed by ICP/MAP and AUC. In contrast, when ASC were infected with LV_AM siRNA, ASC-induced restoration of ICP/MAP and AUC was significantly diminished. CONCLUSIONS These results suggested that ASC have potentials to restore erectile function and that AM produced by ASC when cultured especially in EGM plays a major role in the restoration of erectile function. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e407 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hiroaki Nishimatsu Tokyo, Japan More articles by this author Etsu Suzuki Kawasaki, Japan More articles by this author Shintaro Kumano Tokyo, Japan More articles by this author Akira Nomiya Tokyo, Japan More articles by this author Motofumi Suzuki Tokyo, Japan More articles by this author Tetsuya Fujimura Tokyo, Japan More articles by this author Hiroshi Fukuhara Tokyo, Japan More articles by this author Yutaka Enomoto Tokyo, Japan More articles by this author Akira Ishikawa Tokyo, Japan More articles by this author Haruki Kume Tokyo, Japan More articles by this author Yasuhiko Igawa Tokyo, Japan More articles by this author Yukio Homma Tokyo, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Effect and mechanism of adrenomedullin on apoptosis of renal tubular epithelial cell in rats induced by renal ischemia reperfusion injury

Objective To investigate the effect and mechanism of adrenomedullin (AM) on apoptosis of renal tubular epithelial cell in rats induced by renal ischemia reperfusion injury. Methods Thirty-two Wistar rats were randomly divided into 4 groups: control group, IRI group, empty plasmid group and AM group. One week after removing the right kidney, eukaryotic expression vector encoding rat AM gene was transfected into the left kidney using an ultrasound-microbubble mediated system. After 1 week the transfer efficiency was detected by immunohistochemical method . Renal IRI model induced by clamping left renal arteries for 45 min followed by reperfusion for 24 h. Tubular cell apoptosis was detected by TUNEL assay. Bcl-2, Bax and Fas expressions were examined by RT-PCR. The expressions of caspase-3, caspase-8 and caspase-9 were determined by Western bolt analysis. Results The expression of AM in the AM group was significantly higher than the empty plasmid group (0.51±0.09 vs 0.23±0.05; P 0.05). Conclusion AM can reduce apoptosis of renal tubular epithelial cell induced by renal IRI, the mechanism of which might be achieved by inhibiting caspase-dependent intrinsic and extrinsic pathways. Key words: Adrenomedullin; Reperfusion injury; Apoptosis; Caspase

Attenuation of Renal Ischemia and Reperfusion Injury by Human Adrenomedullin and its Binding Protein

Acute renal failure secondary to ischemia and reperfusion (I/R) injury poses a significant burden on both surgeons and patients. It carries a high morbidity and mortality rate and no specific treatment currently exists. Major causes of renal I/R injury include trauma, sepsis, hypoperfusion, and various surgical procedures. We have demonstrated that adrenomedullin (AM), a novel vasoactive peptide, combined with AM binding protein-1 (AMBP-1), which augments the activity of AM, is beneficial in various disease conditions. However, it remains unknown whether human AM/AMBP-1 provides any beneficial effects in renal I/R injury. The objective of our study therefore was to determine whether administration of human AM/AMBP-1 can prevent and/or minimize damage in a rat model of renal I/R injury. Male adult rats were subjected to renal I/R injury by bilateral renal pedicle clamping with microvascular clips for 60 min followed by reperfusion. Human AM (12 microg/kg BW) and human AMBP-1 (40 microg/kg BW) or vehicle (52 microg/kg BW human albumin) were given intravenously over 30 min immediately following the clip removal (i.e., reperfusion). Rats were allowed to recover for 24 h post-treatment, and blood and renal tissue samples were collected. Plasma levels of AM were measured using a radioimmunoassay specific for rat AM. Plasma AMBP-1 was measured by Western analysis. Renal water content and serum levels of systemic markers of tissue injury were measured. Serum and renal TNF-alpha levels were also assessed. At 24 h after renal I/R injury, plasma levels of AM were significantly increased while plasma AMBP-1 was markedly decreased. Renal water content and systemic markers of tissue injury (e.g., creatinine, BUN, AST, and ALT) were significantly increased following renal I/R injury. Serum and renal TNF-alpha levels were also increased post injury. Administration of human AM/AMBP-1 decreased renal water content, and plasma levels of creatinine, BUN, AST, and ALT. Serum and renal TNF-alpha levels were also significantly decreased after AM/AMBP-1 treatment. Treatment with human AM/AMBP-1 in renal I/R injury significantly attenuated organ injury and the inflammatory response. Thus, human AM combined with human AMBP-1 may be developed as a novel treatment for patients with acute renal I/R injury.

Mechanisms involved in the regional haemodynamic effects of intermedin (adrenomedullin 2) compared with adrenomedullin in conscious rats

Intermedin (IMD) is a newly identified member of the calcitonin family of peptides that shares structural and functional homology with adrenomedullin (AM). In vivo cardiovascular effects of AM have been described, but relatively little is known of the in vivo actions of IMD. The purpose of this study was to compare the regional haemodynamic effects of IMD with those of AM in conscious rats, and investigate possible underlying mechanisms. Measurements of blood pressure, heart rate and renal, mesenteric and hindquarters haemodynamics were made in conscious, chronically-instrumented rats. IMD caused tachycardia and vasodilatation in all three vascular beds, associated with modest hypotension. At an equimolar dose (1 nmol.kg(-1)), most of the cardiovascular effects of IMD were greater than those of AM. The AM receptor antagonist, AM(22-52), was equally effective in attenuating the renal and mesenteric vasodilator effects of IMD (1 nmol.kg(-1)) and AM (3 nmol.kg(-1)), but inhibition of NO synthase was more effective at reducing the vasodilator effects of IMD than AM. Vascular K(ATP) channel blockade with U-37883A did not inhibit the vasodilator effects of either peptide. In vivo, the regional haemodynamic profile of IMD resembles that of AM, and some of the vasodilator effects of IMD are mediated by AM receptors and NO, but not by K(ATP) channels. The cardiovascular effects of AM have been implicated in various pathological conditions, but whether or not endogenous IMD fulfils a similar role remains to be determined.

Adrenomedullin and Oviduct Function in Human and Rats.

Adrenomedullin and Oviduct Function in Human and Rats.

Reversing Established Sepsis in Rats with Human Vasoactive Hormone Adrenomedullin and its Binding Protein

We recently demonstrated that early administration of rat adrenomedullin (AM), a vasoactive peptide, in combination with its binding protein (human AMBP-1) produces various beneficial effects in sepsis. Human AM is a 52-amino acid peptide, but rat AM differs from human AM, having only 50 amino acid residues, with two amino acid deletions and six substitutions. It remains unknown whether a combination of human AM and human AMBP-1 (AM/AMBP-1) is also beneficial in sepsis and, if so, whether human AM/AMBP-1 reverses established sepsis in rats. To test the effects of human AM/AMBP-1, we induced sepsis in male adult rats by cecal ligation and puncture (CLP). At 10 h after CLP (i.e., severe sepsis), human AM (12-48 microg/kg body weight) was administered in combination with human AMBP-1 (40-160 microg/kg body weight). Vehicle-treated animals received a nonspecific human plasma protein (albumin). Blood and intestinal samples were collected at 20 h for various measurements. In additional groups of septic animals, the gangrenous cecum was surgically excised at 20 h after CLP. The 10-day survival was recorded. Our results showed that tissue injury, as evidenced by increased levels of transaminases and lactate, was present at 20 h after CLP. Proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6 were significantly elevated. Gut barrier dysfunction, manifested by increased mucosal permeability to hydrophilic macromolecules and increased bacterial translocation to mesenteric lymph nodes, also occurred at 20 h after CLP. Administration of human AM/AMBP-1 in established sepsis markedly attenuated tissue injury, reduced proinflammatory cytokine levels, ameliorated intestinal-barrier dysfunction, and improved the survival rate from 47% to 67%-80%. Thus, human AM/AMBP-1 can be further developed as a safe and effective therapy for patients with established sepsis.

Human Adrenomedullin Up-regulates Interleukin-13 Receptor α2 Chain in Prostate CancerIn vitroandIn vivo: A Novel Approach to Sensitize Prostate Cancer to Anticancer Therapy

Interleukin-13 (IL-13) receptor alpha2 (IL-13Ralpha2), a high-affinity IL-13 binding subunit and a tumor antigen, is amplified in a variety of human tumor cell lines and tumors in vivo. By cDNA microarray, we have shown that gene transfer of human and rat adrenomedullin (AM) up-regulates IL-13Ralpha2 in a human prostate tumor cell line. Here, we show that IL-13Ralpha2 mRNA and protein are also up-regulated in PC-3 prostate tumor cells by recombinant AM (rAM) and human synthetic AM peptide in a dose-dependent manner in vitro and in vivo in mouse prostate tumor model. The 8- to 10-fold up-regulation of IL-13Ralpha2 by rAM or AM peptide in prostate tumor cells in vitro and in vivo increased their sensitivity to IL-13PE cytotoxin consisting of IL-13 and a truncated form of Pseudomonas exotoxin. Immunodeficient mice with established prostate tumors transfected with AM or treated with AM peptide showed reduction in tumor size by intratumoral administration of IL-13PE in a dose-dependent manner. At the highest dose (three 100 mug/kg/d every alternate day), >70% reduction of tumor size was observed compared with controls (P <or= 0.01). These results indicate that two completely unrelated hormones (AM and IL-13) are closely related to each other and that we have identified a novel role of AM in sensitizing certain types of prostate tumors to IL-13R-directed therapeutic agent.

Human adrenomedullin combined with human adrenomedullin binding protein-1 is protective in gut ischemia and reperfusion injury in the rat

Previous studies have demonstrated that co-administration of rat adrenomedullin (AM) and human AM binding protein-1 (AMBP-1) has various beneficial effects following adverse circulatory conditions. In order to reduce rat proteins to elicit possible immune responses in humans, we determined the effect of human AM combined with human AMBP-1 after intestinal ischemia and reperfusion (I/R). Intestinal ischemia was induced in the rat by occluding the superior mesenteric artery for 90 min. At 60 min after the beginning of reperfusion, human AM/AMBP-1 at 3 different dosages was administered intravenously over 30 min. At 240 min after the treatment, blood and tissue samples were harvested and measured for pro-inflammatory cytokines (i.e., TNF-α and IL-6), myeloperoxidase activities in the gut and lungs, and cleaved caspase-3 expression in the lungs, as well as serum levels of hepatic enzymes and lactate. In additional groups of animals, a 10-day survival study was conducted. Results showed that administration of human AM/AMBP-1 reduced pro-inflammatory cytokines, attenuated organ injury, and improved the survival rate in a seemingly dose–response fashion. Co-administration of the highest dose of human AM/AMBP-1 in this study had the optimal therapeutic effect in the rat model of intestinal I/R.

Coexpression of Adrenomedullin and Its Receptors in the Reproductive System of the Rat: Effects on Steroid Secretion in Rat Ovary1

The present study demonstrates the expression of adrenomedullin (ADM) in the reproductive system of the female rat and its effect on the secretion of estradiol and progesterone. Ovarian ADM and Adm mRNA levels were decreased at estrus, whereas oviductal Adm mRNA levels were low at proestrus. Both tissues were shown to coexpress mRNAs encoding the calcitonin receptor-like receptor and receptor activity-modifying protein 1 (Ramp1), Ramp2, and Ramp3. Gel filtration chromatography of ovarian extracts showed two peaks, with the predominant one eluting at the position of authentic rat ADM (1-50) at estrus and at the position of ADM precursor at diestrus. Positive ADM immunostaining was localized in the granulosa and theca cells of the follicle and corpora lutea of the ovary. Adrenomedullin inhibited FSH-induced estradiol secretion in 2-day-old follicles and also suppressed eCG-stimulated progesterone release in corpora lutea. The inhibitory effect of ADM on the follicles and the corpora lutea was abolished by calcitonin gene-related peptide (8-37) and ADM (22-52), respectively. The presence of ADM and the gene expression of ADM and its receptor components in the female reproductive system suggest a paracrine effect of ADM on ovarian steroidogenesis.

Human vasoactive hormone adrenomedullin and its binding protein rescue experimental animals from shock

We recently discovered that vascular responsiveness to adrenomedullin (AM), a vasoactive hormone, decreases after hemorrhage, which is markedly improved by the addition of its binding protein AMBP-1. One obstacle hampering the development of AM/AMBP-1 as resuscitation agents in trauma victims is the potential immunogenicity of rat proteins in humans. Although less potent than rat AM, human AM has been shown to increase organ perfusion in rats. We therefore hypothesized that administration of human AM/AMBP-1 improves organ function and survival after severe blood loss in rats. To test this, male Sprague-Dawley rats were bled to and maintained at an MAP of 40 mmHg for 90 min. They were then resuscitated with an equal volume of shed blood in the form of Ringer's lactate (i.e., low-volume resuscitation) over 60 min. At 15 min after the beginning of resuscitation, human AM/AMBP-1 (12/40 or 48/160 microg/kg BW) were administered intravenously over 45 min. Various pathophysiological parameters were measured 4h after resuscitation. In additional groups of animals, a 12-day survival study was conducted. Our result showed that tissue injury as evidenced by increased levels of transaminases, lactate, and creatinine, was present at 4h after hemorrhage and resuscitation. Moreover, pro-inflammatory cytokines TNF-alpha and IL-6 were also significantly elevated. Administration of AM/AMBP-1 markedly attenuated tissue injury, reduced cytokine levels, and improved the survival rate from 29% (vehicle) to 62% (low-dose) or 70% (high-dose). However, neither human AM alone nor human AMBP-1 alone prevented the significant increase in ALT, AST, lactate and creatinine at 4h after the completion of hemorrhage and resuscitation. Moreover, the half-life of human AM and human AMBP-1 in rats was 35.8 min and 1.68 h, respectively. Thus, administration of human AM/AMBP-1 may be a useful approach for attenuating organ injury, and reducing mortality after hemorrhagic shock.

AMELIORATION OF CARDIAC FUNCTION IN CHRONIC MYOCARDIAL INFARCTED RATS FOLLOWING ADMINISTRATION OF VECTOR pcDNA3.1AM

1. The present study was designed to examine the cardiovascular effects of intravenously administered pcDNA3.1AM, a recombinant non-virus vector carrying a rat adrenomedullin (AM) gene translation fragment, in rats with chronic cardiac dysfunction induced by ligation of the left descending coronary artery. 2. Haemodynamic parameters were recorded by intraventricular catheterization. In situ hybridization and polymerase chain reaction (PCR) were performed to identify the distribution of the introduced vector. The concentration of AM was determined by radioimmunoassay. 3. Progressive cardiac dysfunction was observed following coronary artery ligation, as indicated by a significant reduction in mean arterial pressure (MAP) and increases in both central venous pressure (CVP) and end-diastolic pressure of the left ventricle (LVEDP; P < 0.01). Administration of pcDNA3.1AM significantly attenuated the progressive cardiac dysfunction and lowered the elevated CVP and LVEDP. The introduced vector was widely distributed in different organs, including the lungs, kidney, heart, liver, spleen and brain. However, intense staining of pcDNA3.1 AM was observed in the lungs and kidneys. The introduced vector was localized mainly in the endothelial cells of blood vessels. Radioimmunoassay showed elevated levels of AM in the plasma and lung and heart after surgery, but there was no significant further increase in the concentration of AM after pcDNA3.1AM delivery. 4. The present study has provided some novel findings on the potential beneficial effects of AM gene delivery on chronic cardiac function in rats. Expression of AM by a non-virus vector may also have therapeutic value against cardiac dysfunction in vivo.