Receptor activity-modifying proteins of adrenomedullin (RAMP2/3): Roles in the pathogenesis of ARDS.

Acute respiratory distress syndrome (ARDS) is a life-threatening lung condition characterized by widespread inflammation and pulmonary edema. Adrenomedullin (AM), a bioactive peptide with various functions, is expected to be applied in treating ARDS. Its functions are regulated primarily by two receptor activity-modifying proteins, RAMP2 and RAMP3, which bind to the AM receptor calcitonin receptor-like receptor (CLR). However, the roles of RAMP2 and RAMP3 in ARDS remain unclear. We generated a mouse model of ARDS via intratracheal administration of lipopolysaccharide (LPS), and analyzed the pathophysiological significance of RAMP2 and RAMP3. RAMP2 expression declined with LPS administration, whereas RAMP3 expression increased at low doses and decreased at high doses of LPS. After LPS administration, drug-inducible vascular endothelial cell-specific RAMP2 knockout mice (DI-E-RAMP2-/-) showed reduced survival, increased lung weight, and had more apoptotic cells in the lungs. DI-E-RAMP2-/- mice exhibited reduced expression of Epac1 (which regulates vascular endothelial cell barrier function), while RAMP3 was upregulated in compensation. In contrast, after LPS administration, RAMP3-/- mice showed no significant changes in survival, lung weight, or lung pathology, although they exhibited significant downregulation of iNOS, TNF-α, and NLRP3 during the later stages of inflammation. Based on transcriptomic analysis, RAMP2 contributed more to the circulation-regulating effects of AM, whereas RAMP3 contributed more to its inflammation-regulating effects. These findings indicate that, while both RAMP2 and RAMP3 participate in ARDS pathogenesis, their functions differ distinctly. Further elucidation of the pathophysiological significance and functional differences between RAMP2 and RAMP3 is critical for the future therapeutic application of AM in ARDS.

Rational design of highly stabilized and selective adrenomedullin analogs.

The peptide hormone adrenomedullin (ADM) consists of 52 amino acids with a disulfide bond and an amidated C-terminus. Due to the vasodilatory and cardioprotective effects, the agonistic activity of the peptide on the adrenomedullin 1 receptor (AM1 R) is of high pharmacological interest. However, the wild-type peptide shows low metabolic stability leading to rapid degradation in the cardiovascular system. Previous work by our group has identified proteolytic cleavage sites and demonstrated stabilization of ADM by lipidation, cyclization, and N-methylation. Nevertheless, these ADM analogs showed reduced activity and subtype selectivity toward the closely related calcitonin gene-related peptide receptor (CGRPR). Here, we report on the rational development of ADM derivatives with increased proteolytic stability and high receptor selectivity. Stabilizing motifs, including lactamization and lipidation, were evaluated regarding AM1 R and CGRPR activation. Furthermore, the central DKDK motif of the peptide was replaced by oligoethylene glycol linkers. The modified peptides were synthesized by Fmoc/t-Bu solid-phase peptide synthesis and receptor activation of AM1 R and CGRPR was measured by cAMP reporter gene assay. Peptide stability was tested in human blood plasma and porcine liver homogenate and analyzed by RP-HPLC and MALDI-ToF mass spectrometry. Combination of the favorable lactam, lipidation, ethylene glycol linker, and previously described disulfide mimetic resulted in highly stabilized analogs with a plasma half-life of more than 144 h. The compounds display excellent AM1 R activity and wild-type-like selectivity toward CGRPR. Additionally, dose-dependent vasodilatory effects of the ADM derivatives lasted for several hours in rodents. Thus, we successfully developed an ADM analog with long-term in vivo activity.

Reassessing the adrenomedullin scavenging function of ACKR3 in lymphatic endothelial cells.

Atypical chemokine receptor 3 (ACKR3) is a scavenger of the chemokines CXCL11 and CXCL12 and of several opioid peptides. Additional evidence indicates that ACKR3 binds two other non-chemokine ligands, namely the peptide hormone adrenomedullin (AM) and derivatives of the proadrenomedullin N-terminal 20 peptide (PAMP). AM exhibits multiple functions in the cardiovascular system and is essential for embryonic lymphangiogenesis in mice. Interestingly, AM-overexpressing and ACKR3-deficient mouse embryos both display lymphatic hyperplasia. Moreover, in vitro evidence suggested that lymphatic endothelial cells (LECs), which express ACKR3, scavenge AM and thereby reduce AM-induced lymphangiogenic responses. Together, these observations have led to the conclusion that ACKR3-mediated AM scavenging by LECs serves to prevent overshooting AM-induced lymphangiogenesis and lymphatic hyperplasia. Here, we further investigated AM scavenging by ACKR3 in HEK293 cells and in human primary dermal LECs obtained from three different sources in vitro. LECs efficiently bound and scavenged fluorescent CXCL12 or a CXCL11/12 chimeric chemokine in an ACKR3-dependent manner. Conversely, addition of AM induced LEC proliferation but AM internalization was found to be independent of ACKR3. Similarly, ectopic expression of ACKR3 in HEK293 cells did not result in AM internalization, but the latter was avidly induced upon co-transfecting HEK293 cells with the canonical AM receptors, consisting of calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying protein (RAMP)2 or RAMP3. Together, these findings indicate that ACKR3-dependent scavenging of AM by human LECs does not occur at ligand concentrations sufficient to trigger AM-induced responses mediated by canonical AM receptors.

Adrenomedullin 2/intermedin is a slow off-rate, long-acting endogenous agonist of the adrenomedullin2 G protein-coupled receptor.

Adrenomedullin 2/intermedin (AM2/IMD), adrenomedullin (AM), and calcitonin gene-related peptide (CGRP) have functions in the cardiovascular, lymphatic, and nervous systems by activating three heterodimeric receptors comprising the class B GPCR CLR and a RAMP1, -2, or -3 modulatory subunit. CGRP and AM prefer the RAMP1 and RAMP2/3 complexes, respectively, whereas AM2/IMD is thought to be relatively nonselective. Accordingly, AM2/IMD exhibits overlapping actions with CGRP and AM, so the rationale for this third agonist for the CLR-RAMP complexes is unclear. Here, we report that AM2/IMD is kinetically selective for CLR-RAMP3, known as the AM2R, and we define the structural basis for its distinct kinetics. In live cell biosensor assays, AM2/IMD-AM2R elicited longer-duration cAMP signaling than the other peptide-receptor combinations. AM2/IMD and AM bound the AM2R with similar equilibrium affinities, but AM2/IMD had a slower off-rate and longer receptor residence time, thus explaining its prolonged signaling capacity. Peptide and receptor chimeras and mutagenesis were used to map the regions responsible for the distinct binding and signaling kinetics to the AM2/IMD mid-region and the RAMP3 extracellular domain (ECD). Molecular dynamics simulations revealed how the former forms stable interactions at the CLR ECD-transmembrane domain interface and how the latter augments the CLR ECD binding pocket to anchor the AM2/IMD C terminus. These strong binding components only combine in the AM2R. Our findings uncover AM2/IMD-AM2R as a cognate pair with unique temporal features, reveal how AM2/IMD and RAMP3 collaborate to shape CLR signaling, and have significant implications for AM2/IMD biology.

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.

Targeting the adrenomedullin-2 receptor for the discovery and development of novel anti-cancer agents.

INTRODUCTION: Adrenomedullin (AM) is a peptide responsible for many physiological processes including vascular health and hormone regulation. Dysregulation of AM signaling can stimulate cancers by promoting proliferation, angiogenesis and metastasis. Two AM receptors contribute to tumor progression in different ways. Adrenomedullin-1 receptor (AM1R) regulates blood pressure and blocking AM signaling via AM1R would be clinically unacceptable. Therefore, antagonizing adrenomedullin-2 receptor (AM2R) presents as an avenue for anti-cancer drug development. AREAS COVERED: We review the literature to highlight AM's role in cancer as well as delineating the specific roles AM1R and AM2R mediate in the development of a pro-tumoral microenvironment. We highlight the importance of exploring the residue differences between the receptors that led to the development of first-in-class selective AM2R small molecule antagonists. We also summarize the current approaches targeting AM and its receptors, their anti-tumor effects and their limitations. EXPERT OPINION: As tool compounds, AM2R antagonists will allow the dissection of the functions of CGRPR (calcitonin gene-related peptide receptor), AM1R and AM2R, and has considerable potential as a first-in-class oncology therapy. Furthermore, the lack of detectable side effects and good drug-like pharmacokinetic properties of these AM2R antagonists support the promise of this class of compounds as potential anti-cancer therapeutics.

Adrenomedullin and its receptors are expressed in mouse pancreatic ß-cells and suppresses insulin synthesis and secretion.

Gestational diabetes mellitus (GDM) is associated with defective pancreatic ß-cell adaptation in pregnancy, but the underlying mechanism remains obscure. Our previous studies demonstrated that GDM women display increased plasma adrenomedullin (ADM) levels, and non-obese GDM mice show decreased serum concentrations of insulin and the number of ß-cells in pancreas islets. The aims of this study is to examine if ADM and its receptors are expressed in female mouse pancreas, and if so, whether insulin secretion is regulated by ADM in mouse ß-cell line, NIT-1 cells and isolated mouse pancreatic islets. Present study shows that ADM and its receptor components CRLR, RAMPs are present in mouse pancreatic islets and co-localized with insulin. The expressions of ADM, CRLR and RAMP2 in islets from pregnant mice are reduced compared to that of non-pregnant mice. NIT-1-ß cells express ADM and its receptor mRNA, and glucose dose-dependently stimulates expressions. Furthermore, ADM inhibits NIT-1-ß cell growth, and this inhibition is reversed by ADM antagonist, ADM22-52. The glucose-induced insulin secretion was suppressed by ADM in NIT-1-ß cells and isolated pancreatic islets from pregnant mice. These inhibitory effects are accompanied by upregulation of endoplasmic reticulum (ER) stress biomarker genes in NIT-1-ß cells. This study unveils that reduced ADM and its receptors may play a role in ß-cell adaptation during pregnancy, while increased plasma ADM in GDM may contribute to the ß-cells dysfunction, and blockade of ADM may reverse ß-cell insulin production.

Peptide ligand interaction with maltose-binding protein tagged to the calcitonin gene-related peptide receptor: The inhibitory role of receptor N-glycosylation.

Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are peptide hormones and their receptors play a critical role in migraine progression and blood pressure control, respectively. CGRP and AM receptors are structurally related since they are the complex of the calcitonin receptor-like receptor (CLR) with the different types of receptor activity-modifying protein (RAMP). Several crystal structures of the CGRP and AM receptor extracellular domain (ECD) used maltose-binding protein (MBP) as a tag protein to facilitate crystallization. Unexpectedly, the recent crystal structures of CGRP receptor ECD showed that the N-terminal tag MBP located in proximity of bound/mutated peptide ligands. This study provided evidence that MBP N-terminally tagged to the CGRP receptor ECD formed chemical interaction with the mutated peptide ligands. Interestingly, N-glycosylation of the CGRP receptor ECD was predicted to prevent MBP docking to the mutated peptide ligands. I found that the N-glycosylation of CLR ECD N123 was the most critical for inhibiting MBP interaction with the mutated peptide ligands. The MBP tag protein interaction was also dependent on the sequence of the peptide ligands. In contrast to the CGRP receptor, the MBP tag was not involved in peptide ligand binding at AM receptor ECD. Here, I provided evidence that N-glycosylation of the CGRP receptor ECD inhibited the tag protein interaction suggesting an additional function of N-glycosylation in the MBP-fused CGRP receptor ECD. This study reveals the importance of using tag protein-free versions of the CGRP receptor for the accurate assessment of peptide binding affinity.

Pharmacological characterisation of mouse calcitonin and calcitonin receptor-like receptors reveals differences compared with human receptors.

BACKGROUND AND PURPOSE: The calcitonin (CT) receptor family is complex, comprising two receptors (the CT receptor [CTR] and the CTR-like receptor [CLR]), three accessory proteins (RAMPs) and multiple endogenous peptides. This family contains several important drug targets, including CGRP, which is targeted by migraine therapeutics. The pharmacology of this receptor family is poorly characterised in species other than rats and humans. To facilitate understanding of translational and preclinical data, we need to know the receptor pharmacology of this family in mice. EXPERIMENTAL APPROACH: Plasmids encoding mouse CLR/CTR and RAMPs were transiently transfected into Cos-7 cells. cAMP production was measured in response to agonists in the absence or presence of antagonists. KEY RESULTS: We report the first synthesis and characterisation of mouse adrenomedullin, adrenomedullin 2 and ßCGRP and of mouse CTR without or with mouse RAMPs. Receptors containing m-CTR had subtly different pharmacology than human receptors; they were promiscuous in their pharmacology, both with and without RAMPs. Several peptides, including mouse αCGRP and mouse adrenomedullin 2, were potent agonists of the m-CTR:m-RAMP3 complex. Pharmacological profiles of receptors comprising m-CLR:m-RAMPs were generally similar to those of their human counterparts, albeit with reduced specificity. CONCLUSION AND IMPLICATIONS: Mouse receptor pharmacology differed from that in humans, with mouse receptors displaying reduced discrimination between ligands. This creates challenges for interpreting which receptor may underlie an effect in preclinical models and thus translation of findings from mice to humans. It also highlights the need for new ligands to differentiate between these complexes. LINKED ARTICLES: This article is part of a themed issue on Advances in Migraine and Headache Therapy (BJP 75th Anniversary).. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.3/issuetoc.

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.

CGRP, adrenomedullin and adrenomedullin 2 display endogenous GPCR agonist bias in primary human cardiovascular cells.

Agonist bias occurs when different ligands produce distinct signalling outputs when acting at the same receptor. However, its physiological relevance is not always clear. Using primary human cells and gene editing techniques, we demonstrate endogenous agonist bias with physiological consequences for the calcitonin receptor-like receptor, CLR. By switching the receptor-activity modifying protein (RAMP) associated with CLR we can "re-route" the physiological pathways activated by endogenous agonists calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and adrenomedullin 2 (AM2). AM2 promotes calcium-mediated nitric oxide signalling whereas CGRP and AM show pro-proliferative effects in cardiovascular cells, thus providing a rationale for the expression of the three peptides. CLR-based agonist bias occurs naturally in human cells and has a fundamental purpose for its existence. We anticipate this will be a starting point for more studies into RAMP function in native environments and their importance in endogenous GPCR signalling.

Temporal and spatial expression of adrenomedullin and its receptors in the porcine uterus and peri-implantation conceptuses.

Adrenomedullin (ADM) is an evolutionarily conserved multifunctional peptide hormone that regulates implantation, embryo spacing, and placentation in humans and rodents. However, the potential roles of ADM in implantation and placentation in pigs, as a litter-bearing species, are not known. This study determined abundances of ADM in uterine luminal fluid, and the patterns of expression of ADM and its receptor components (CALCRL, RAMP2, RAMP3, and ACKR3) in uteri from cyclic and pregnant gilts, as well as conceptuses (embryonic/fetus and its extra-embryonic membranes) during the peri-implantation period of pregnancy. Total recoverable ADM was greater in the uterine fluid of pregnant compared with cyclic gilts between Days 10 and 16 post-estrus and was from uterine luminal epithelial (LE) and conceptus trophectoderm (Tr) cells. Uterine expression of CALCRL, RAMP2, and ACKR3 were affected by day (P < 0.05), pregnant status (P < 0.01) and/or day x status (P < 0.05). Within porcine conceptuses, the expression of CALCRL, RAMP2, and ACKR3 increased between Days 10 and 16 of pregnancy. Using an established porcine trophectoderm (pTr1) cell line, it was determined that 10-7 M ADM stimulated proliferation of pTr1 cells (P < 0.05) at 48 h, and increased phosphorylated mechanistic target of rapamycin (p-MTOR) and 4E binding protein 1 (p-4EBP1) by 6.1- and 4.9-fold (P < 0.0001), respectively. These novel results indicate a significant role for ADM in uterine receptivity for implantation and conceptus growth and development in pigs. They also provide a framework for future studies of ADM signaling to affect proliferation and migration of Tr cells, spacing of blastocysts, implantation, and placentation in pigs.

Development of a novel human adrenomedullin derivative: human serum albumin-conjugated adrenomedullin.

Adrenomedullin is a biologically active peptide with multiple functions. Here, we have developed a novel human serum albumin-adrenomedullin (HSA-AM) conjugate, which was synthesized by the covalent attachment of a maleimide derivative of adrenomedullin to the 34th cysteine residue of HSA via a linker. Denaturing gel electrophoresis and western blotting for HSA-AM yielded a single band with adrenomedullin immunoreactivity at the position corresponding to a molecular weight (MW) of 73 kDa. Following gel-filtration chromatography, the purified HSA-AM showed a single main peak corresponding with an MW of 73 kDa, indicating that HSA-AM is a monomer. Both adrenomedullin and HSA-AM stimulated the intracellular accumulation of cyclic AMP (cAMP) in HEK-293 cells stably expressing the adrenomedullin 1 receptor. The pEC50 values for adrenomedullin and HSA-AM were 8.660 and 7.208 (equivalent to 2.19 and 61.9 nM as EC50), respectively. The bioavailability of HSA-AM compared with that of adrenomedullin was much improved after subcutaneous administration in the rat, which was probably due to the superior resistance of HSA-AM towards endogenous proteases and its reduced clearance from the blood. HSA-AM may be a promising drug candidate for clinical application.

Discovery of a First-In-Class Small Molecule Antagonist against the Adrenomedullin-2 Receptor: Structure-Activity Relationships and Optimization.

Class B G-protein-coupled receptors (GPCRs) remain an underexploited target for drug development. The calcitonin receptor (CTR) family is particularly challenging, as its receptors are heteromers comprising two distinct components: the calcitonin receptor-like receptor (CLR) or calcitonin receptor (CTR) together with one of three accessory proteins known as receptor activity-modifying proteins (RAMPs). CLR/RAMP1 forms a CGRP receptor, CLR/RAMP2 forms an adrenomedullin-1 (AM1) receptor, and CLR/RAMP3 forms an adrenomedullin-2 (AM2) receptor. The CTR/RAMP complexes form three distinct amylin receptors. While the selective blockade of AM2 receptors would be therapeutically valuable, inhibition of AM1 receptors would cause clinically unacceptable increased blood pressure. We report here a systematic study of structure-activity relationships that has led to the development of first-in-class AM2 receptor antagonists. These compounds exhibit therapeutically valuable properties with 1000-fold selectivity over the AM1 receptor. These results highlight the therapeutic potential of AM2 antagonists.

Adrenomedullin 2 and 5 activate the calcitonin receptor-like receptor (clr) - Receptor activity-modifying protein 3 (ramp3) receptor complex in Xenopus tropicalis.

The adrenomedullin (AM) family is involved in diverse biological functions, including cardiovascular regulation and body fluid homeostasis, in multiple vertebrate lineages. The AM family consists of AM1, AM2, and AM5 in tetrapods, and the receptor for mammalian AMs has been identified as the complex of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 2 (RAMP2) or RAMP3. However, the receptors for AM in amphibians have not been identified. In this study, we identified the cDNAs encoding calcrl (clr), ramp2, and ramp3 receptor components from the western clawed frog (Xenopus tropicalis). Messenger RNAs of amphibian clr and ramp2 were highly expressed in the heart, whereas that of ramp3 was highly expressed in the whole blood. In HEK293T cells expressing clr-ramp2, cAMP response element luciferase (CRE-Luc) reporter activity was activated by am1. In HEK293T cells expressing clr-ramp3, CRE-Luc reporter activity was increased by the treatment with am2 at the lowest dose, but with am5 and am1 at higher dose. Our results provided new insights into the roles of AM family peptides through CLR-RAMP receptor complexes in the tetrapods.

Adrenomedullin Attenuates Inflammation in White Adipose Tissue of Obese Rats Through Receptor-Mediated PKA Pathway.

OBJECTIVE: Adrenomedullin (ADM) possesses therapeutic potential for inflammatory diseases. Consequently, the effects of ADM on inflammation in visceral white adipose tissue (vWAT) of obese rats or in adipocytes were explored in this study. METHODS: Male rats were fed a high-fat diet for 12 weeks to induce obesity, and obese rats were implanted with osmotic minipumps providing constant infusion of ADM (300 ng/kg per hour) and continued to be fed a high-fat diet for 4 weeks. RESULTS: When compared with the control group, endogenous protein expression of ADM and ADM receptors in vWAT and in lipopolysaccharide (LPS)-treated adipocytes was markedly increased. ADM significantly decreased the protein expression of the inflammatory mediators TNFα, IL-1ß, cyclooxygenase-2, and inducible nitric oxide synthase in vWAT of obese rats and in adipocytes stimulated by LPS. It also inhibited the activation of the inflammatory signaling pathways MAPK and NF-κB induced by LPS in adipocytes. These effects of ADM in adipocytes were inhibited by the administration of ADM receptor antagonist and cAMP-dependent protein kinase (PKA) activation inhibitor. CONCLUSIONS: ADM can inhibit inflammation in WAT in obesity, which may be mediated by the activation of ADM receptors and PKA.

Biochemical characterization of G protein coupling to calcitonin gene-related peptide and adrenomedullin receptors using a native PAGE assay.

Calcitonin gene-related peptide (CGRP), adrenomedullin (AM), and adrenomedullin 2/intermedin (AM2/IMD) have overlapping and unique functions in the nervous and circulatory systems including vasodilation, cardioprotection, and pain transmission. Their actions are mediated by the class B calcitonin-like G protein-coupled receptor (CLR), which heterodimerizes with three receptor activity-modifying proteins (RAMP1-3) that determine its peptide ligand selectivity. How the three agonists and RAMPs modulate CLR binding to transducer proteins remains poorly understood. Here, we biochemically characterized agonist-promoted G protein coupling to each CLR·RAMP complex. We adapted a native PAGE method to assess the formation and thermostabilities of detergent-solubilized fluorescent protein-tagged CLR·RAMP complexes expressed in mammalian cells. Addition of agonist and the purified Gs protein surrogate mini-Gs (mGs) yielded a mobility-shifted agonist·CLR·RAMP·mGs quaternary complex gel band that was sensitive to antagonists. Measuring the apparent affinities of the agonists for the mGs-coupled receptors and of mGs for the agonist-occupied receptors revealed that both ligand and RAMP control mGs coupling and defined how agonist engagement of the CLR extracellular and transmembrane domains affects transducer recruitment. Using mini-Gsq and -Gsi chimeras, we observed a coupling rank order of mGs > mGsq > mGsi for each receptor. Last, we demonstrated the physiological relevance of the native gel assays by showing that they can predict the cAMP-signaling potencies of AM and AM2/IMD chimeras. These results highlight the power of the native PAGE assay for membrane protein biochemistry and provide a biochemical foundation for understanding the molecular basis of shared and distinct signaling properties of CGRP, AM, and AM2/IMD.

Adrenomedullin insufficiency alters macrophage activities in fallopian tube: a pathophysiologic explanation of tubal ectopic pregnancy.

Ectopic pregnancy is the major cause of maternal morbidity and mortality in the first trimester of pregnancy. Tubal ectopic pregnancy (TEP) accounts for nearly 98% of all ectopic pregnancies. TEP is usually associated with salpingitis but the underlying mechanism in salpingitis leading to TEP remains unclear. Adrenomedullin (ADM) is a peptide hormone abundantly expressed in the fallopian tube with potent anti-inflammatory activities. Its expression peaks at the early luteal phase when the developing embryo is being transported through the fallopian tube. In the present study, we demonstrated reduced expression of ADM in fallopian tubes of patients with salpingitis and TEP. Using macrophages isolated from the fallopian tubes of these women, our data revealed that the salpingistis-associated ADM reduction contributed to aggravated pro-inflammatory responses of the tubal macrophages resulting in production of pro-inflammatory and pro-implantation cytokines IL-6 and IL-8. These cytokines activated the expression of implantation-associated molecules and Wnt signaling pathway predisposing the tubal epithelium to an adhesive and receptive state for embryo implantation. In conclusion, this study provided evidence for the role of ADM in the pathogenesis of TEP through regulating the functions of tubal macrophages.

Investigation of the therapeutic mechanism of subthreshold micropulse laser irradiation in retina.

PURPOSE: Subthreshold micropulse laser irradiation has been used for the treatment of retinal edema; however, there are few reports about the mechanism of its therapeutic effect. In this study, we compared threshold short pulse and subthreshold micropulse laser irradiation in mice and investigated their mechanism. METHODS: Nine to 12-week-old male C57BL/6J mice were used in this study. After general anesthesia, threshold short pulse or subthreshold micropulse laser irradiation was performed on the right eye using IQ577. Enucleation was performed 24 h after the laser irradiation, and histological and gene expression analyses were carried out. RESULTS: Coagulation spots and atrophy of the retinal pigment epithelium were observed after threshold short pulse laser irradiation but not after subthreshold micropulse laser irradiation. Twenty-four hours after laser, aquaporin (AQP) 1, 2, 7, and 11 levels were significantly elevated by 1.7- to 3-fold in the threshold short pulse laser group compared with non-treated control group. AQP 3 was increased significantly and prominently by 100-fold. VEGF-A and VEGFR2 were upregulated 1.5- and 2.3-fold, respectively. In the subthreshold micropulse laser group, AQP 3 was increased by 6-fold compared with the non-treated control group. Angiopoietin-1 and the adrenomedullin (AM) receptor CLR were decreased by 0.6-fold and 0.5-fold, respectively. CONCLUSION: Threshold short pulse laser irradiation caused retinal damage and prominent changes in the expression of various genes. Contrarily, subthreshold micropulse laser irradiation did not induce retinal damage; it upregulated AQP 3, which might have improved retinal edema by drainage of subretinal fluid.

Adrenomedullin has a cytoprotective role against endoplasmic reticulum stress for pancreatic ß-cells in autocrine and paracrine manners.

AIMS/INTRODUCTION: Pancreatic ß-cells are sensitive to endoplasmic reticulum (ER) stress, which has a major role in the context of ß-cell death. Adrenomedullin (ADM) has been shown to exert a cytoprotective effect under various pathophysiological conditions. Several studies have suggested that thiazolidinediones have protective effects on ß-cells. During the course to elucidate the molecular mechanisms by which pioglitazone prevents ß-cell death, ADM emerged as a candidate. Here, we studied the regulation of ADM expression, including the effects of pioglitazone, and its role in pancreatic islets. MATERIALS AND METHODS: We analyzed ADM expression in islet cell lines treated with pioglitazone. The effects of ER stress on ADM and ADM receptor expressions were investigated by analyzing thapsigargin-treated MIN6 cells and islets isolated from Wfs1-/- and db/db mice. To study the anti-apoptotic effect of ADM, ER stress-exposed MIN6 cells were treated with ADM peptides or transfected with ADM expression plasmid. RESULTS: Pioglitazone increased the production and secretion of ADM in islets through peroxisome-proliferator activated receptor-γ-dependent mechanisms. Thapsigargin treatment increased expressions of both ADM and ADM receptor, composed of Ramp2, Ramp3 and Crlr, in MIN6 cells. ADM and ADM receptor expressions were also increased in isolated islets from Wfs1-/- and db/db mice. ADM peptides and ADM overexpression protected MIN6 cells from thapsigargin-induced apoptosis. CONCLUSIONS: ER stress stimulates ADM production and secretion in islets. ADM signaling might protect ß-cells from ER stress-induced apoptosis, and might be one of the self-protective mechanisms. ß-Cell protection by pioglitazone is partly through induction of ADM. ADM-based therapy could be a novel strategy for treating diabetes.