The pharmacology of adrenomedullin receptors and their relationship to CGRP receptors

Adrenomedullin (AM) has two specific receptors formed by the calcitonin-receptor-like receptor (CL) and receptor activity-modifying protein (RAMP) 2 or 3. These are known as AM1 and AM2 receptors, respectively. In addition, AM has appreciable affinity for the CGRP1 receptor, composed of CL and RAMP1. The AM1 receptor has a high degree of selectivity for AM over CGRP and other peptides, and AM 22-52 is an effective antagonist at this receptor. By contrast, the AM2 receptor shows less specificity for AM, having appreciable affinity for βCGRP. Here, CGRP8-37 is either equipotent or more effective as an antagonist than AM22-52, depending on the species from which the receptor components are derived. Thus, under the appropriate circumstances it seems that βCGRP might be able to activate both CGRP 1 and AM2 receptors and AM could activate both AM 1 and AM2 receptors as well as CGRP1 receptors. Current peptide antagonists are not sufficiently selective to discriminate between these three receptors. The CGRP-selectivity of RAMP1 and RAMP3 may be conferred by a putative disulfide bond from the N-terminus to the middle of the extracellular domain of these molecules. This is not present in RAMP2. Copyright © 2004 Humana Press Inc. All rights of any nature whatsoever reserved.

CL/RAMP2 and CL/RAMP3 produce pharmacologically distinct adrenomedullin receptors:a comparison of effects of adrenomedullin 22-52, CGRP 8-37 and BIBN4096BS

1. Adrenomedullin (AM) has two known receptors formed by the calcitonin receptor-like receptor (CL) and receptor activity-modifying protein (RAMP) 2 or 3: We report the effects of the antagonist fragments of human AM and CGRP (AM 22-52 and CGRP 8-37) in inhibiting AM at human (h), rat (r) and mixed species CL/RAMP2 and CL/RAMP3 receptors transiently expressed in Cos 7 cells or endogenously expressed as rCL/rRAMP2 complexes by Rat 2 and L6 cells. 2. AM 22-52 (10 μM) antagonised AM at all CL/RAMP2 complexes (apparent pA 2 values: 7.34±0.14 (hCL/hRAMP2), 7.28±0.06 (Rat2), 7.00±0.05 (L6), 6.25±0.17(rCL/hRAMP2)). CGRP 8-37 (10 μM) resembled AM 22-52 except on the rCL/hRAMP2 complex, where it did not antagonise AM (apparent PA 2 values: 7.04±0.13 (hCL/hRAMP2), 6.72±0.06 (Rat2), 7.03±0. 12 (L6)). 3. On CL/RAMP3 receptors, 10 μM CGRP 8-37 was an effective antagonist at all combinations (apparent pA 2 values: 6.96±0.08 (hCL/hRAMP3), 6.18±0.18 (rCL/rRAMP3), 6.48±0.20 (rCL/ hRAMP3)). However, 10 μm AM 22-52 only antagonised AM at the hCL/hRAMP3 receptor (apparent PA 2 6.73±0.14). 4. BIBN4096BS (10 μM) did not antagonise AM at any of the receptors. 5. Where investigated (all-rat and rat/human combinations), the agonist potency order on the CL/ RAMP3 receptor was AM∼βCGRP>αCGRP. 6. rRAMP3 showed three apparent polymorphisms, none of which altered its coding sequence. 7. This study shows that on CL/RAMP complexes, AM 22-52 has significant selectivity for the CL/ RAMP2 combination over the CL/RAMP3 combination. On the mixed species receptor, CGRP 8-37 showed the opposite selectivity. Thus, depending on the species, it is possible to discriminate pharmacologically between CL/RAMP2 and CL/RAMP3 AM receptors.

Desensitisation of adrenomedullin and CGRP receptors

Adrenomedullin (AM), a potent vasoactive peptide, is elevated in certain disease states such as sepsis. Its role as a physiologically relevant peptide has been confirmed with the advent of the homozygous lethal AM peptide knockout mouse. So far, there have been few and conflicting studies which examine the regulatory role of AM at the receptor level. In this article, we discuss the few studies that have been presented on the desensitisation of AM receptors and also present novel data on the desensitisation of endogenous AM receptors in Rat-2 fibroblasts. © 2003 Elsevier Science B.V. All rights reserved.

The role of the 8-18 helix of CGRP 8-37 in mediating high affinity binding to CGRP receptors; coulombic and steric interactions

1. The role of individual residues in the 8-18 helix of CGRP 8-37 in promoting high-affinity binding to CGRP 1 receptors expressed on rat L6 and human SK-N-MC cells has been examined. The relative potencies of various derivatives were estimated from their ability to inhibit the human αCGRP-mediated increase in cyclic AMP production and the binding of [ 125I]-human αCGRP. 3. Arg 11 and Arg 18 were replaced by serines to give [Ser 11.18]CGRP 8-37. These bound with pKi values <6 to SK-N-MC cells and had apparent pA 2 values of 5.81 ± 0.04 and 5.31 ± 0.11 on SK-N-MC and L6 cells. CGRP 8-37 had a pKi of 8.22 on SK-N-MC cells and pK b values on the above cell lines of 8.95±0.04 and 8.76±0.04. 3. The arginines were replaced with glutamic acid residues. [Glu 11]CGRP 8-37 had a pK b of 7.14±0.14 on SK-N-MC cells (pKi=7.05±0.05) and 6.99±0.08 on L6 cells. [Glu 18]CGRP 8-37 had a pK b of 7.10±0.0.08 on SK-N-MC cells (pKi=6.91±0.23) and 7.12±0.09 on L6 cells. 4. Leu 12, Leu 15 and Leu 16 were replaced by benzoyl-phenylalanine (bpa) residues. On SK-N-MC cells, the apparent pA 2 values of [bpa 12]-, [bpa 15]- and [bpa 16]CGRP 8-37 were respectively 7.43±0.23, 8.34±0.11 and 5.66±0.16 (pKi values of 7.14±0.17, 7.66±0.21 and <6): on L6 cells they were 7.96±0.36, 8.28±0.21 and 6.09±0.04 (all n=3). 5. It is concluded that the Arg 11 and Arg 18 are involved in specific electrostatic interactions with other residues, either on the CGRP 1 receptors or elsewhere on CGRP 8-37. Leu 16 is in a conformationally restricted site when CGRP 8-37 binds to CGRP 1 receptors, unlike Leu 12 and Leu 15.

Interaction of calcitonin-gene-related peptide with its receptors

The receptor for calcitonin-gene-related peptide (CGRP) is a heterodimer formed by calcitonin-receptor-like receptor (CRLR), a type II (family B) G-protein-coupled receptor, and receptor-activity-modifying protein 1 (RAMP1), a single-membrane-pass protein. It is likely that the first seven or so amino acids of CGRP (which form a disulphide-bonded loop) interact with the transmembrane domain of CRLR to cause receptor activation. The rest of the CGRP molecule falls into three domains. Residues 28-37 and 8-18 are normally required for high-affinity binding, while residues 19-27 form a hinge region. The 28-37 region is almost certainly in direct contact with the receptor; 8-18 may make additional receptor contacts or may stabilize an appropriate conformation of 28-37. It is likely that these regions of CGRP interact both with CRLR and with the extracellular domain of RAMP1.

Adrenomedullin: receptor and signal transduction

Adrenomedullin is a vascular tissue peptide and a member of the calcitonin family of peptides, which includes calcitonin calcitonin-gene-related peptide (CGRP) and amylin. Its many biological actions are mediated via CGRP type 1 (CGRP(1)) receptors and by specific adrenomedullin receptors. Although the pharmacology of these receptors is distinct, they are both represented in molecular terms by the type II family G-protein-coupled receptor, calcitonin-receptor-like receptor (CRLR). The specificity here is defined by co-expression of receptor-activity-modifying proteins (RAMPs). CGRP(1) receptors are represented by CRLR and RAMP1, and specific adrenomedullin receptors by CRLR and RAMP2 or 3. Here we discuss how CRLR/RAMP2 relates to adrenomedullin binding, pharmacology and pathophysiology, and how chemical cross-linking of receptor-ligand complexes in tissue relates to that in CRLR/RAMP2-expressing cells. CRLR, like other type II family G-protein-coupled receptors, signals via G(s) and adenylate cyclase activation. We demonstrated that adrenomedullin signalling in cell lines expressing specific adrenomedullin receptors followed this expected pattern.

Comparison of the expression of calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) with CGRP and adrenomedullin binding in cell lines

1. The calcitonin receptor-like receptor (CRLR) and specific receptor activity modifying proteins (RAMPs) together form receptors for calcitonin gene-related peptide (CGRP) and/or adrenomedullin in transfected cells. 2. There is less evidence that innate CGRP and adrenomedullin receptors are formed by CRLR/RAMP combinations. We therefore examined whether CGRP and/or adrenomedullin binding correlated with CRLR and RAMP mRNA expression in human and rat cell lines known to express these receptors. Specific human or rat CRLR antibodies were used to examine the presence of CRLR in these cells. 3. We confirmed CGRP subtype 1 receptor (CGRP(1)) pharmacology in SK-N-MC neuroblastoma cells. L6 myoblast cells expressed both CGRP(1) and adrenomedullin receptors whereas Rat-2 fibroblasts expressed only adrenomedullin receptors. In contrast we could not confirm CGRP(2) receptor pharmacology for Col-29 colonic epithelial cells, which, instead were CGRP(1)-like in this study. 4. L6, SK-N-MC and Col-29 cells expressed mRNA for RAMP1 and RAMP2 but Rat-2 fibroblasts had only RAMP2. No cell line had detectable RAMP3 mRNA. 5. SK-N-MC, Col-29 and Rat-2 fibroblast cells expressed CRLR mRNA. By contrast, CRLR mRNA was undetectable by Northern analysis in one source of L6 cells. Conversely, a different source of L6 cells had mRNA for CRLR. All of the cell lines expressed CRLR protein. Thus circumstances where CRLR mRNA is apparently absent by Northern analysis do not exclude the presence of this receptor. 6. These data strongly support CRLR, together with appropriate RAMPs as binding sites for CGRP and adrenomedullin in cultured cells.

International Union of Pharmacology. XXXII. The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors

The calcitonin family of peptides comprises calcitonin, amylin two calcitonin gene-related peptides (CGRPs), and adrenomedullin. The first calcitonin receptor was cloned in 1991. Its pharmacology is complicated by the existence of several splice variants. The receptors for the other members the family are made up of subunits. The calcitonin-like receptor (CL receptor) requires a single transmembrane domain protein, termed receptor activity modifying protein, RAMP1, to function as a CGRP receptor. RAMP2 and -3 enable the same CL receptor to behave as an adrenomedullin receptor. Although the calcitonin receptor does not require RAMP to bind and respond to calcitonin, it can associate with the RAMPs, resulting in a series of receptors that typically have high affinity for amylin and varied affinity for CGRP. This review aims to reconcile what is observed when the receptors are reconstituted in vitro with the properties they show in native cells and tissues. Experimental conditions must be rigorously controlled because different degrees of protein expression may markedly modify pharmacology in such a complex situation. Recommendations, which follow International Union of Pharmacology guidelines, are made for the nomenclature of these multimeric receptors.

Characterization of receptors for calcitonin gene-related peptide and adrenomedullin on the guinea-pig vas deferens

1. The receptors which mediate the effects of calcitonin gene-related peptide (CGRP), amylin and adrenomedullin on the guinea-pig vas deferens have been investigated. 2. All three peptides cause concentration dependant inhibitions of the electrically stimulated twitch response (pD 2s for CGRP, amylin and adrenomedullin of 7.90 ± 0.11, 7.70 ± 0.19 and 7.25 ± 0.10 respectively). 3. CGRP 8-37 (1 μM) and AC187 (10 μM) showed little antagonist activity against adrenomedullin. 4. Adrenomedullin 22-52 by itself inhibited the electrically stimulated contractions of the vas deferens and also antagonized the responses to CGRP, amylin and adrenomedullin. 5. [ 125I]-adrenomedullin labelled a single population of binding sites in vas deferens membranes with a pIC 50 of 8.91 and a capacity of 643 fmol mg -1. Its selectivity profile was adrenomedullin > AC187 > CGRP = amylin. It was clearly distinct from a site labelled by [ 125I]-CGRP (pIC 50 = 8.73, capacity = 114 fmol mg -1, selectivity CGRP > amylin = AC187 > adrenomedullin). [ 125I]-amylin bound to two sites with a total capacity of 882 fmol mg -1. 6. Although CGRP has been shown to act at a CGRP 2 receptor on the vas deferens with low sensitivity to CGRP 8-37, this antagonist displaced [ 125I]-CGRP with high affinity from vas deferens membranes. This affinity was unaltered by increasing the temperature from 4°C to 25°C, suggesting the anomalous behaviour of CGRP 8-37 is not due to temperature differences between binding and functional assays.

Performance of five different electrospray ionisation sources in conjunction with rapid monolithic column liquid chromatography and fast MS/MS scanning

The performances of five different ESI sources coupled to a polystyrene-divinylbenzene monolithic column were compared in a series of LC-ESI-MS/MS analyses of Escherichia coli outer membrane proteins. The sources selected for comparison included two different modifications of the standard electrospray source, a commercial low-flow sprayer, a stainless steel nanospray needle and a coated glass Picotip. Respective performances were judged on sensitivity and the number and reproducibility of significant protein identifications obtained through the analysis of multiple identical samples. Data quality varied between that of a ground silica capillary, with 160 total protein identifications, the lowest number of high quality peptide hits obtained (3012), and generally peaks of lower intensity; and a stainless steel nanospray needle, which resulted in increased precursor ion abundance, the highest-quality peptide fragmentation spectra (5414) and greatest number of total protein identifications (259) exhibiting the highest MASCOT scores (average increase in score of 27.5% per identified protein). The data presented show that, despite increased variability in comparative ion intensity, the stainless steel nanospray needle provides the highest overall sensitivity. However, the resulting data were less reproducible in terms of proteins identified in complex mixtures -- arguably due to an increased number of high intensity precursor ion candidates.

Effects of engine cooling water temperature on performance and exhaust emission characteristics of a multi-cylinder CI engine operated with Jatropha-Butanol blend

Renewable alternatives such as biofuels and optimisation of the engine operating parameters can enhance engine performance and reduce emissions. The temperature of the engine coolant is known to have significant influence on engine performance and emissions. Whereas much existing literature describes the effects of coolant temperature in engines using fossil derived fuels, very few studies have investigated these effects when biofuel is used as an alternative fuel. Jatropha oil is a non-edible biofuel which can substitute fossil diesel for compression ignition (CI) engine use. However, due to the high viscosity of Jatropha oil, technique such as transesterification, preheating the oil, mixing with other fuel is recommended for improved combustion and reduced emissions. In this study, Jatropha oil was blended separately with ethanol and butanol, at ratios of 80:20 and 70:30. The fuel properties of all four blends were measured and compared with diesel and jatropha oil. It was found that the 80% jatropha oil + 20% butanol blend was the most suitable alternative, as its properties were closest to that of diesel. A 2 cylinder Yanmar engine was used; the cooling water temperature was varied between 50°C and 95°C. In general, it was found that when the temperature of the cooling water was increased, the combustion process enhanced for both diesel and Jatropha-Butanol blend. The CO2 emissions for both diesel and biofuel blend were observed to increase with temperature. As a result CO, O2 and lambda values were observed to decrease when cooling water temperature increased. When the engine was operated using diesel, NOX emissions correlated in an opposite manner to smoke opacity; however, when the biofuel blend was used, NOX emissions and smoke opacity correlated in an identical manner. The brake thermal efficiencies were found to increase slightly as the temperature was increased. In contrast, for all fuels, the volumetric efficiency was observed to decrease as the coolant temperature was increased. Brake specific fuel consumption was observed to decrease as the temperature was increased and was higher on average when the biofuel was used, in comparison to diesel. The study concludes that the effects of engine coolant temperature on engine performance and emission characteristics differ between biofuel blend and fossil diesel operation. The coolant temperature needs to be optimised depending on the type of biofuel for optimum engine performance and reduced emissions.

Synthesis of gold nanoparticles within a supramolecular gel-phase network

Gold nanoparticles with diameters of ca. 13 nm were synthesised by UV irradiation of a supramolecular organogel into which HAuCl4 and tetraoctylammonium bromide had been diffused—the gel network plays an essential role in nanoparticle stabilisation. © The Royal Society of Chemistry 2005.

Synthesis and characterization of silica-supported L-Lysine-based dendritic branches

Two series of novel modified silicas have been prepared in which individual dendritic branches have been attached to aminopropylsilica using standard peptide coupling methodology. The dendritic branches are composed of enantiomerically pure l-lysine building blocks, and hence, the modified silicas have the potential to act as chiral stationary phases in chromatography. In one series of modified silicas, the surface of the dendritic branch consists of Boc carbamate groups, whereas the other has benzoyl amide surface groups. Different coupling reagents have been investigated in order to maximize the loading onto the solid phase. The new supported dendritic materials have been fully characterized with properties of the bulk material determined by elemental analysis, 13C NMR, and IR spectroscopy, whereas XPS provides important information about the surface of the modified silica exposed to the incident X-rays, the key region in which potential chromatographic performance of these materials will take place. Although the bulk analyses indicate that loading of the dendritic branch onto silica decreases with increasing dendritic generation (and consequently steric bulk), XPS indicates that the optimum surface coverage is actually obtained at the second generation of dendritic growth.

Copper complexes as a source of redox active MRI contrast agents

The study reports an advance in designing copper-based redox sensing MRI contrast agents. Although the data demonstrate that copper(II) complexes are not able to compete with lanthanoids species in terms of contrast, the redox-dependent switch between diamagnetic copper(I) and paramagnetic copper(II) yields a novel redox-sensitive contrast moiety with potential for reversibility.

The early career researcher's toolkit:translating tissue engineering, regenerative medicine and cell therapy products

Although the importance of translation for the development of tissue engineering, regenerative medicine and cell-based therapies is widely recognized, the process of translation is less well understood. This is particularly the case among some early career researchers who may not appreciate the intricacies of translational research or make decisions early in development which later hinders effective translation. Based on our own research and experiences as early career researchers involved in tissue engineering and regenerative medicine translation, we discuss common pitfalls associated with translational research, providing practical solutions and important considerations which will aid process and product development. Suggestions range from effective project management, consideration of key manufacturing, clinical and regulatory matters and means of exploiting research for successful commercialization.