Renal depletion of myo-inositol is associated with its increased degradation in animal models of metabolic disease.

Renal depletion of myo-inositol (MI) is associated with the pathogenesis of diabetic nephropathy in animal models, but the underlying mechanisms involved are unclear. We hypothesized that MI depletion was due to changes in inositol metabolism and therefore examined the expression of genes regulating de novo biosynthesis, reabsorption, and catabolism of MI. We also extended the analyses from diabetes mellitus to animal models of dietary-induced obesity and hypertension. We found that renal MI depletion was pervasive across these three distinct disease states in the relative order: hypertension (-51%)>diabetes mellitus (-35%)>dietary-induced obesity (-19%). In 4-wk diabetic kidneys and in kidneys derived from insulin-resistant and hypertensive rats, MI depletion was correlated with activity of the MI-degrading enzyme myo-inositol oxygenase (MIOX). By contrast, there was decreased MIOX expression in 8-wk diabetic kidneys. Immunohistochemistry localized the MI-degrading pathway comprising MIOX and the glucuronate-xylulose (GX) pathway to the proximal tubules within the renal cortex. These findings indicate that MI depletion could reflect increased catabolism through MIOX and the GX pathway and implicate a common pathological mechanism contributing to renal oxidative stress in metabolic disease.

Receptor activity-modifying protein-dependent effects of mutations in the calcitonin receptor-like receptor: implications for adrenomedullin and calcitonin gene-related peptide pharmacology.

BACKGROUND AND PURPOSE: Receptor activity-modifying proteins (RAMPs) define the pharmacology of the calcitonin receptor-like receptor (CLR). The interactions of the different RAMPs with this class B GPCR yield high-affinity calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors. However, the mechanism for this is unclear. EXPERIMENTAL APPROACH: Guided by receptor models, we mutated residues in the N-terminal helix of CLR, RAMP2 and RAMP3 hypothesized to be involved in peptide interactions. These were assayed for cAMP production with AM, AM2 and CGRP together with their cell surface expression. Binding studies were also conducted for selected mutants. KEY RESULTS: An important domain for peptide interactions on CLR from I32 to I52 was defined. Although I41 was universally important for binding and receptor function, the role of other residues depended on both ligand and RAMP. Peptide binding to CLR/RAMP3 involved a more restricted range of residues than that to CLR/RAMP1 or CLR/RAMP2. E101 of RAMP2 had a major role in AM interactions, and F111/W84 of RAMP2/3 was important with each peptide. CONCLUSIONS AND IMPLICATIONS: RAMP-dependent effects of CLR mutations suggest that the different RAMPs control accessibility of peptides to binding residues situated on the CLR N-terminus. RAMP3 appears to alter the role of specific residues at the CLR-RAMP interface compared with RAMP1 and RAMP2.

PACAP receptor pharmacology and agonist bias: analysis in primary neurons and glia from the trigeminal ganglia and transfected cells.

BACKGROUND AND PURPOSE: A major challenge in the development of new medicines targeting GPCRs is the ability to quantify drug action in physiologically relevant models. Primary cell models that closely resemble the clinically relevant in vivo site of drug action are important translational tools in drug development. However, pharmacological studies in these models are generally very limited due to the methodology used. EXPERIMENTAL APPROACH: We used a neuropeptide system to demonstrate the applicability of using highly sensitive signalling assays in primary cells. We quantified the action of pituitary adenylate cyclase-activating peptide (PACAP)-38, PACAP-27 and vasoactive intestinal polypeptide in primary cultures of neurons and glia derived from rat trigeminal ganglia (TG), comparing our observations to transfected cells. KEY RESULTS: PACAP-responsive receptors in rat trigeminal neurons, glia and transfected PAC1n receptors were pharmacologically distinct. PACAP-38, but not PACAP-27, activated ERK in glia, while both forms stimulated cellular cAMP production. PACAP(6-38) also displayed cell-type-dependent, agonist-specific, antagonism. CONCLUSIONS AND IMPLICATIONS: The complexity of PACAP pharmacology in the TG may help to direct, more effectively, the development of disease treatments targeting the PACAP receptor. We suggest that these methodologies are broadly applicable to other primary cell types of human or animal origin, and that our approach may allow more thorough characterization of ligand properties in physiologically relevant cell types.

CGRP in the trigeminovascular system: a role for CGRP, adrenomedullin and amylin receptors?

UNLABELLED: The neuropeptide calcitonin gene-related peptide (CGRP) is reported to play an important role in migraine. It is expressed throughout the trigeminovascular system. Antagonists targeting the CGRP receptor have been developed and have shown efficacy in clinical trials for migraine. However, no CGRP antagonist is yet approved for treating this condition. The molecular composition of the CGRP receptor is unusual because it comprises two subunits; one is a GPCR, the calcitonin receptor-like receptor (CLR). This associates with receptor activity-modifying protein (RAMP) 1 to yield a functional receptor for CGRP. However, RAMP1 also associates with the calcitonin receptor, creating a receptor for the related peptide amylin but this also has high affinity for CGRP. Other combinations of CLR or the calcitonin receptor with RAMPs can also generate receptors that are responsive to CGRP. CGRP potentially modulates an array of signal transduction pathways downstream of activation of these receptors, in a cell type-dependent manner. The physiological significance of these signalling processes remains unclear but may be a potential avenue for refining drug design. This complexity has prompted us to review the signalling and expression of CGRP and related receptors in the trigeminovascular system. This reveals that more than one CGRP responsive receptor may be expressed in key parts of this system and that further work is required to determine their contribution to CGRP physiology and pathophysiology. LINKED ARTICLES: This article is part of a themed section on Neuropeptides. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.170.issue-7.

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

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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. KEY RESULTS: 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)) . CONCLUSIONS AND IMPLICATIONS: 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.

On a potential global role for vitamin K-dependent gamma-carboxylation in animal systems. Evidence for a gamma-glutamyl carboxylase in Drosophila.

The vitamin K-dependent gamma-carboxylation of glutamate to gamma-carboxyglutamate was originally well characterized in the mammalian blood clotting cascade. gamma-Carboxyglutamate has also been found in a number of other mammalian proteins and in neuropeptides from the venoms of marine snails belonging to the genus Conus, suggesting wider prevalence of gamma-carboxylation. We demonstrate that an open reading frame from a Drosophila melanogaster cDNA clone encodes a protein with vitamin K-dependent gamma-carboxylase activity. The open reading frame, 670 amino acids in length, is truncated at the C-terminal end compared with mammalian gamma-carboxylase, which is 758 amino acids. The mammalian gene has 14 introns; in Drosophila there are two much shorter introns but in positions precisely homologous to two of the mammalian introns. In addition, a deletion of 6 nucleotides is observed when cDNA and genomic sequences are compared. In situ hybridization to fixed embryos indicated ubiquitous presence of carboxylase mRNA throughout embryogenesis. Northern blot analysis revealed increased mRNA levels in 12-24-h embryos. The continued presence of carboxylase mRNA suggests that it plays an important role during embryogenesis. Although the model substrate FLEEL is carboxylated by the enzyme, a substrate containing the propeptide of a Conus carboxylase substrate, conantokin G, is poorly carboxylated. Its occurrence in vertebrates, molluscan systems (i.e. Conus), and Drosophila and the apparently strong homology between the three systems suggest that this is a highly conserved and widely distributed post-translational modification in biological systems.

The T-superfamily of conotoxins.

We report the discovery and initial characterization of the T-superfamily of conotoxins. Eight different T-superfamily peptides from five Conus species were identified; they share a consensus signal sequence, and a conserved arrangement of cysteine residues (- -CC- -CC-). T-superfamily peptides were found expressed in venom ducts of all major feeding types of Conus; the results suggest that the T-superfamily will be a large and diverse group of peptides, widely distributed in the 500 different Conus species. These peptides are likely to be functionally diverse; although the peptides are small (11-17 amino acids), their sequences are strikingly divergent, with different peptides of the superfamily exhibiting varying extents of post-translational modification. Of the three peptides tested for in vivo biological activity, only one was active on mice but all three had effects on fish. The peptides that have been extensively characterized are as follows: p5a, GCCPKQMRCCTL*; tx5a, gammaCCgammaDGW(+)CCT( section sign)AAO; and au5a, FCCPFIRYCCW (where gamma = gamma-carboxyglutamate, W(+) = bromotryptophan, O = hydroxyproline, T( section sign) = glycosylated threonine, and * = COOH-terminal amidation). We also demonstrate that the precursor of tx5a contains a functional gamma-carboxylation recognition signal in the -1 to -20 propeptide region, consistent with the presence of gamma-carboxyglutamate residues in this peptide.

Discoloration of a compomer by stains.

This study evaluated the color stability of a polyacid-modified composite (compomer) upon exposure to stains. Five disks were prepared for immersion in each of five stains: coffee, chlorhexidine, cola, red wine, and water as a control. Color measurements were made on a reflection spectrophotometer at baseline, after 24-hour incubation, and after 24, 48, and 72 hour immersion in each stain. At 24 hours, perceptible color changes occurred for specimens in red wine and coffee. After 48 hours, perceptible color changes occurred for specimens in cola. Chlorhexidine and water caused no perceptible color changes. A compomer is susceptible to staining by coffee, red wine, and cola.

Conantokin-G precursor and its role in gamma-carboxylation by a vitamin K-dependent carboxylase from a Conus snail.

Conantokin-G isolated from the marine snail Conus geographus is a 17-amino acid gamma-carboxyglutamate (Gla)-containing peptide that inhibits the N-methyl-D-aspartate receptor. We describe the cloning and sequence of conantokin-G cDNA and the possible role of the propeptide sequence. The cDNA encodes a 100amino acid peptide. The N-terminal 80 amino acids constitute the prepro-sequence, and the mature peptide is derived from the remaining C-terminal residues after proteolysis, C-terminal amidation, and a unique post-translational modification, gamma-carboxylation of glutamate residues to Gla. Mature conantokin-G peptide containing Glu residues (E.Con-G) in place of Gla is a poor substrate for the vitamin K-dependent gamma-glutamyl carboxylase (apparent Km = 3.4 mM). Using peptides corresponding to different segments of the propeptide we investigated a potential role for the propeptide sequences in gamma-carboxylation. Propeptide segment -20 to -1 covalently linked to E.Con-G or the synthetic pentapeptide FLEEL increased their apparent affinities 2 orders of magnitude. These substrates are not efficiently carboxylated by the bovine microsomal gamma-glutamyl carboxylase, suggesting differences in specificities between the Conus and the mammalian enzyme. However, the role of propeptide in enhancing the efficiency of carboxylation is maintained.