Metabolic and skeletal homeostasis are maintained in full locus GPRC6A knockout mice.

The G protein-coupled receptor class C, group 6, subtype A (GPRC6A) is suggested to have a physiological function in glucose and bone metabolism, although the precise role lacks consensus due to varying findings in different knockout (KO) mouse models and inconsistent findings on the role of osteocalcin, a proposed GPRC6A agonist. We have further characterized a full locus GPRC6A KO model with respect to energy metabolism, including a long-term high-dose glucocorticoid metabolic challenge. Additionally, we analyzed the microarchitecture of tibiae from young, middle-aged and aged GPRC6A KO mice and wildtype (WT) littermates. Compared to WT, vehicle-treated KO mice presented with normal body composition, unaltered insulin sensitivity and basal serum insulin and glucose levels. Corticosterone (CS) treatment resulted in insulin resistance, abnormal fat accrual, loss of lean mass and suppression of serum osteocalcin levels in both genotypes. Interestingly, serum osteocalcin and skeletal osteocalcin mRNA levels were significantly lower in vehicle-treated GPRC6A KO mice compared to WT animals. However, WT and KO age groups did not differ in long bone mass and structure assessed by micro-computed tomography. We conclude that GPRC6A is not involved in glucose metabolism under normal physiological conditions, nor does it mediate glucocorticoid-induced dysmetabolism in mice. Moreover, GPRC6A does not appear to possess a direct, non-compensable role in long bone microarchitecture under standard conditions.

Synthesis and pharmacological evaluation of N-benzyl substituted 4-bromo-2,5-dimethoxyphenethylamines as 5-HT2A/2C partial agonists.

N-Benzyl substitution of phenethylamine 5-HT2A receptor agonists has dramatic effects on binding affinity, receptor selectivity and agonist activity. In this paper we examine how affinity for the 5-HT2A/2C receptors are influenced by N-benzyl substitution of 4-bromo-2,5-dimethoxyphenethylamine derivatives. Special attention is given to the 2' and 3'-position of the N-benzyl as such compounds are known to be very potent. We found that substitutions in these positions are generally well tolerated. The 2'-position was further examined using a range of substituents to probe the hydrogen bonding requirements for optimal affinity and selectivity, and it was found that small changes in the ligands in this area had a profound effect on their affinities. Furthermore, two ligands that lack a 2'-benzyl substituent were also found to have high affinity contradicting previous held notions. Several high-affinity ligands were identified and assayed for functional activity at the 5-HT2A and 5-HT2C receptor, and they were generally found to be less efficacious agonists than previously reported N-benzyl phenethylamines.

Structure-based discovery of antagonists for GluN3-containing N-methyl-D-aspartate receptors.

NMDA receptors are ligand-gated ion channels that assemble into tetrameric receptor complexes composed of glycine-binding GluN1 and GluN3 subunits (GluN3A-B) and glutamate-binding GluN2 subunits (GluN2A-D). NMDA receptors can assemble as GluN1/N2 receptors and as GluN3-containing NMDA receptors, which are either glutamate/glycine-activated triheteromeric GluN1/N2/N3 receptors or glycine-activated diheteromeric GluN1/N3 receptors. The glycine-binding GluN1 and GluN3 subunits display strikingly different pharmacological selectivity profiles. However, the pharmacological characterization of GluN3-containing receptors has been hampered by the lack of methods and pharmacological tools to study GluN3 subunit pharmacology in isolation. Here, we have developed a method to study the pharmacology of GluN3 subunits in recombinant diheteromeric GluN1/N3 receptors by mutating the orthosteric ligand-binding pocket in GluN1. This method is suitable for performing compound screening and characterization of structure-activity relationship studies on GluN3 ligands. We have performed a virtual screen of the orthosteric binding site of GluN3A in the search for antagonists with selectivity for GluN3 subunits. In the subsequent pharmacological evaluation of 99 selected compounds, we identified 6-hydroxy-[1,2,5]oxadiazolo[3,4-b]pyrazin-5(4H)-one (TK80) a novel competitive antagonist with preference for the GluN3B subunit. Serendipitously, we also identified [2-hydroxy-5-((4-(pyridin-3-yl)thiazol-2-yl)amino]benzoic acid (TK13) and 4-(2,4-dichlorobenzoyl)-1H-pyrrole-2-carboxylic acid (TK30), two novel non-competitive GluN3 antagonists. These findings demonstrate that structural differences between the orthosteric binding site of GluN3 and GluN1 can be exploited to generate selective ligands.

Interactions between calcium and phosphorus in the regulation of the production of fibroblast growth factor 23 in vivo.

Calcium and phosphorus homeostasis are highly interrelated and share common regulatory hormones, including FGF23. However, little is known about calcium's role in the regulation of FGF23. We sought to investigate the regulatory roles of calcium and phosphorus in FGF23 production using genetic mouse models with targeted inactivation of PTH (PTH KO) or both PTH and the calcium-sensing receptor (CaSR; PTH-CaSR DKO). In wild-type, PTH KO, and PTH-CaSR DKO mice, elevation of either serum calcium or phosphorus by intraperitoneal injection increased serum FGF23 levels. In PTH KO and PTH-CaSR DKO mice, however, increases in serum phosphorus by dietary manipulation were accompanied by severe hypocalcemia, which appeared to blunt stimulation of FGF23 release. Increases in dietary phosphorus in PTH-CaSR DKO mice markedly decreased serum 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] despite no change in FGF23, suggesting direct regulation of 1,25(OH)(2)D(3) synthesis by serum phosphorus. Calcium-mediated increases in serum FGF23 required a threshold level of serum phosphorus of about 5 mg/dl. Analogously, phosphorus-elicited increases in FGF23 were markedly blunted if serum calcium was less than 8 mg/dl. The best correlation between calcium and phosphorus and serum FGF23 was found between FGF23 and the calcium × phosphorus product. Since calcium stimulated FGF23 production in the PTH-CaSR DKO mice, this effect cannot be mediated by the full-length CaSR. Thus the regulation of FGF23 by both calcium and phosphorus appears to be fundamentally important in coordinating the serum levels of both mineral ions and ensuring that the calcium × phosphorus product remains within a physiological range.

L-Arginine improves multiple physiological parameters in mice exposed to diet-induced metabolic disturbances.

L-Arginine (L-Arg) is a conditionally essential amino acid and a natural constituent of dietary proteins. Studies in obese rats and type 2 diabetic humans have indicated that dietary supplementation with L-Arg can diminish gain in white adipose tissue (WAT) and improve insulin sensitivity. However, the effects of L-Arg on glucose homeostasis, body composition and energy metabolism remain unclear. In addition, no studies have, to our knowledge, examined whether L-Arg has beneficial effects as a dietary supplement in the mouse model. In the present study, we investigated the effects of L-Arg supplementation to male C57BL/6 mice on an array of physiological parameters. L-Arg supplemented mice were maintained on a low-protein diet and body composition, appetite regulation, glucose tolerance, insulin sensitivity and energy expenditure were evaluated. A significant reduction in epididymal WAT was observed in L-Arg supplemented mice compared with mice fed an isocaloric control diet. Surprisingly, the L-Arg supplemented animals were hyperphagic corresponding to a highly significant decrease in feed efficiency, as body weight developed in a similar pattern in both experimental groups. Glucose homeostasis experiments revealed a major effect of L-Arg supplementation on glucose tolerance and insulin sensitivity, interestingly, independent of a parallel regulation in whole-body adiposity. Increased L-Arg ingestion also raised energy expenditure; however, no concurrent effect on locomotor activity, substrate metabolism or expression of uncoupling proteins (UCP1 and UCP2) in adipose tissues was displayed. In conclusion, dietary L-Arg supplementation substantially affects an array of metabolic-associated parameters including a reduction in WAT, hyperphagia, improved insulin sensitivity and increased energy expenditure in mice fed a low-protein diet.

Implementation of a fluorescence-based screening assay identifies histamine H3 receptor antagonists clobenpropit and iodophenpropit as subunit-selective N-methyl-D-aspartate receptor antagonists.

N-Methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that mediate a slow, Ca(2+)-permeable component of excitatory synaptic transmission in the central nervous system and play a pivotal role in synaptic plasticity, neuronal development, and several neurological diseases. We describe a fluorescence-based assay that measures NMDA receptor-mediated changes in intracellular calcium in a BHK-21 cell line stably expressing NMDA receptor NR2D with NR1 under the control of a tetracycline-inducible promoter (Tet-On). The assay selectively identifies allosteric modulators by using supramaximal concentrations of glutamate and glycine to minimize detection of competitive antagonists. The assay is validated by successfully identifying known noncompetitive, but not competitive NMDA receptor antagonists among 1800 screened compounds from two small focused libraries, including the commercially available library of pharmacologically active compounds. Hits from the primary screen are validated through a secondary screen that used two-electrode voltage-clamp recordings on recombinant NMDA receptors expressed in Xenopus laevis oocytes. This strategy identified several novel modulators of NMDA receptor function, including the histamine H3 receptor antagonists clobenpropit and iodophenpropit, as well as the vanilloid receptor transient receptor potential cation channel, subfamily V, member 1 (TRPV1) antagonist capsazepine. These compounds are noncompetitive antagonists and the histamine H3 receptor ligand showed submicromolar potency at NR1/NR2B NMDA receptors, which raises the possibility that compounds can be developed that act with high potency on both glutamate and histamine receptor systems simultaneously. Furthermore, it is possible that some actions attributed to histamine H3 receptor inhibition in vivo may also involve NMDA receptor antagonism.

The four human gamma-aminobutyric acid (GABA) transporters: pharmacological characterization and validation of a highly efficient screening assay.

The neurotransmission mediated by gamma-aminobutyric acid (GABA) in the mammalian brain is terminated by a family of four GABA transporters (GATs). Inhibition of GATs is currently used in the treatment of epilepsy and these proteins are generally considered as important drug targets. In this study, we perform the first elaborate pharmacological characterization of all four human GAT subtypes. We conduct the experiments in parallel in a [3H]GABA uptake assay using 14 standard GAT substrates and inhibitors. This setup enables direct comparison of the absolute values of inhibitory activities of the compounds between the different GAT subtypes. The results are overall in agreement with data reported by other groups for the orthologous murine GATs. However, there do seem to be some minor variations among species. In contrast to the several subtype selective ligands identified for the GAT-1 subtype, no subtype selective ligands have been reported for the three remaining GATs. Given the potential therapeutic relevance of the individual GAT subtypes, a search for novel structures displaying selectivities for specific GAT subtypes is important. In this study, we validate our [3H]GABA uptake assay for use in high throughput screening. We find that the assay is categorized by high Z'-factors (Z' > 0.5) for all four GAT subtypes, demonstrating that the assay is excellent for a high throughput screen. This [3H]GABA uptake assay therefore enables future high throughput screening of compound libraries at the four human GATs.

Pharmacological characterization of ligands at recombinant NMDA receptor subtypes by electrophysiological recordings and intracellular calcium measurements.

Generation of in vitro cellular assays using fluorescence measurements at heterologously expressed NMDA receptors would speed up the process of ligand characterization and enable high-throughput screening. The major drawback to the development of such assays is the cytotoxicity caused by Ca(2+)-flux into the cell via NMDA receptors upon prolonged activation by agonists present in the culture medium. In the present study, we established four cell lines with stable expression of NMDA receptor subtypes NR1/NR2A, NR1/NR2B, NR1/NR2C, or NR1/NR2D in BHK-21 cells. To assess the usefulness of the stable cell lines in conjunction with intracellular calcium ([Ca(2+)](i)) measurements for evaluation of NMDA receptor pharmacology, several ligands were characterized using this method. The results were compared to parallel data obtained by electrophysiological recordings at NMDA receptors expressed in Xenopus oocytes. This comparison showed that agonist potencies determined by [Ca(2+)](i) measurements and electrophysiological recordings correlated well, meaning that the stable cell lines in conjunction with [Ca(2+)](i) measurements provide a useful tool for characterization of NMDA receptor ligands. The agonist series of conformationally constrained glutamate analogues (2S,3R,4S)-alpha-(carboxycyclopropyl)glycine (CCG), 1-aminocyclobutane-r-1,cis-3-dicarboxylic acid (trans-ACBD), and (+/-)-1-aminocyclopentane-r-1,cis-3-dicarboxylic acid (cis-ACPD), as well as the highly potent agonist tetrazolylglycine were among the characterized ligands that were assessed with respect to subtype selectivity at NMDA receptors. However, none of the characterized agonists displays more than 2-3 fold selectivity towards a specific NMDA receptor subtype. Thus, the present study provides a broad pharmacological characterization of structurally diverse ligands at recombinant NMDA receptor subtypes.

Cloning and characterization of a functional human gamma-aminobutyric acid (GABA) transporter, human GAT-2.

Plasma membrane gamma-aminobutyric acid (GABA) transporters act to terminate GABA neurotransmission in the mammalian brain. Intriguingly four distinct GABA transporters have been cloned from rat and mouse, whereas only three functional homologs of these transporters have been cloned from human. The aim of this study therefore was to search for this fourth missing human transporter. Using a bioinformatics approach, we successfully identified and cloned the full-length cDNA of a so far uncharacterized human GABA transporter (GAT). The predicted protein displays high sequence similarity to rat GAT-2 and mouse GAT3, and in accordance with the nomenclature for rat GABA transporters, we therefore refer to the transporter as human GAT-2. We used electrophysiological and cell-based methods to demonstrate that this protein is a functional transporter of GABA. The transport was saturable and dependent on both Na(+) and Cl(-). Pharmacologically the transporter is distinct from the other human GABA transporters and similar to rat GAT-2 and mouse GAT3 with high sensitivity toward GABA and beta-alanine. Furthermore the GABA transport inhibitor (S)-SNAP-5114 displayed some inhibitory activity at the transporter. Expression analysis by reverse transcription-PCR showed that GAT-2 mRNA is present in human brain, kidney, lung, and testis. The finding of the human GAT-2 demonstrates for the first time that the four plasma membrane GABA transporters identified in several mammalian species are all conserved in human. Furthermore the availability of human GAT-2 enables the use of all human clones of the GABA transporters in drug development programs and functional characterization of novel inhibitors of GABA transport.

Functional characterisation of homomeric ionotropic glutamate receptors GluR1-GluR6 in a fluorescence-based high throughput screening assay.

We have constructed stable HEK293 cell lines expressing the rat ionotropic glutamate receptor subtypes GluR1(i), GluR2Q(i), GluR3(i), GluR4(i), GluR5Q and GluR6Q and characterised the pharmacological profiles of the six homomeric receptors in a fluorescence-based high throughput screening assay using Fluo-4/AM as a fluorescent Ca2+ indicator. In this assay, the pharmacological properties of nine standard GluR ligands correlated nicely with those previously observed in electrophysiology studies of GluRs expressed in Xenopus oocytes or mammalian cells. The potencies and efficacies displayed by the agonists (S)-glutamate, (S)-quisqualate, kainate, (RS)-AMPA, (RS)-ATPA, (RS)-ACPA] and (S)-4-AHCP at the six GluRs were in concordance with electrophysiological studies. Furthermore, the Ki values exhibited by the competitive antagonists NBQX and (RS)-ATPO were also in agreement with findings of previous studies. Finally, the effects of various concentrations of Ca2+ in the assay buffer and of the allosteric modulators cyclothiazide and concanavalin A on GluR signalling were examined. This study represents the most elaborate functional characterisation of multiple AMPA and KA receptor subtypes in the same assay reported to date. We propose that high throughput screening of compound libraries at the six GluR-HEK293 cell lines could be helpful in the search for structurally and pharmacologically novel ligands acting at the receptors.

Molecular cloning, expression, and sequence analysis of GPRC6A, a novel family C G-protein-coupled receptor.

By similarity searching of the human genome sequence using known family C G-protein-coupled receptors (GPCRs) as query sequences, we have identified a putative novel human gene product of unknown function (located on chromosome band 6q22.31). The transcript, entitled GPRC6A (isoform 1), was cloned from a human kidney cDNA (DNA complementary to RNA) library and shown to encode a protein of 926 amino acids (aa). Protein sequence analysis revealed the presence of a seven-transmembrane (7TM) domain and an unusually long amino-terminal domain (ATD) of 590 amino acids. These traits, along with a significant homology to the human calcium-sensing receptor (CaR, 34% aa sequence identity), the taste receptor 1 (T1R1, 28%), and the metabotropic glutamate receptor 1 (mGluR1, 24%), places GPRC6A in family C of the GPCRs. Interestingly, GPRC6A bears the highest resemblance with an odorant goldfish 5.24 receptor (45%) which suggests that GPRC6A is the human orthologue of this receptor. GPRC6A is widely expressed in brain and peripheral tissues with highest levels in kidney, skeletal muscle, testis, and leucocytes. All three isoforms are expressed in mammalian cells, but are poorly expressed on the cell surface. In this work, we report the existence of two additional GPRC6A isoforms (2 and 3) carrying in-frame deletions in the ATD. Except for the kidney, where isoforms 1 and 2 appear equally expressed, isoforms 2 and 3 are generally less abundant than isoform 1. Analysis of the intron-exon composition of the GPRC6A gene confirms that isoforms 2 and 3 are naturally occurring splice variants.

Three distinct epitopes on the extracellular face of the glucagon receptor determine specificity for the glucagon amino terminus.

The glucagon and glucagon-like peptide-1 (GLP-1) receptors are homologous family B seven-transmembrane (7TM) G protein-coupled receptors, and they selectively recognize the homologous peptide hormones glucagon (29 amino acids) and GLP-1 (30-31 amino acids), respectively. The amino-terminal extracellular domain of the glucagon and GLP-1 receptors (140-150 amino acids) determines specificity for the carboxyl terminus of glucagon and GLP-1, respectively. In addition, the glucagon receptor core domain (7TM helices and connecting loops) strongly determines specificity for the glucagon amino terminus. Only 4 of 15 residues are divergent in the glucagon and GLP-1 amino termini; Ser2, Gln3, Tyr10, and Lys12 in glucagon and the corresponding Ala8, Glu9, Val16, and Ser18 in GLP-1. In this study, individual substitution of these four residues of glucagon with the corresponding residues of GLP-1 decreased the affinity and potency at the glucagon receptor relative to glucagon. Substitution of distinct segments of the glucagon receptor core domain with the corresponding segments of the GLP-1 receptor rescued the affinity and potency of specific glucagon analogs. Site-directed mutagenesis identified the Asp385 --> Glu glucagon receptor mutant that specifically rescued Ala2-glucagon. The results show that three distinct epitopes of the glucagon receptor core domain determine specificity for the N terminus of glucagon. We suggest a glucagon receptor binding model in which the extracellular ends of TM2 and TM7 are close to and determine specificity for Gln3 and Ser2 of glucagon, respectively. Furthermore, the second extracellular loop and/or proximal segments of TM4 and/or TM5 are close to and determine specificity for Lys12 of glucagon.