Variants in the 1q21 risk region are associated with a visual endophenotype of autism and schizophrenia

Deficits in sensitivity to visual stimuli of low spatial frequency and high temporal frequency (so-called frequency-doubled gratings) have been demonstrated both in schizophrenia and in autism spectrum disorder (ASD). Such basic perceptual functions are ideal candidates for molecular genetic study, because the underlying neural mechanisms are well characterized; but they have sometimes been overlooked in favor of cognitive and neurophysiological endophenotypes, for which neural substrates are often unknown. Here, we report a genome-wide association study of a basic visual endophenotype associated with psychological disorder. Sensitivity to frequency-doubled gratings was measured in 1060 healthy young adults, and analyzed for association with genotype using linear regression at 642758 single nucleotide polymorphism (SNP) markers. A significant association (P=7.9×10) was found with the SNP marker rs1797052, situated in the 5′-untranslated region of PDZK1; each additional copy of the minor allele was associated with an increase in sensitivity equivalent to more than half a standard deviation. A permutation procedure, which accounts for multiple testing, showed that the association was significant at the α=0.005 level. The region on chromosome 1q21.1 surrounding PDZK1 is an established susceptibility locus both for schizophrenia and for ASD, mirroring the common association of the visual endophenotype with the two disorders. PDZK1 interacts with N-methyl-d-aspartate receptors and neuroligins, which have been implicated in the etiologies of schizophrenia and ASD. These findings suggest that perceptual abnormalities observed in two different disorders may be linked by common genetic elements. © 2013 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

CoMFA and homology-based models of the glycine binding site of N-methyl-D-aspartate receptor

Homology modeling was used to build 3D models of the N-methyl-D-aspartate (NMDA) receptor glycine binding site on the basis of an X-ray structure of the water-soluble AMPA-sensitive receptor. The docking of agonists and antagonists to these models was used to reveal binding modes of ligands and to explain known structure-activity relationships. Two types of quantitative models, 3D-QSAR/CoMFA and a regression model based on docking energies, were built for antagonists (derivatives of 4-hydroxy-2-quinolone, quinoxaline-2,3-dione, and related compounds). The CoMFA steric and electrostatic maps were superimposed on the homology-based model, and a close correspondence was marked. The derived computational models have permitted the evaluation of the structural features crucial for high glycine binding site affinity and are important for the design of new ligands.

Modulation of contractile function through NPY receptors during development of cardiomyocyte hypertrophy.

Severity of left ventricular hypertrophy (LVH) correlates with elevated plasma levels of neuropeptide Y (NPY) in hypertension. NPY elicits positive and negative contractile effects in cardiomyocytes through Y(1) and Y(2) receptors, respectively. This study tested the hypothesis that NPY receptor-mediated contraction is altered during progression of LVH. Ventricular cardiomyocytes were isolated from spontaneously hypertensive rats (SHRs) pre-LVH (12 weeks), during development (16 weeks), and at established LVH (20 weeks) and age-matched normotensive Wistar Kyoto (WKY) rats. Electrically stimulated (60 V, 0.5 Hz) cell shortening was measured using edge detection and receptor expression determined at mRNA and protein level. The NPY and Y(1) receptor-selective agonist, Leu(31)Pro(34)NPY, stimulated increases in contractile amplitude, which were abolished by the Y(1) receptor-selective antagonist, BIBP3226 [R-N(2)-(diphenyl-acetyl)-N-(4-hydroxyphenyl)methyl-argininamide)], confirming Y(1) receptor involvement. Potencies of both agonists were enhanced in SHR cardiomyocytes at 20 weeks (2300- and 380-fold versus controls). Maximal responses were not attenuated. BIBP3226 unmasked a negative contraction effect of NPY, elicited over the concentration range (10(-12) to 3 x 10(-9) M) in which NPY and PYY(3-36) attenuated the positive contraction effects of isoproterenol, the potencies of which were increased in cardiomyocytes from SHRs at 20 weeks (175- and 145-fold versus controls); maximal responses were not altered. Expression of NPY-Y(1) and NPY-Y(2) receptor mRNAs was decreased (55 and 69%) in left ventricular cardiomyocytes from 20-week-old SHRs versus age-matched WKY rats; parallel decreases (32 and 80%) were observed at protein level. Enhancement of NPY potency, producing (opposing) contractile effects on cardiomyocytes together with unchanged maximal response despite reduced receptor number, enables NPY to contribute to regulating cardiac performance during compensatory LVH.

Partial agonism, neutral antagonism, and inverse agonism at the human wild-type and constitutively active cholecystokinin-2 receptors

Cholecystokinin receptor-2 (CCK2R) is a G protein receptor that regulates a number of physiological functions. Activation of CCK2R and/or expression of a constitutively active CCK2R variant may contribute to human diseases, including digestive cancers. Search for antagonists of the CCK2R has been an important challenge during the last few years, leading to discovery of a set of chemically distinct compounds. However, several early-discovered antagonists turned out to be partial agonists. In this context, we carried out pharmacological characterization of six CCK2R antagonists using COS-7 cells expressing the human CCK2R or a CCK2R mutant having a robust constitutive activity on inositol phosphates production, and we investigated the molecular mechanisms which, at a CCK2R binding site, account for these features. Results indicated that three compounds, 3R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3- yl)-N'-(3-methylphenyl)urea (L365,260), 4-{[2-[[3-(lH-indol-3-yl)-2- methyl-1-oxo-2-[[[1.7.7-trimethyl-bicyclo[2.2.1]hept-2-yl)-oxy]carbonyl]amino] propyl]amino]-1-phenylethyl]amino-4-oxo-[lS-la.2[S*(S*)]4a]} -butanoate N-methyl-D-glucamine (PD135, 158), and (R)-1-naphthalenepropanoic acid, b-[2-[[2-(8-azaspiro-[4.5]dec-8-ylcarbonyl)-4,6-dimethylphenyl]amino]-2- oxoethyl] (CR2945), were partial agonists; one molecule, 1-[(R)-2,3-dihydro-1- (2,3-dihydro-1-(2-methylphenacyl)-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl] -3-(3-methylphenyl)urea (YM022), was a neutral antagonist; and two compounds, N-(+)-[1-(adamant-1-ylmethyl)-2,4-dioxo-5-phenyl2,3,4,5-tetrahydro-1H-1, 5-benzodiazepin-3-yl]-N'-phenylurea (GV150,013X) and ([(N-[methoxy-3 phenyl] N-[N-methyl N-phenyl carbamoylmethyl], carbomoylmethyl)-3 ureido]-3-phenyl)2-propionic acid (RPR101,048), were inverse agonists. Furthermore, target- and pharmacophore-based docking of ligands followed by molecular dynamic simulation experiments resulted in consistent motion of aromatic residues belonging to a network presumably important for activation, thus providing the first structural explanations for the different pharmacological profiles of tested compounds. This study confirms that several referenced so-called antagonists are in fact partial agonists, and because of this undesired activity, we suggest that newly generated molecules should be preferred to efficiently block CCK2R-related physiological effects. Furthermore, data on the structural basis for the different pharmacological features of CCK2R ligands will serve to further clarify CCK2R mechanism of activation. Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics.

A Novel Selective Muscarinic Acetylcholine Receptor Subtype 1 Antagonist Reduces Seizures without Impairing Hippocampus-Dependent Learning

Previous studies suggest that selective antagonists of specific subtypes of muscarinic acetylcholine receptors (mAChRs) may provide a novel approach for the treatment of certain central nervous system (CNS) disorders, including epileptic disorders, Parkinson's disease, and dystonia. Unfortunately, previously reported antagonists are not highly selective for specific mAChR subtypes, making it difficult to definitively establish the functional roles and therapeutic potential for individual subtypes of this receptor subfamily. The M 1 mAChR is of particular interest as a potential target for treatment of CNS disorders. We now report the discovery of a novel selective antagonist of M-1 mAChRs, termed VU0255035 [N-(3-oxo-3-(4-(pyridine-4-yl)piperazin-1-yl)propyl)benzo[c][1,2,5]thiadiazole-4-sulfonamide]. Equilibrium radioligand binding and functional studies demonstrate a greater than 75-fold selectivity of VU0255035 for M-1 mAChRs relative to M-2-M-5. Molecular pharmacology and mutagenesis studies indicate that VU0255035 is a competitive orthosteric antagonist of M-1 mAChRs, a surprising finding given the high level of M-1 mAChR selectivity relative to other orthosteric antagonists. Whole-cell patch-clamp recordings demonstrate that VU0255035 inhibits potentiation of N-methyl-D-aspartate receptor currents by the muscarinic agonist carbachol in hippocampal pyramidal cells. VU0255035 has excellent brain penetration in vivo and is efficacious in reducing pilocarpine-induced seizures in mice. We were surprised to find that doses of VU0255035 that reduce pilo-carpine-induced seizures do not induce deficits in contextual freezing, a measure of hippocampus-dependent learning that is disrupted by nonselective mAChR antagonists. Taken together, these data suggest that selective antagonists of M-1 mAChRs do not induce the severe cognitive deficits seen with nonselective mAChR antagonists and could provide a novel approach for the treatment certain of CNS disorders.

Artificial receptors for the extraction of nucleoside metabolite 7-methylguanosine from aqueous media made by molecular imprinting

A series of imprinted polymers targeting nucleoside metabolites, prepared using a template analogue approach, are presented. These were prepared following selection of the optimum functional monomer by solution association studies using 1H-NMR titrations whereby methacrylic acid was shown to be the strongest receptor with and affinity constant of 621 ± 51 L mol-1 vs. 110 ± 16 L mol-1 for acrylamide. The best performing polymers were prepared using methanol as porogenic co-solvent and although average binding site affinities were marginally reduced, 2.3×104 L mol-1 vs. 2.7×104 L mol-1 measured for a polymer prepared in acetonitrile, these polymers contained the highest number of binding sites, 5.27 μmol g-1¬¬ vs. 1.64 μmol g-1, while they also exhibited enhanced selectivity for methylated guanosine derivatives. When applied as sorbents in the extraction of nucleoside derivative cancer biomarkers from synthetic urine samples, significant sample clean-up and recoveries of up to 90% for 7-methylguanosine were achieved.

Inositol 1,4,5-trisphosphate receptors modulate Ca2+ sparks and Ca2+ store content in vas deferens myocytes.

Spontaneous Ca2+ sparks were observed in fluo 4-loaded myocytes from guinea pig vas deferens with line-scan confocal imaging. They were abolished by ryanodine (100 microM), but the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) blockers 2-aminoethoxydiphenyl borate (2-APB; 100 microM) and intracellular heparin (5 mg/ml) increased spark frequency, rise time, duration, and spread. Very prolonged Ca2+ release events were also observed in approximately 20% of cells treated with IP3R blockers but not under control conditions. 2-APB and heparin abolished norepinephrine (10 microM; 0 Ca2+)-evoked Ca2+ transients but increased caffeine (10 mM; 0 Ca2+) transients in fura 2-loaded myocytes. Transients evoked by ionomycin (25 microM; 0 Ca2+) were also enhanced by 2-APB. Ca2+ sparks and transients evoked by norepinephrine and caffeine were abolished by thimerosal (100 microM), which sensitizes the IP3R to IP3. In cells voltage clamped at -40 mV, spontaneous transient outward currents (STOCs) were increased in frequency, amplitude, and duration in the presence of 2-APB. These data are consistent with a model in which the Ca2+ store content in smooth muscle is limited by tonic release of Ca2+ via an IP3-dependent pathway. Blockade of IP3Rs elevates sarcoplasmic reticulum store content, promoting Ca2+ sparks and STOC activity.

Carbachol triggers RyR-dependent Ca(2+) release via activation of IP(3) receptors in isolated rat gastric myocytes.

Possible interactions between different intracellular Ca(2+) release channels were studied in isolated rat gastric myocytes using agonist-evoked Ca(2+) signals. Spontaneous, local Ca(2+) transients were observed in fluo-4-loaded cells with linescan confocal imaging. These were blocked by ryanodine (100 microM) but not by the inositol 1,4,5-trisphosphate receptor (IP(3)R) blocker, 2-aminoethoxydiphenyl borate (100 microM), identifying them as Ca(2+) sparks. Caffeine (10 mM) and carbachol (10 microM) initiated Ca(2+) release at sites which co-localized with each other and with any Ca(2+) spark sites. In fura-2-loaded cells extracellular 2-aminoethoxydiphenyl borate and intracellular heparin (5 mg ml(-1)) both inhibited the global cytoplasmic [Ca(2+)] transient evoked by carbachol, confirming that it was IP(3)R-dependent. 2-Aminoethoxydiphenyl borate and heparin also increased the response to caffeine. This probably reflected an increased Ca(2+) store content since 2-aminoethoxydiphenyl borate more than doubled the amplitude of transients evoked by ionomycin. Ryanodine completely abolished carbachol and caffeine responses but only reduced ionomycin transients by 30 %, suggesting that blockade of carbachol transients by ryanodine was not simply due to store depletion. Double labelling of IP(3)Rs and RyRs demonstrated extensive overlap in their distribution. These results suggest that carbachol stimulates Ca(2+) release through co-operation between IP(3)Rs and RyRs, and implicate IP(3)Rs in the regulation of Ca(2+) store content.