[³H]Chiba-1001(methyl-SSR180711) has low in vitro binding affinity and poor in vivo selectivity to nicotinic alpha-7 receptor in rodent brain.

Neuronal nicotinic acetylcholine receptor (nAChR) agonists active at the alpha-7 (α-7) receptor subtype are potential therapeutics for cognitive deficits in schizophrenia, Alzheimer's disease, and other mental disorders. SSR180711, an α-7 selective partial agonist, has been shown to improve preclinical cognition. A novel positron emission tomography (PET) radioligand, ¹¹C-Chiba1001, is a close analog of SSR180711.

Absence of direct effects on the dopamine D2 receptor by mGluR2/3-selective receptor agonists LY 354,740 and LY 379,268.

We previously reported the absence of high-affinity binding of the group II metabotropic glutamate receptor agonists LY 354,740 and LY 379,268 to the D2L dopamine receptor. A rebuttal to our findings has since been reported (see Introduction section); this study represents our response. Analysis by LCMS of LY 354,740 and LY 379,268 used in this study revealed the correct molecular mass for these compounds.

D2 receptor occupancy in conscious rat brain is not significantly distinguished with [3H]-MNPA, [3H]-(+)-PHNO, and [3H]-raclopride.

Positron emission tomography (PET) antagonist ligands such as [(11)C]-raclopride are commonly used to study dopamine D2 receptor (D2) binding of antipsychotics. It has been suggested that agonist radioligands bind preferentially to the high-affinity state of D2 receptor and may provide a more relevant means of assessing D2 occupancy. The main objective of this study was to determine if D2 receptor occupancy (RO) could be differentiated with agonist and antagonist radioligands in vivo.

Whole-mount analysis reveals normal numbers of dopaminergic neurons following misexpression of alpha-Synuclein in Drosophila.

Previously published reports have suggested that misexpression of alpha-Synuclein in the Drosophila central nervous system causes neurodegeneration and progressive age-dependent locomotor dysfunction similar to pathologic and clinical manifestations of Parkinson's disease. The number of dopaminergic (DA) neurons in these studies was assessed using immunohistochemistry with an anti-tyrosine hydroxylase antibody on sequential paraffin sections of fly brains. In contrast, we do not observe any DA cell loss in alpha-Synuclein expressing fly brains when using whole-mount immunohistochemistry as an assay.

Parkin suppresses wild-type alpha-synuclein-induced toxicity in SHSY-5Y cells.

Current hypotheses concerning the mechanism of neuronal cell death in Parkinson's disease (PD) and related synucleopathies propose a functional interaction between parkin and alpha-synuclein (alphaS). Recently parkin was shown to suppress mutant alphaS-induced toxicity in primary neurons, providing a basis for an association between these proteins and neuronal loss [Neuron 36 (2000) 1007-1019]. We have asked if a similar association could be made between wild-type (wt) alphaS and parkin.

Linear non-competitive inhibition of solubilized human gamma-secretase by pepstatin A methylester, L685458, sulfonamides, and benzodiazepines.

Cerebral deposition of amyloid beta-protein (A beta) is believed to play a key role in the pathogenesis of Alzheimer's disease. Because A beta is produced from the processing of amyloid beta-protein precursor (APP) by beta- and gamma-secretases, these enzymes are considered important therapeutic targets for identification of drugs to treat Alzheimer's disease. Unlike beta-secretase, which is a monomeric aspartyl protease, gamma-secretase activity resides as part of a membrane-bound, high molecular weight, macromolecular complex.

Homogeneous pharmacologic and cell-based screens provide diverse strategies in drug discovery: somatostatin antagonists as a case study.

The development of high throughput, homogeneous pharmacologic and functional assays and their implementation in screening combinatorial libraries has increased the pace of stochastic drug discovery in recent years. New, noninvasive approaches involving radiometric proximity assays, an array of fluorescence-based technologies, and reporter gene constructs in mammalian and nonmammalian systems are providing more options for the selection of specific therapeutic targets. The increasing sophistication of homogeneous assay designs has also served as a springboard to better lead validation in drug discovery initiatives.

A linear hexapeptide somatostatin antagonist blocks somatostatin activity in vitro and influences growth hormone release in rats.

Somatostatin (SRIF) is the main inhibitory peptide regulating growth hormone (GH) secretion. It has been difficult to establish the role of endogenous SRIF release in the absence of pure SRIF antagonists. Although several SRIF antagonists have recently been described, none have been shown to possess in vivo activity in the absence of added SRIF.

Purification of the growth hormone releasing hormone receptor with a C-terminal, biotinylated affinity ligand.

The receptor for growth hormone-releasing hormone (GHRH) has been purified from bovine pituitary tissue and HEK293 cells transfected with human or porcine receptor using a retrievable biotinylated GHRH analog. Custom synthesized [His1, Nle27, Biotin-Lys41]-human GHRH-(1-41)-NH2 (GHRHb) bound to pituitary membranes with affinity comparable to human GHRH. GHRHb which has the biotinyl group on the C-terminus of the peptide allowed simultaneous binding to both the receptor and streptavidin agarose.

A rapid and sensitive binding assay for growth hormone releasing factor.

A binding assay for growth hormone releasing factor (GRF) has been developed using scintillation proximity assay (SPA) technology. Binding conditions were validated by several criteria. Equilibrium binding was attained within three hours at 22 degrees C in crude membrane fractions of HEK293 (293-P2) and GH4C1 (GH4-P1) cells transfected with the porcine GRF receptor.

Selective uptake of estrogenic compounds by Saccharomyces cerevisiae: a mechanism for antiestrogen resistance in yeast expressing the mammalian estrogen receptor.

Estrogen antagonists such as ICI164,384 do not inhibit 17 beta-estradiol (E2)-dependent gene activity in yeast expressing the mammalian estrogen receptor although these compounds bind to receptors isolated from these cells. Various explanations have been offered for antiestrogen resistance in yeast systems including differences in cell-specific components and lack of permeability of the yeast cell wall to these compounds. We have used a strain of Saccharomyces cerevisiae transformed with the human estrogen receptor gene, and two estrogen response elements linked to a lacZ reporter gene to study the pharmacology of estrogen agonists and antagonists.

Photoaffinity cross-linking to the pituitary receptor for growth hormone-releasing factor.

Photoaffinity cross-linking methods presented here demonstrate a 55-kilodalton (kDa) GH-releasing factor (GRF) receptor in ovine pituitary membranes and in cell lines expressing the cloned human pituitary receptor complementary DNA. Covalent cross-linking of photoprobe to this high affinity site is strongly competed by 1 nM GRF. Competition shows strong specificity for GRF over related peptides.

Molecular cloning and expression of a human anterior pituitary receptor for growth hormone-releasing hormone.

GH-releasing hormone (GHRH), acting through the GHRH receptor (GHRH-R), plays a pivotal role in the regulation of GH synthesis and secretion in the pituitary. It is possible that GHRH may serve other roles in other tissues. Here we report the cloning of a cDNA encoding a human GHRH-R from an acromegalic pituitary cDNA library.

Purification of a pituitary receptor for somatostatin. The utility of biotinylated somatostatin analogs.

A somatostatin (SRIF) receptor and its associated Gi regulatory proteins was purified from GH4C1 rat pituitary cells by: 1) saturation of the membrane-bound receptor with biotinyl-NH-[Leu8,D-Trp22,Tyr25] SRIF28 (bio-S28); 2) solubilization of receptor-ligand (R.L) complex with deoxycholate-lysophosphatidylcholine (D.L); 3) adsorption of solubilized receptor-ligand complex to immobilized streptavidin; and 4) elution of receptor and G-protein by GTP.

Partial amino acid sequence of a somatostatin receptor isolated from GH4C1 pituitary cells.

A somatostatin receptor isolated from GH4C1 rat pituitary tumor-derived cells was cleaved with cyanogen bromide or cyanogen bromide+trypsin to obtain sequenceable fragments. Five unique amino acid sequences ranging from 6 to 27 amino acid residues were obtained. The sequence was identical to sequence recently reported for one of two somatostatin receptors cloned from human pancreas [Yamada et al.

Ionophore bromo-A23187 reveals cellular calcium stores in single pituitary somatotropes.

Free cytosolic calcium concentration, [Ca2+]i, in single rat pituitary cells can be measured with the fluorescent, calcium-sensitive probe fura-2 and digital image analysis. A reverse hemolytic plaque assay (RHPA) identifies somatotropes in the mixed population of pituitary cells. Previous studies showed that growth hormone releasing factor (GRF) stimulates growth hormone (GH) release from pituitary somatotropes by increasing the influx of calcium into the cell.

Cross-linking of a growth hormone releasing factor-binding protein in anterior pituitary cells.

Growth hormone-releasing factor (GRF) stimulates the release of growth hormone from the anterior pituitary and is related to the peptides of the glucagon/secretin family. Although the mechanism of action of this hormone has been studied in considerable detail, little is known concerning the GRF receptor itself. We have attempted to label the GRF receptor by chemically coupling the 125I-GRF analog [His1, Nle27]-hGRF(1-32)-NH2 (GRFa) (where Nle is norleucine) to plated rat anterior pituitary cells with the protein cross-linker disuccinimidyl suberate (DSS) (0.

Protein kinase C potentiates corticotropin releasing factor stimulated cyclic AMP in pituitary.

The hypophysiotrophic hormone corticotropin releasing factor (CRF) stimulates the anterior pituitary corticotroph to export stress hormones such as adrenocorticotrophic hormone (ACTH). In rat anterior pituitary cells, CRF-induced elevation of cyclic AMP was profoundly potentiated (by an order of magnitude) by stimulators of protein kinase C. This effect occurred within minutes, was concentration dependent, and exhibited the appropriate pharmacological specificity to attribute the effects to protein kinase C.

Pertussis toxin mediates ADP-ribosylation of pituitary membrane proteins.

Pertussis toxin catalyzes the ADP-ribosylation of the inhibitory subunit (Ni) of adenylate cyclase. Despite several studies which demonstrate that pertussis toxin influences cyclic AMP accumulation and hormone secretion in normal anterior pituitary cells, the target protein(s) for this toxin in these cells has not been identified. We have examined pertussis toxin mediated ADP-ribosylation in membrane preparations of tumor-derived (235-1, GH4C1, GH3) and normal anterior pituitary cells.

Stimulation of calmodulin by cadmium ion.

Cd2+, a serious environmental pollutant in certain industrial regions, accumulates in mammalian tissues with a very slow turnover. Using various criteria, we studied the ability of Cd2+ to substitute for Ca2+ in calmodulin (CaM), a ubiquitous Ca2+-binding protein that mediates many of the Ca2+ effects. CaM bound Cd2+ with a Kd of 4.

Activation of calmodulin by various metal cations as a function of ionic radius.

The active form of calmodulin is a Ca2+ . calmodulin complex. The purpose of this investigation was to determine whether other metal cations substitute for Ca2+ to activate calmodulin.

Calcium/calmodulin-dependent phosphorylation of vimentin in rat sertoli cells.

Ca2+-dependent protein phosphorylation and the role of calmodulin in this process was investigated in subcellular fractions of primary cultures of rat Sertoli cells. Significant Ca2+/calmodulin-dependent protein phosphorylation in Sertoli cells was restricted to the cytosol fraction. The calmodulin dependence of these effects was confirmed by using the calmodulin inhibitor trifluoperazine.

Temporary sterility produced in male mice by 5-thio-D-glucose.

When 5-thio-D-glucose was fed to male mice at daily dose levels greater than 30 mg/kg sperm development was completely inhibited within 3 weeks and remained so without impairment of libido for the experimental period of 7 weeks. Removal of this substance from the diet resulted in a resumption of sperm development and fertility within 5 to 8 weeks. Normal litters were sired by males which had recovered after this treatment.