Membrane Binding Assays Research Articles

Distribution and differential regulation of galanin receptor subtypes in rat brain: effects of seizure activity

Galanin, acting at the GalR1-3 subtypes of galanin receptors, is involved in the regulation of cognition, mood, feeding, seizure activity and pain. The understanding of galanin's effects in molecular and cellular terms has been hampered by the lack of receptor subtype selective ligands and antibodies. Previous in situ hybridization data showed that GalR1 and GalR2 receptors are abundant in the rat brain, while the distribution of GalR3 is contradictory and most studies demonstrated a low expression levels in the rat brain. The distribution of galanin receptor subtypes at protein level is unknown. In the present study, we report the regional distribution of the galanin receptors: GalR1 and non-GalR1 receptors, using a recently synthesized high affinity GalR2/3 selective ligand, galanin (2-11), and galanin (1-29), as competitors, in saturating (125)I-galanin membrane binding assay. We show that paraventricular nucleus (PVN) express predominantly GalR1, whereas areas like the dorsal raphe nucleus (DRN), hippocampus and amygdala express both the GalR1 and non-GalR1 receptors. We speculate that the GalR2/3 binding sites detected by galanin (2-11) binding in our study probably represent mostly GalR2 receptors. In addition, we show regionally specific and subtype specific regulation of galanin receptors. Status epilepticus (SE), known to deplete galanin from axonal projections of locus coeruleus and septum/diagonal band neurons in the hippocampus and to induce galanin expression in a subset of hippocampal cells, down regulates GalR2 receptor mRNA and proteins by 30% without altering the GalR1 receptors.

Renal endothelin receptor type B upregulation in rats with low or high renin hypertension.

To evaluate the role of endothelin-1 (ET-1) in hypertension, we investigated density and distribution of ETA and ETB receptors in hearts and kidneys of deoxycorticosterone acetate (DOCA)-salt and 1 kidney -- 1 clip (1K1C) hypertensive rats. Five groups of uninephrectomized Wistar rats were put on a low salt diet. Three groups of rats drank tap water and two groups received saline. One group of each regimen received DOCA subcutaneously and two corresponding groups without DOCA served as controls. The fifth group of rats had the renal artery clipped to induce 1K1C hypertension. At 6 weeks, mean arterial pressure (MAP) was recorded and membrane binding assays using 125I-ET-1 were carried out. MAP was increased from control 122 +/- 3 to 155 +/- 6 and 218 +/- 11 mmHg in DOCA-salt and 1K1C rats, respectively, and cardiac weight index was increased. ETA receptors were predominantly expressed in the heart, whereas ETB receptors were predominant in the kidney. In the kidneys, the density of the ETB receptor subtype was upregulated in DOCA-salt and 1K1C rats from 160 +/- 8 to 217 +/- 12 and 190 +/- 2 fmol/mg (P < 0.05), respectively, and ETA tended to be downregulated (P = 0.057). Plasma renin activity was decreased in DOCA-salt rats from 17 +/- 3 to 0.17 +/- 0.01 ng/ml per h and increased in 1K1C rats on low salt diet to 30 +/- 5 ng/ml per h. Since ETB is the predominant endothelin receptor in the kidneys, upregulation of the ETB receptor mediating vasodilation and downregulation of the ETA receptor mediating vasoconstriction would be compatible with a mainly renal counter-regulatory effect of endothelin-1 to hypertension. Both low and high renin models of hypertension may be affected.

ALpha1-adrenoceptor antagonist properties of CGP 12177A and other beta-adrenoceptor ligands: evidence against beta(3)- or atypical beta-adrenoceptors in rat aorta.

1. The alpha(1)-adrenoceptor antagonist properties of the beta-adrenoceptor nonconventional partial agonist, CGP 12177A, was investigated in functional assays in rat aorta and in radioligand binding assays in rat cerebral cortical membranes. In addition, binding affinities of other beta-adrenoceptor ligands were measured to investigate any correlation between alpha(1)-adrenoceptor affinity and relaxant potency in phenylephrine-constricted rings. 2. In functional studies, CGP 12177A produced parallel rightward shifts of the phenylephrine CRC with no reduction in the maximum responses. Schild regression analysis gave a straight line with a slope of 0.95 (95% CL: 0.87-1.04), suggesting reversible competitive antagonism, and gave a pK(B) value of 5.26. In contrast, CGP 12177A (<or=300 microm) had no effect on contraction induced by the thromboxane-mimetic, U46619. 3. In binding studies, CGP 12177A competed monophasically with [(3)H]prazosin binding (Hill slope, 0.95, 95% CL: 0.76-1.13), giving a pK(i) value of 5.48, in good agreement with the pK(B) from functional studies. 4. Competition experiments with various other beta-adrenoceptor ligands showed that they all displaced [(3)H]prazosin in a manner consistent with one-site competition. pK(i) values were as follows: SR 59230A, 6.25; cyanopindolol, 6.33; bupranolol, 6.35; alprenolol, 5.90; propranolol, 5.80; BRL 37344, 5.50; ICI 118551, 5.55; CGP 20712A, 5.26. The pK(i) values correlated well with the pEC(50) values for relaxation of phenylephrine-constricted rat aorta obtained previously (r(2)=0.984, P<0.0001). 5. In conclusion, relaxant effects of CGP 12177A and other beta-adrenoceptor ligands in phenylephrine-constricted rat aorta can be attributed to alpha(1)-adrenoceptor blockade and are unrelated to effects at beta(3)-adrenoceptors or atypical beta-adrenoceptors.

The [ 3H]dofetilide binding assay is a predictive screening tool for hERG blockade and proarrhythmia: Comparison of intact cell and membrane preparations and effects of altering [K +] o

Introduction: The human ether-a-go-go-related gene (hERG) encodes a potassium channel responsible for the cardiac delayed rectifier current (I Kr) involved in ventricular repolarization. Drugs that block hERG have been associated with QT interval prolongation and serious, sometimes fatal, cardiac arrhythmias (including torsade de pointes). While displacement of [ 3H]dofetilide, a potent methanesulfonanilide hERG blocker, from cells heterologously expressing hERG has been suggested as a screening assay, questions have been raised about its predictive value. Methods: To validate the utility of this assay as a screening tool, we performed a series of saturation and competition binding studies using [ 3H]dofetilide as ligand and either intact cells or membrane preparations from HEK 293 cells stably transfected with hERG K + channels. The object of these experiments was to (1) compare binding K i values for 22 hERG blockers using intact cells or membrane homogenates to determine whether maintaining cell integrity enhanced assay reliability; (2) evaluate the ability of different K + concentrations (2, 5, 10, 20, and 60 mM) to modulate hERG binding; and (3) to establish the predictive value of the assay by comparing K i values from binding studies at 5 and 60 mM [K +] o to functional IC 50 values for hERG current block using 56 structurally diverse drugs. Results: We found (a) comparable K i values in the intact cell and isolated membrane binding assays, although there were some differences in rank order; (b) increasing [K +] o lowered the K d and increased the B max for [ 3H]dofetilide, particularly in the membrane assay; and (c) good correlation between binding K i values and functional IC 50 values for hERG current block. Discussion: In conclusion, increasing K + concentrations results in an increase in both [ 3H]dofetilide affinity for hERG and available binding sites, particularly when using membrane homogenates. There are no meaningful differences between K i values when comparing intact cell versus membrane assay, neither are there meaningful trends with increasing [K +] o within assays. There is good correlation between binding K i values and functional (whole-cell patch clamp) IC 50 values at both 5 and 60 mM K + concentrations ( R 2 values of .824 and .863, respectively). The simplicity, predictability, and adaptability to high-throughput platforms make the [ 3H]dofetilide membrane binding assay a useful tool for screening and ranking compounds for their potential to block the hERG K + channel.

The 63H9dofetilide binding assay is a predictive screening tool for hERG blockade and proarrhythmia: Comparison of intact cell and membrane preparations and effects of altering 6K+9o

Introduction: The human ether-a-go-go-related gene (hERG) encodes a potassium channel responsible for the cardiac delayed rectifier current (IKr) involved in ventricular repolarization. Drugs that block hERG have been associated with QT interval prolongation and serious, sometimes fatal, cardiac arrhythmias (including torsade de pointes). While displacement of [3H]dofetilide, a potent methanesulfonanilide hERG blocker, from cells heterologously expressing hERG has been suggested as a screening assay, questions have been raised about its predictive value.Methods: To validate the utility of this assay as a screening tool, we performed a series of saturation and competition binding studies using [3H]dofetilide as ligand and either intact cells or membrane preparations from HEK 293 cells stably transfected with hERG K+ channels. The object of these experiments was to (1) compare binding Ki values for 22 hERG blockers using intact cells or membrane homogenates to determine whether maintaining cell integrity enhanced assay reliability; (2) evaluate the ability of different K+ concentrations (2, 5, 10, 20, and 60 mM) to modulate hERG binding; and (3) to establish the predictive value of the assay by comparing Ki values from binding studies at 5 and 60 mM [K+]o to functional IC50 values for hERG current block using 56 structurally diverse drugs.Results: We found (a) comparable Ki values in the intact cell and isolated membrane binding assays, although there were some differences in rank order; (b) increasing [K+]o lowered the Kd and increased the Bmax for [3H]dofetilide, particularly in the membrane assay; and (c) good correlation between binding Ki values and functional IC50 values for hERG current block.Discussion: In conclusion, increasing K+ concentrations results in an increase in both [3H]dofetilide affinity for hERG and available binding sites, particularly when using membrane homogenates. There are no meaningful differences between Ki values when comparing intact cell versus membrane assay, neither are there meaningful trends with increasing [K+]o within assays. There is good correlation between binding Ki values and functional (whole-cell patch clamp) IC50 values at both 5 and 60 mM K+ concentrations (R2 values of .824 and .863, respectively). The simplicity, predictability, and adaptability to high-throughput platforms make the [3H]dofetilide membrane binding assay a useful tool for screening and ranking compounds for their potential to block the hERG K+ channel.

A chimeric opioid peptide with mixed mu agonist/delta antagonist properties.

There is evidence to indicate that opioid compounds with mixed mu agonist/delta antagonist properties are analgesics with low propensity to produce tolerance and physical dependence. A chimeric peptide containing the potent and selective mu agonist H-Dmt-D-Arg-Phe-Lys-NH2 ([Dmt1]DALDA) (Dmt=2',6'-dimethyltyrosine) and the potent and selective delta antagonist H-Tyr-TicPsi[CH2-NH]Cha-Phe-OH (TICP[Psi]) (Cha=cyclohexylalanine), connected 'tail-to-tail' via a short linker, was synthesized using a combination of solid-phase and solution techniques. The resulting peptide, H-Dmt-->D-Arg-->Phe-->Lys-NH-CH2-CH2-NH-Phe<--Cha[NH-CH2]PsiTic<--Tyr-H, showed the expected mu agonist/delta antagonist profile in the guinea-pig ileum and mouse vas deferens assays. Its mu and delta receptor binding affinities were in the low nanomolar range, as determined in rat brain membrane binding assays.

Gamma-hydroxybutyric acid, unlike gamma-aminobutyric acid, does not stimulate Gi/Go proteins in rat brain membranes.

gamma-Hydroxybutyric acid is a naturally occurring substance that may act as a neurotransmitter or neuromodulator to elicit several biological effects. Although the existence of a specific gamma-hydroxybutyric acid receptor has been postulated, the receptor protein itself has not been cloned yet. The current study was designed to elucidate whether gamma-hydroxybutyric acid receptors are functionally coupled with heterotrimeric G-proteins, especially Gi/Go family, by means of high-affinity GTPase activity and guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS) binding assays in rat brain membranes. The stimulatory effects of GABAB receptor activation were always determined in parallel as a positive control. The selective GABAB receptor agonist (+/-)-baclofen stimulated the high-affinity GTPase activity in cerebral cortical, hippocampal, and striatal membranes, whereas gamma-hydroxybutyric acid was inactive up to 1 mM in these brain regions. The optimum assay conditions for [35S]GTPgammaS binding to detect a receptor-mediated activation of G-proteins at the greatest signal to noise ratio were then probed as to the concentrations of constituents in the assay mixture (GDP, MgCl2, and NaCl) and incubation period. Even under such an optimized experimental condition, [35S]GTPgammaS binding was not altered by gamma-hydroxybutyric acid in the membranes prepared from cerebral cortex or hippocampus. On the other hand, the specific [35S]GTPgammaS binding was increased by GABAB receptor agonists in a concentration-dependent manner, which was competitively inhibited by CGP54626, a selective GABAB receptor antagonist. These results indicate that gamma-hydroxybutyric acid receptors, if any, are not associated with G-proteins, at least Gi/Go family.

Agonist binding and Gq-stimulating activities of the purified human P2Y1 receptor.

The human P2Y1 receptor (P2Y1-R) was purified after high-level expression from a recombinant baculovirus in Sf9 insect cells. Quantification by protein staining and with a radioligand binding assay using the high-affinity P2Y1-R antagonist [3H]MRS2279 ([3H]2-chloro-N6-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bis-phosphate) indicated a nearly homogenous preparation of receptor protein. Ki values determined in [3H]MRS2279 binding assays for antagonists with the purified P2Y1-R were in good agreement with the Ki and KB values determined for these molecules in membrane binding and activity assays, respectively. Availability of P2Y1-R in purified form allowed direct determination of nucleotide agonist affinities under conditions not compromised by nucleotide metabolism/interconversion, and an order of affinities of 2-methylthio-ADP (2MeSADP) > ADP = 2-methylthioATP = adenosine-5'-O-(3-thio)triphosphate = adenosine-5'-O(2-thiodiphosphate) >> ATP was obtained. The signaling activity of the purified P2Y1-R was quantified after reconstitution in proteoliposomes with heterotrimeric G proteins. Steady-state GTP hydrolysis in vesicles reconstituted with P2Y1-R and Galpha(q)beta(1)gamma(2) was stimulated by the addition of either 2MeADP or RGS4 alone and was increased by up to 50-fold in their combined presence. EC50 values of agonists for activation of the purified P2Y1-R were similar to their respective Ki values determined in radioligand binding experiments with the purified receptor. Moreover, ATP exhibited 20-fold higher EC50 and Ki values than did ADP and was a partial agonist relative to ADP and 2MeSADP under conditions in which no metabolism of the nucleotide occurred. Both RGS4 and PLC-beta1 were potent and efficacious GTPase-activating proteins for Galphaq and Galpha11 in P2Y1-R-containing vesicles. These results illustrate that the binding and signaling properties of the human P2Y1-R can be studied with purified proteins under conditions that circumvent the complications that occur in vivo.

Differential ligand efficacy at h5-HT 1A receptor-coupled G-protein subtypes: a commentary

Activation of heterotrimeric G-protein involves the exchange by α subunits of GDP for GTP. The binding of the hydrolysis-resistant GTP analogue, guanosine-5′- O-(3-[ 35S]thio)-triphosphate ([ 35S]GTPγS), provides a measure of agonist, antagonist and inverse agonist actions at G-protein-coupled receptors. However, classical [ 35S]GTPγS binding methods do not distinguish among the subtypes of G-proteins activated. This is a limitation in view of (i) promiscuity of receptor coupling to multiple families of G-proteins, and (ii) the differential influence of ligands on distinct signal transduction pathways. A recent development of [ 35S]GTPγS binding methodology employing antibody capture and scintillation proximity assays (SPA) permits the targeting of specific Gα subtypes in both recombinant and native tissues. When applied to human serotonin 1A receptor (h5-HT 1A) expressed in Chinese hamster ovary (CHO) cells, this methodology revealed surprising patterns of Gα i3 subunit activation. For example, low but not high concentrations of high-efficacy h5-HT 1A agonists direct receptor signalling to Gα i3. In contrast, partial agonists favour h5-HT 1A receptor signalling to Gα i3 over a wide concentration range. Further, alterations in buffer sodium concentration reversed these actions of agonists: stimulation of Gα i3 activation was observed at high sodium concentration, but inhibition was observed at low sodium concentration, suggestive of protean efficacy, i.e. a switch from positive to negative efficacy dependent on receptor tone. These results indicate that complex changes in both magnitude and direction of response to receptor ligands can occur for specific G-protein subtypes. Interestingly, the inverse agonist, spiperone, inhibited constitutive h5-HT 1A receptor-mediated Gα i3 activation under all conditions tested, consistent with the hypothesis that it selectively stabilises distinct receptor conformation(s). Further, whereas classical [ 35S]GTPγS binding assays in CHO cell membranes failed to demonstrate negative efficacy for the neuroleptic, haloperidol, this compound exhibited robust inverse agonism for the activation of Gα i3 subunits. These data suggest that haloperidol may selectively inhibit constitutive activation of this G-protein subtype and, thus, exhibit inverse agonist-mediated trafficking of receptor signalling.

The Molecular Basis for the pH-activation Mechanism in the Channel-forming Bacterial Colicin E1

The in vitro activity of the channel-forming bacteriocins such as colicin E1 in model membranes requires the specific activation of the protein by an acidic environment in the presence of a membrane potential. Acid activation of the C-terminal domain results in the formation of an insertion-competent intermediate with an enhanced ability to penetrate and perforate cell membranes. We report novel findings of this activation process through the design and study of mutant proteins involving the replacement of conserved Asp residues Asp-408, Asp-410, and Asp-423 within helices 5a and 4 in the colicin E1 channel domain that resulted in enhanced membrane binding, bilayer insertion rates, and ion channel activities at near neutral pH values. This activation process involves the destabilization of a critical salt bridge (Asp-410 and Lys-406) and H-bonds (Asp-408 and Ser-405 main chain; Asp-423 and Lys-420 main chain). The helix-to-coil transition of this motif was identified previously by time-resolved Trp fluorescence measurements (Merrill, A. R., Steer, B. A., Prentice, G. A., Weller, M. J., and Szabo, A. G. (1997) Biochemistry 36, 6874-6884), and here we use this approach to demonstrate that disruption of the helical structure of helices 4 and 5a results in a shift in this equilibrium to favor the coil state. Finally, we show that the essential components of the pH trigger motif are conserved among the channel-forming colicins and that it likely exists within other bacterial proteins and may even have evolved into more sophisticated devices in a number of microbial species.

Export of β-Lactamase Is Independent of the Signal Recognition Particle

In Escherichia coli, three different types of proteins engage the SecY translocon of the inner bacterial membrane for translocation or insertion: 1) polytopic membrane proteins that prior to their insertion into the membrane are targeted to the translocon using the bacterial signal recognition particle (SRP) and its receptor; 2) secretory proteins that are targeted to and translocated across the SecY translocon in a SecA- and SecB-dependent reaction; and 3) membrane proteins with large periplasmic domains, requiring SRP for targeting and SecA for the translocation of the periplasmic moiety. In addition to its role as a targeting device for membrane proteins, a function of the bacterial SRP in the export of SecB-independent secretory proteins has also been postulated. In particular, beta-lactamase, a hydrolytic enzyme responsible for cleavage of the beta-lactam ring containing antibiotics, is considered to be recognized and targeted by SRP. To examine the role of the SRP pathway in beta-lactamase targeting and export, we performed a detailed in vitro analysis. Chemical cross-linking and membrane binding assays did not reveal any significant interaction between SRP and beta-lactamase nascent chains. More importantly, membrane vesicles prepared from mutants lacking a functional SRP pathway did block the integration of SRP-dependent membrane proteins but supported the export of beta-lactamase in the same way as that of the SRP-independent protein OmpA. These data demonstrate that in contrast to previous results, the bacterial SRP is not involved in the export of beta-lactamase and further suggest that secretory proteins of Gram-negative bacteria in general are not substrates of SRP.

Dihydroergotamine and its metabolite, 8'-hydroxy-dihydroergotamine, as 5-HT1A receptor agonists in the rat brain.

1 In addition to stopping migraine attacks, dihydroergotamine (DHE) is an efficient drug for migraine prophylaxis. Whether 5-HT(1A) receptors could contribute to the latter action was assessed by investigating the effects of DHE and its metabolite, 8'-OH-DHE, on these receptors in the rat brain. 2 Membrane binding assays with [(3)H]8-OH-DPAT and [(3)H]WAY 100635 as radioligands showed that both DHE (IC(50)=28-30 nM) and 8'-OH-DHE (IC(50)=8-11 nM) are high-affinity 5-HT(1A) receptor ligands. 3 Both DHE and 8'-OH-DHE enhanced the specific binding of [(35)S]GTP-gamma-S to the dorsal raphe nucleus and the hippocampus in brain sections, but to a lower extent than 5-carboxamido-tryptamine (5-CT) in the latter area. 4 Both DHE (EC(50)=10.9+/-0.3 nM) and 8'-OH-DHE (EC(50)=30.4+/-0.8 nM) inhibited the firing of serotoninergic neurons in the dorsal raphe nucleus within brain stem slices. 5 Intracellular recording showed that 8'-OH-DHE was more potent than DHE to hyperpolarize CA1 pyramidal cells in rat hippocampal slices. 6 Both the stimulatory effects of DHE and 8'-OH-DHE on [(35)S]GTP-gamma-S binding and their electrophysiological effects were completely prevented by the selective 5-HT(1A) receptor antagonist WAY 100635. 7 As expected of 5-HT(1A) receptor partial agonists, DHE and 8'-OH-DHE prevented any subsequent hyperpolarization of CA1 pyramidal cells by 5-HT or 5-CT. 8 Through their actions at 5-HT(1A) auto- (in the dorsal raphe nucleus) and hetero-(notably in the hippocampus) receptors, DHE, and even more its metabolite 8'-OH-DHE, can exert both an inhibitory influence on neuronal excitability and anxiolytic effects which might contribute to their antimigraine prophylactic efficiency.

Characterization of the binding of [3H][Dmt1]H-Dmt-D-Arg-Phe-Lys-NH2, a highly potent opioid peptide.

The dermorphin-derived peptide [Dmt1]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2; Dmt, 2',6'-dimethyltyrosine) labels mu-opioid receptors with high affinity and selectivity in receptor binding assays. In previous studies, [Dmt1]DALDA displayed a mechanism of action distinct from that of morphine, as evidenced by its insensitivity to antisense probes reducing morphine analgesia and incomplete cross tolerance to morphine. In an effort to further elucidate the unusual mechanism of action, [3H][Dmt1]DALDA has been synthesized and its binding profile studied. [3H][Dmt1]DALDA binding was high affinity (KD = 0.22 nM) and showed a regional distribution consistent with mu-receptors with highest levels in calf striatal membranes. [3H][Dmt1]DALDA binding was far less sensitive than [3H][d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) to the effects of divalent and sodium cations and guanine nucleotides, although NaCl and guanosine 5'-(beta,gamma-imido)triphosphate together reduced specific [3H][Dmt1]DALDA binding levels by almost 75%. Competition studies confirmed the mu-selectivity of the binding, with Ki values that were not appreciably different from those seen against [3H]DAMGO. In guanosine 5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS) binding assays in brain and spinal cord membranes, [Dmt1]DALDA was more potent than DAMGO, but showed plateaus suggestive of a partial agonist. [Dmt1]DALDA bound to mu-opioid receptor clone 1 (MOR-1) and its splice variants with high affinity. Unlike [3H]DAMGO, [3H][Dmt1]DALDA seemed to label both agonist and antagonist conformations of MOR-1 expressed in Chinese hamster ovary cells. In [35S]GTPgammaS assays [Dmt1]DALDA showed high efficacy with all the MOR-1 variants, but its potency (EC50) varied markedly among some of the splice variants despite similar affinities in receptor binding assays. Although [3H][Dmt1]DALDA is a very potent mu-selective analgesic, its binding characteristics and its ability to stimulate GTPgammaS binding differed from that of the classical mu-opioid peptide DAMGO.

1,3-dioxolane-based ligands as a novel class of alpha1-adrenoceptor antagonists.

1,3-Dioxolane-based compounds (2-14) were synthesized, and the pharmacological profiles at alpha(1)-adrenoceptor subtypes were assessed by functional experiments in isolated rat vas deferens (alpha(1A)), spleen (alpha(1B)), and aorta (alpha(1D)). Compound 9, with a pA(2) of 7.53, 7.36, and 8.65 at alpha(1A), alpha(1B), and alpha(1D), respectively, is the most potent antagonist of the series, while compound 10 with a pA(2) of 8.37 at alpha(1D) subtype and selectivity ratios of 162 (alpha(1D)/alpha(1A)) and 324 (alpha(1D)/alpha(1B)) is the most selective. Binding assays in CHO cell membranes expressing human cloned alpha(1)-adrenoceptor subtypes confirm the pharmacological profiles derived from functional experiments, although the selectivity values are somewhat lower. Therefore, it is concluded that 1,3-dioxolane-based ligands are a new class of alpha(1)-adrenoceptor antagonists.

The effects of Δ 9-tetrahydrocannabinol physical dependence on brain cannabinoid receptors

The effects of chronic Δ 9-tetrahydrocannabinol on cannabinoid receptor levels and receptor–G-protein coupling were investigated. Male Sprague–Dawley rats were infused continuously with low or high dose regimens of Δ 9-tetrahydrocannabinol or vehicle for 4 days. Following treatment, rats were sacrificed for cannabinoid CB 1 receptor binding analysis or challenged with the cannabinoid CB 1 receptor antagonist, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H-pyrazole-3-carboxamide HCl (SR141716A). The rats receiving Δ 9-tetrahydrocannabinol exhibited antagonist-precipitated withdrawal signs. Each brain region (cerebellum, cortex, hippocampus and basal ganglia) from high-dose rats showed 30–70% decreases in [ 3H] (−)- cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]- trans-4-(3-hydroxyphenyl)cyclohexanol (WIN55212-2) B max values, indicating receptor down-regulation. Most regions showed decreased WIN55212-2-stimulated [ 35S]guanosine-5′- O-3-thiotriphosphate (GTPγS) binding, indicating desensitization of cannabinoid CB 1 receptors. Additional receptor binding assays in cerebellar membranes showed a significantly greater decrease in agonist than in antagonist B max values, indicating a lower fraction of coupled receptors after treatment. Concentration–effect analysis of five agonists revealed that the treatment resulted in greater decreases in the efficacy of low-efficacy agonists.

Visualization of Sigma1 Receptors in Eyes by ex vivo Autoradiography and in vivo Positron Emission Tomography

.Sigma receptors are present on the neurons of the central nervous system and in peripheral organs. They have also been demonstrated in ocular tissues by in vitro membrane binding assays. We have investigated whether sigma1 receptors can be demonstrated in rat eyes by ex vivo autoradiography using [11C]SA4503, a selective radioligand. We also tested whether in vivo positron emission tomography (PET) can be used to show sigma1 receptors in rabbit eyes. In rats, a high accumulation of [11C]SA4503 was found in the iris–ciliary body and retina. A carrier-loading experiment showed that the receptor-specific binding of [11C]SA4503 was approximately 75% of the total binding in the brain. Sigma1 receptors were also detected in the projecting terminals of the retina to the superior colliculus. PET showed radioactivity in the anterior segment including the iris–ciliary body and retina, and pretreatment or displacement by a sigma receptor ligand (haloperidol), suggested that the PET signal reflects radioligand-receptor binding. The high density of sigma1 receptors in the iris–ciliary body and retina was confirmed by ex vivo autoradiography. In conclusion, the iris–ciliary body and retina are rich in sigma1 receptors, and PET may be used to investigate the in vivo distribution of these neuroreceptors in the eye.

Molecular pharmacology of the human prostaglandin D2 receptor, CRTH2.

1. The recombinant human prostaglandin D(2) (PGD(2)) receptor, hCRTH2, has been expressed in HEK293(EBNA) and characterized with respect to radioligand binding and signal transduction properties. High and low affinity binding sites for PGD(2) were identified in the CRTH2 receptor population by saturation analysis with respective equilibrium dissociation constants (K(D)) of 2.5 and 109 nM. This revealed that the affinity of PGD(2) for CRTH2 is eight times less than its affinity for the DP receptor. 2. Equilibrium competition binding assays revealed that of the compounds tested, only PGD(2) and several related metabolites bound with high affinity to CRTH2 (K(i) values ranging from 2.4 to 34.0 nM) with the following rank order of potency: PGD(2)>13,14-dihydro-15-keto PGD(2)>15-deoxy-Delta(12,14)-PGJ(2)>PGJ(2)>Delta(12)-PGJ(2)>15(S)-15 methyl-PGD(2). This is in sharp contrast with the rank order of potency obtained at DP : PGD(2)>PGJ(2)>Delta(12)-PGJ(2)>15-deoxy-Delta(12,14)-PGJ(2) >>>13,14-dihydro-15-keto-PGD(2). 3. Functional studies demonstrated that PGD(2) activation of recombinant CRTH2 results in decrease of intracellular cAMP in a pertussis toxin-sensitive manner. Therefore, we showed that CRTH2 can functionally couple to the G-protein G(alphai/o). PGD(2) and related metabolites were tested and their rank order of potency followed the results of the membrane binding assay. 4. By Northern blot analysis, we showed that, besides haemopoietic cells, CRTH2 is expressed in many other tissues such as brain, heart, thymus, spleen and various tissues of the digestive system. In addition, in situ hybridization studies revealed that CRTH2 mRNA is expressed in human eosinophils. Finally, radioligand binding studies demonstrated that two eosinophilic cell lines, butyric acid-differentiated HL-60 and AML 14.3D10, also endogenously express CRTH2.

Enhanced stability of wild-type and constitutively active alpha(2A)-adrenoceptors by ligands with agonist, silent and inverse agonist properties.

The hypothesis that prolonged treatment of a constitutively active receptor with inverse agonists may lead to increased receptor density was tested for the alpha(2)-adrenoceptor (AR) inverse agonist (+)-RX 811059 at both the wild-type (WT) and Thr(373)Lys alpha(2A) ARs in CHO-K1 cells by monitoring [(3)H]RX 821002 and [(35)S]GTPgammaS binding responses. One-hundred micromolar KCl instead of NaCl in the [(35)S]GTPgammaS membrane binding assay favoured the detection of a high-magnitude constitutive alpha(2A) AR activity. Under this condition, (+)-RX 811059 was an inverse agonist [ E(max) (% vs. basal): Thr(373)Lys alpha(2A) AR (-52+/-2) > WT alpha(2A) AR (-31+/-6)] while atipamezole was a silent neutral antagonist for both WT and Thr(373)Lys alpha(2A) ARs. The B(max) value of [(3)H]RX 821002 binding sites to membranes of transfected CHO-K1 cells was <90% for the Thr(373)Lys alpha(2A) AR compared with the WT alpha(2A) AR (9.1+/-1.4 pmol/mg protein); K(d) values were similar (1.16+/-0.19 nM and 1.51+/-0.15 nM, respectively). Forty-eight-hours' pre-treatment of cells with either 0.1 microM (+)-RX 811059, 1 microM atipamezole or 1 microM of the efficacious agonist d-medetomidine increased the amount of [(3)H]RX 821002 binding sites of both WT (52%-59%) and mutant (306%-447%) Thr(373)Lys alpha(2A) ARs. The same alpha(2) AR ligands also prevented the loss of [(3)H]RX 821002 binding sites as induced by incubation of transfected CHO-K1 cellular membranes at 37 degrees C for 4 h (WT alpha(2A) AR) and 2 h (Thr(373)Lys alpha(2A) AR); 0.1 microM (+)-RX 811059 and 1 microM atipamezole caused an increase compared with the control amount of [(3)H]RX 821002 binding sites to the Thr(373)Lys alpha(2A) AR by 73% and 50%, respectively. In conclusion, no relationship was found between inverse agonism and alpha(2A) AR up-regulation. It is suggested that this is due to structural stabilisation of the alpha(2A) AR, irrespective of the nature of the ligand.

Brain opioid receptor density reflects behavioral and heart rate responses in pigs

Results from our previous research indicate that long-term tether-housed pigs with high and low levels of stereotypies show differences in the density of endogenous opioid receptors in the hippocampus and hypothalamus. It was not clear whether differences in opioid receptor density were induced by the chronic stress of tether housing or stereotypy performance, or were already present before the animals were tethered. The latter possibility was tested in the present experiment. We used a group of 18 nonstereotyping pigs that had no experience with tether housing and investigated whether the animals differed in the density of endogenous opioid receptors in the brain and, if so, whether these differences were related to the animals' reactions to acute challenges. The pigs were subjected to two tests: an open field test and a tethering test. Behavioral reactions as well as heart rate responses were measured. Opioid receptor densities were determined postmortem in the hippocampus and hypothalamus using a membrane binding assay with [ 3H]naloxone as a ligand. Animals differed widely in their responses to the two tests. In support of our hypothesis, we found a relationship between behavioral and heart rate responses and densities of naloxone binding sites in the hippocampus and hypothalamus. The data suggest that endogenous opioid systems in the brain contribute to differences in stress responding between individual pigs.

Structure-activity relationships in 1,4-benzodioxan-related compounds. 7. Selectivity of 4-phenylchroman analogues for alpha(1)-adrenoreceptor subtypes.

WB4101 (1)-related compounds 5-10 were synthesized, and their biological profile at alpha(1)-adrenoreceptor (AR) subtypes and 5-HT(1A) serotoninergic receptors was assessed by binding assays in Chinese hamster ovary and HeLa cell membranes expressing the human cloned receptors. Moreover, their receptor selectivity was further determined in functional experiments in isolated rat prostate (alpha(1A)), vas deferens (alpha(1A)), aorta (alpha(1D)), and spleen (alpha(1B)). In functional assays, compound 5 was the most potent at alpha(1D)-ARs with a reversed selectivity profile (alpha(1D) > alpha(1A) > alpha(1B)) relative to both prototype 1 and phendioxan (2) (alpha(1A) > alpha(1D) > alpha(1B)), whereas compound 8, bearing a carbonyl moiety at position 1, was the most potent at alpha(1A)-ARs with a selectivity profile similar to that of prototypes. The least potent of the series was the trans isomer 6, suggesting that optimum alpha(1)-AR blocking activity in this series is associated with a cis relationship between the 2-side chain and the 4-phenyl ring rather than a trans relationship as previously observed for the 2-side chain and the 3-phenyl ring in 2 and related compounds. Binding affinity results were not in complete agreement with the selectivity profiles deriving from functional experiments. Although a firm explanation was not available, neutral and negative antagonism and receptor dimerization were considered as two possibilities to account for the difference between binding and functional affinities. Finally, compound 5 was selected for a modeling study in comparison with 1, mephendioxan (3), and open phendioxan (4) to achieve information on the physicochemical interactions that account for its high affinity toward alpha(1d/D)-ARs.