Autoradiographic localization of nicotinic receptor binding in rat brain using [3H]methylcarbamylcholine, a novel radioligand

Light microscopic autoradiography was used to visualize the neuroanatomical distribution of nicotinic receptors in rat brain using a novel radioligand, [3H]methylcarbamylcholine (MCC). Specific [3H]MCC binding to slide-mounted tissue sections of rat brain was saturable, reversible and of high affinity. Data analysis revealed a single population of [3H]MCC binding sites with a Kd value of 1.8 nM and Bmax of 20.1 fmol/mg protein. Nicotinic agonists and antagonists competed for [3H]MCC binding sites in slide-mounted brain sections with much greater potency than muscarinic drugs. The rat brain areas containing the highest densities of [3H]MCC binding were in thalamic regions, the medial habenular nucleus and the superior colliculus. Moderate densities of [3H]MCC binding were seen over the anterior cingulate cortex, the nucleus accumbens, the zona compacta of substantia nigra and ventral tegmental area. Low densities of [3H]MCC binding were found in most other brain regions. These data suggest that [3H]MCC selectively labels central nicotinic receptors and that these receptors are concentrated in the thalamus, the medial habenular nucleus and the superior colliculus of the rat brain.     

Species differences in cannabinoid receptor 2 (CNR2 gene): identification of novel human and rodent CB2 isoforms, differential tissue expression and regulation by cannabinoid receptor ligands

Cannabinoids, endocannabinoids and marijuana activate two well-characterized cannabinoid receptors (CB-Rs), CB1-Rs and CB2-Rs. The expression of CB1-Rs in the brain and periphery has been well studied, but neuronal CB2-Rs have received much less attention than CB1-Rs. Many studies have now identified and characterized functional glial and neuronal CB2-Rs in the central nervous system. However, many features of CB2-R gene structure, regulation and variation remain poorly characterized in comparison with the CB1-R. In this study, we report on the discovery of a novel human CB2 gene promoter transcribing testis (CB2A) isoform with starting exon located ca 45 kb upstream from the previously identified promoter transcribing the spleen isoform (CB2B). The 5' exons of both CB2 isoforms are untranslated 5'UTRs and alternatively spliced to the major protein coding exon of the CB2 gene. CB2A is expressed higher in testis and brain than CB2B that is expressed higher in other peripheral tissues than CB2A. Species comparison found that the CB2 gene of human, rat and mouse genomes deviated in their gene structures and isoform expression patterns. mCB2A expression was increased significantly in the cerebellum of mice treated with the CB-R mixed agonist, WIN55212-2. These results provide much improved information about CB2 gene structure and its human and rodent variants that should be considered in developing CB2-R-based therapeutic agents.     

Bacterial expression of functional, biotinylated peripheral cannabinoid receptor CB2

A biotin-protein ligase recognition site (BRS) was inserted into a polypeptide comprised of the maltose-binding protein, the peripheral cannabinoid receptor (CB2), thioredoxin A, and a polyhistidine tag at the carboxy terminus. Expression levels of the recombinant receptor in Escherichia coli BL21(DE3) cells were approximately 1mg per liter of bacterial culture. The biotinylated CB2-fusion fully retained its ligand-binding capacity. Introduction of the BRS at the C-terminus of the CB2 fusion protein (construct CB2-109) resulted in its complete in vivo biotinylation; the biotinylated protein was streptavidin-binding competent. Positioning of the BRS near the N-terminus of CB2 (CB2-112) resulted in a very low level of biotinylation in vivo. However, the detergent solubilized and purified CB2-112 fusion protein were successfully biotinylated in vitro by action of a BirA biotin-protein ligase. The biotinylated CB2-112 fusion protein was cleaved by the tobacco etch virus protease at specifically inserted sites, and deposited onto monomeric avidin agarose beads. Biotinylation of the recombinant CB2 receptor enabled not only purification but also immobilization of the GPCR on a solid support in homogeneous orientation which is beneficial for subsequent structural characterization.     

Kinetic analysis of [35S]dATP alpha S interaction with P2y(1) nucleotide receptor

The kinetics of 2'-deoxyadenosine-5'-O-(1-thiotriphosphate) ([(35)S]dATP alpha S) interaction with membrane fragments of transfected astrocytoma 1321N1 cells, expressing human P2Y(1) receptors, and the same wild-type cells, not expressing P2Y receptors were studied. Binding of this radioligand was observed with both types of membranes, but sites showing slow on-rate were found only on the transfected cells. These "slow" binding sites behaved as a kinetically homogeneous population and their interaction with the radioligand was shown to occur in two steps, R+A(K(A))<==>RA(k(i))<==>(k(-i))(RA), including the relatively slow isomerization of the complex RA into (RA). Evidence was obtained to assign the isomerized ("slow") binding sites on the transfected cells as P2Y(1) receptor sites, differentiated from other binding sites of non-receptor origin by kinetic analysis, and characterised by the kinetic parameters K(A)=59 +/- 19 nM, k(i)=(9.0 +/- 0.8)10(-3)s(-1) and k(-i)=(3.9 +/- 0.7)10(-3)s(-1). [(35)S]dATP alpha S binding, with kinetic criteria, can be of value for differentiation of the receptor sites from non-receptor sites and thus provides solid basis for radioligand assay of P2Y(1) receptors.     

Synthesis and biodistribution of [11C]A-836339, a new potential radioligand for PET imaging of cannabinoid type 2 receptors (CB2)

Recently, A-836339 [2,2,3,3-tetramethylcyclopropanecarboxylic acid [3-(2-methoxyethyl)-4,5-dimethyl-3H-thiazol-(2Z)-ylidene]amide] (1) was reported to be a selective CB₂ agonist with high binding affinity. Here we describe the radiosynthesis of [¹¹C]A-836339 ([¹¹C]1) via its desmethyl precursor as a candidate radioligand for imaging CB₂ receptors with positron-emission tomography (PET). Whole body and the regional brain distribution of [¹¹C]1 in control CD1 mice demonstrated that this radioligand exhibits specific uptake in the CB₂-rich spleen and little specific in vivo binding in the control mouse brain. However, [¹¹C]1 shows specific cerebral uptake in the lipopolysaccharide (LPS)-induced mouse model of neuroinflammation and in the brain areas with Aβ amyloid plaque deposition in a mouse model of Alzheimer’s disease (APPswe/PS1dE9 mice). These data establish a proof of principle that CB₂ receptors binding in the neuroinflammation and related disorders can be measured in vivo.     

D-[35S(U)]inositol 1,4,5-trisphosphorothioate, a novel radioligand for the inositol 1,4,5-trisphosphate receptor. Complex binding to rat cerebellar membranes.

D-[35S(U)]myo-inositol 1,4,5-trisphosphorothioate [( 35S]InsPS3), a synthetic, metabolically stable analogue of inositol 1,4,5-trisphosphate (InsP3), binds with high affinity (Kd 58.6 +/- 9.1 nM) to rat cerebellar membranes revealing a high density of specific binding sites (Bmax 21.5 +/- 2.1 pmol/mg of protein). Comparison with [3H]InsP3 binding reveals a higher density of sites labelled by [35S]InsPS3 and complex competition curves for displacement of specific [35S]InsPS3 by InsP3. The results suggest that [35S]InsPS3 labels two sites in rat cerebellar membranes with equal affinity: the InsP3 receptor and a site that displays low affinity for InsP3.     

The [35S]GTPgammaS binding assay: approaches and applications in pharmacology

Receptors of the of seven transmembrane spanning, heterotrimeric G protein coupled family (GPCR) play crucial roles in regulating physiological functions and consequently are targets for the action of many classes of drugs. Activation of receptor by agonist leads to the dissociation of GDP from Galpha of the Galphabetagamma heterotrimer, followed by the binding of GTP to Galpha and subsequent modulation of downstream effectors. The G protein heterotrimer is reformed by GTPase activity of the Galpha subunit, forming Galpha-GDP and so allowing Galpha and Gbetagamma to recombine. The [35S]GTPgammaS assay measures the level of G protein activation following agonist occupation of a GPCR, by determining the binding of the non-hydrolyzable analog [35S]GTPgammaS to Galpha subunits. Thus, the assay measures a functional consequence of receptor occupancy at one of the earliest receptor-mediated events. The assay allows for traditional pharmacological parameters of potency, efficacy and antagonist affinity, with the advantage that agonist measures are not subjected to amplification or other modulation that may occur when analyzing parameters further downstream of the receptor. In general the assay is experimentally more feasible for receptors coupled to the abundant G(i/o) proteins. Nevertheless, [35S]GTPgammaS binding assays are used with GPCRs that couple to the G(s) and G(q) families of G proteins, especially in artificial expression systems, or using receptor-Galpha constructs or immunoprecipitation of [35S]GTPgammaS-labeled Galpha. The relative simplicity of the assay has made it very popular and its use is providing insights into contemporary pharmacological topics including the roles of accessory proteins in signaling, constitutive activity of receptors and agonist specific signaling.     

Design, expression, and characterization of a synthetic human cannabinoid receptor and cannabinoid receptor/ G-protein fusion protein.

We report here the synthesis and characterization of two gene constructs designed to facilitate structure/function studies of the human neuronal cannabinoid receptor, CB1. The first gene, which we call shCB1, is a synthetic gene containing unique restriction sites spaced roughly 50-100 bases apart to facilitate rapid mutagenesis and cloning. A nine amino acid epitope tag (from the rhodopsin C-terminus) is also present in the shCB1 C-terminal tail to enable detection and purification using the monoclonal antibody 1D4. We find that that the shCB1 gene can be transiently expressed in COS cells with yield of approximately 10-15 micro g receptor per 15 cm plate and is wild type like in its ability to bind cannabinoid ligands. Our confocal microscopy studies indicate shCB1 targets to the membrane of HEK293 cells and is internalized in response to agonist. To facilitate functional studies, we also made a chimera in which the C-terminus of shCB1 was fused with the N-terminus of a G-protein alpha subunit, Galphai. The shCB1/Galphai chimera shows agonist stimulated GTPgammaS binding, and thus provides a simplified way to measure agonist induced CB1 activation. Taken together, the shCB1 and shCB1/Galphai gene constructs provide useful tools for biochemical and biophysical examinations of CB1 structure, activation and attenuation.     

3H]desGly-NH2(9)-d(CH2)5[D-Ileu2,Ileu4]AVP: an AVP V2 receptor antagonist radioligand

Binding characteristics of the selective V₂ antagonist radioligand [³H]desGly-NH₂⁹-d(CH₂)₅[D-Ileu²,Ileu⁴]AVP to rat kidney were determined. Binding was specific, saturable and reversible. The peptide bound to a single class of high-affinity binding sites with B_max 69.4±6.8 fmol/mg protein and K_D 2.8±0.3 nM. AVP and other related peptides displaced [³H]desGly-NH₂⁹-d(CH₂)₅[D-Ileu²,Ileu⁴]AVP binding. The order of potency of inhibition was desamino-D-AVP > AVP > d(CH₂)₅[D-Ileu²,Ileu⁴]AVP > oxytocin > d(CH₂)₃[Tyr(Me)²]AVP > d(CH₂)₅[sarcosine⁷]AVP, which is typical of a selective V₂ radioligand. Autoradiographic localization of [³H]desGly-NH₂⁹-d(CH₂)₅[D-Ileu²,Ileu⁴]AVP binding sites in kidney showed dense binding in the inner and outer medulla with less binding in the cortex, which is consistent with known renal V₂ receptor distribution.     

Synthesis and in vivo evaluation of a novel peripheral benzodiazepine receptor PET radioligand

The novel pyrazolopyrimidine ligand, N,N-diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethyl-pyrazolo[1,5-a]pyrimidin-3-yl]-acetamide 1 (DPA-713), has been reported as a potent ligand for the peripheral benzodiazepine receptor (PBR) displaying an affinity of K(i)=4.7 nM. In this study, 1 was successfully synthesised and demethylated to form the phenolic derivative 6 as precursor for labelling with carbon-11 (t(1/2) = 20.4 min). [11C]1 was prepared by O-alkylation of 6 with [11C]methyl iodide. The radiochemical yield of [(11)C]1 was 9% (non-decay corrected) with a specific activity of 36 GBq/micromol at the end of synthesis. The average time of synthesis including formulation was 13.2 min with a radiochemical purity >98%. In vivo assessment of [11C]1 was performed in a healthy Papio hamadryas baboon using positron emission tomography (PET). Following iv administration of [11C]1, significant accumulation was observed in the baboon brain and peripheral organs. In the brain, the radioactivity peaked at 20 min and remained constant for the duration of the imaging experiment. Pre-treatment with the PBR-specific ligand, PK 11195 (5 mg/kg), effectively reduced the binding of [11C]1 at 60 min by 70% in the whole brain, whereas pre-treatment with the central benzodiazepine receptor ligand, flumazenil (1mg/kg), had no inhibitory effect on [11C]1 uptake. These results indicate that accumulation of [11C]1 in the baboon represents selective binding to the PBR. These exceptional in vivo binding properties suggest that [11C]1 may be useful for imaging the PBR in disease states. Furthermore, [11C]1 represents the first ligand of its pharmacological class to be labelled for PET studies and therefore has the potential to generate new information on the pathological role of the PBR in vivo.     

Characterization of [3H]LS‐3‐134, a novel arylamide phenylpiperazine D3 dopamine receptor selective radioligand

LS‐3‐134 is a substituted N‐phenylpiperazine derivative that has been reported to exhibit: (i) high‐affinity binding (K_i value 0.2 nM) at human D3 dopamine receptors, (ii) > 100‐fold D3 versus D2 dopamine receptor subtype binding selectivity, and (iii) low‐affinity binding (K_i > 5000 nM) at sigma 1 and sigma 2 receptors. Based upon a forskolin‐dependent activation of the adenylyl cyclase inhibition assay, LS‐3‐134 is a weak partial agonist at both D2 and D3 dopamine receptor subtypes (29% and 35% of full agonist activity, respectively). In this study, [³H]‐labeled LS‐3‐134 was prepared and evaluated to further characterize its use as a D3 dopamine receptor selective radioligand. Kinetic and equilibrium radioligand binding studies were performed. This radioligand rapidly reaches equilibrium (10–15 min at 37°C) and binds with high affinity to both human (K_d = 0.06 ± 0.01 nM) and rat (K_d = 0.2 ± 0.02 nM) D3 receptors expressed in HEK293 cells. Direct and competitive radioligand binding studies using rat caudate and nucleus accumbens tissue indicate that [³H]LS‐3‐134 selectively binds a homogeneous population of binding sites with a dopamine D3 receptor pharmacological profile. Based upon these studies, we propose that [³H]LS‐3‐134 represents a novel D3 dopamine receptor selective radioligand that can be used for studying the expression and regulation of the D3 dopamine receptor subtype.     

35S]S-[5-(4-benzoylphenyl)pentyl]glutathione: a versatile radioligand targeting GS-X pumps with the ability of photoaffinity labeling.

[35S]S-[5-(4-benzoylphenyl)pentyl]glutathione (GIF-0017) as a biochemical probe targeting the ATP-dependent organic anion transporters GS-X pumps was synthesized by the reaction of [35S]glutathione and excess 4-(5-bromo)pentylbenzophenone under alkaline conditions, with the radiochemical yield of 24-33% after HPLC purification. Photolysis of the mixture of [35S]GIF-0017 and plasma membrane vesicles prepared from the MRP1 cDNA-transfected LLC-PK1 cells resulted in radio-labeling of a 180-kDa membrane protein. Immunoprecipitation and western blotting using an anti-MRP1 monoclonal antibody confirmed that the [35S]GIF-0017-labeled protein was the MRPI/GS-X pump.     

Ligand modulation of [35S]GTPgammaS binding at human alpha(2A), alpha(2B) and alpha(2C) adrenoceptors

Affinities and efficacies of chemically diverse ligands--some of them used as clinical agents--were examined, employing [3H]RX821,002 and [35S]GTPgammaS binding assays, respectively, at human (h) cloned, halpha(2A), halpha(2B) and halpha(2C) adrenoceptors (AR) expressed in Chinese hamster ovary (CHO) cells. As compared to noradrenaline (NA, efficacy defined as 100%), the majority of the 13 agonists tested generally behaved as partial agonists. Amongst 18 antagonists, pK(B) and pK(i) values, which were highly correlated for each alpha(2)-AR subtype, failed to reveal any strikingly selective agents. Inverse agonist properties were not detected for any antagonist, consistent with a lack of constitutive activity suggested by the monophasic inhibition of [35S]GTPgammaS binding by GTPgammaS. These data should facilitate interpretation of experimental and clinical actions of adrenergic agonists. Moreover, they emphasize the continuing need for alpha(2)-AR subtype-selective antagonists in order to define further the roles and therapeutic relevance of halpha(2A)-, halpha(2B)-, and halpha(2C)-AR.     

Biochemistry, pharmacology and physiology of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand

2-Arachidonoylglycerol (2-AG) is a unique molecular species of monoacylglycerol isolated in 1995 from rat brain and canine gut as an endogenous ligand for the cannabinoid receptors. 2-AG is rapidly formed from arachidonic acid-containing phospholipids through increased phospholipid metabolism, such as enhanced inositol phospholipid turnover, in various tissues and cells upon stimulation. 2-AG binds to the cannabinoid receptors (CB1 and CB2) and exhibits a variety of cannabimimetic activities in vitro and in vivo. Notably, anandamide, another endogenous ligand for the cannabinoid receptors, often acts as a partial agonist at these cannabinoid receptors, whereas 2-AG acts as a full agonist in most cases. The results of structure-activity relationship studies suggested that 2-AG rather than anandamide is the true natural ligand for both the CB1 and the CB2 receptors. Evidence is gradually accumulating which shows that 2-AG plays physiologically essential roles in diverse biological systems. For example, several lines of evidence indicate that 2-AG plays an important role as a retrograde messenger molecule in the regulation of synaptic transmission. 2-AG has also been shown to be involved in the regulation of various types of inflammatory reactions and immune responses. In this review, we focused on 2-AG, and summarized information concerning its biosynthesis, metabolism, bioactions and physiological significance, including our latest experimental results.     

7-Azaindolequinuclidinones (7-AIQD): A novel class of cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptor ligands

A series of N-benzyl-7-azaindolequinuclidinone (7-AIQD) analogs have been synthesized and evaluated for affinity toward CB1 and CB2 cannabinoid receptors and identified as a novel class of cannabinoid receptor ligands. Structure–activity relationship (SAR) studies indicate that 7-AIQD analogs are dual CB1/CB2 receptor ligands exhibiting high potency with somewhat greater selectivity towards CB2 receptors compared to the previously reported indolequinuclidinone (IQD) analogs. Initial binding assays showed that 7-AIQD analogs 8b, 8d, 8f, 8g and 9b (1 μM) produced more that 50% displacement of the CB1/CB2 non-selective agonist CP-55,940 (0.1 nM). Furthermore, Ki values determined from full competition binding curves showed that analogs 8a, 8b and 8g exhibit high affinity (110, 115 and 23.7 nM, respectively) and moderate selectivity (26.3, 6.1 and 9.2-fold, respectively) for CB2 relative to CB1 receptors. Functional studies examining modulation of G-protein activity demonstrated that 8a acts as a neutral antagonist at CB1 and CB2 receptors, while 8b exhibits inverse agonist activity at these receptors. Analogs 8f and 8g exhibit different intrinsic activities, depending on the receptor examined. Molecular docking and binding free energy calculations for the most active compounds (8a, 8b, 8f, and 8g) were performed to better understand the CB2 receptor-selective mechanism at the atomic level. Compound 8g exhibited the highest predicted binding affinity at both CB1 and CB2 receptors, and all four compounds were shown to have higher predicted binding affinities with the CB2 receptor compared to their corresponding binding affinities with the CB1 receptor. Further structural optimization of 7-AIQD analogs may lead to the identification of potential clinical agents.     

Increased expression of peripheral benzodiazepine receptor (PBR) in dimethylbenz[a]anthracene-induced mammary tumors in rats

Expression of peripheral benzodiazepine receptors (PBR) has been found in every tissue examined; however, it is most abundant in steroid-producing tissues. Although the primary function of PBR is the regulation of steroidogenesis, its existence in nonsteroidogenic tissues as well as in other cellular compartments including the nucleus suggests that there may be other roles for PBR. Our laboratory reported earlier a significant increase of PBR density in the nucleus of DMBA-induced malignant submandibular glands of rats, suggesting a role of PBR in nuclear events of peripheral tissues. Since then numerous studies have demonstrated the abundance of PBR in tumors. Numerous studies implicate a role for cholesterol in the mechanisms underlying cell proliferation and cancer progression. Based on studies with a battery of human breast cancer cell lines and several human tissue biopsies, Hardwick et al. suggested that PBR expression, nuclear localization, and PBR-mediated cholesterol transport into the nucleus are involved in human breast cancer cell proliferation and aggressive phenotype expression. The purpose of the present study is to confirm this hypothesis by developing an animal breast cancer model and correlating the above events with the breast cancer. Weanling rats were maintained on a diet containing animal protein (casein) for 30 days and then a single dose of DMBA in sesame oil (80 mg/kg) was administered by gavage to the animals. Control animals received the vehicle only. After 122 days of DMBA administration, the animals were sacrificed. All tumors were detected by palpation. B_max of PBRs was 52.6% and 128.4% higher in the non-aggressive and aggressive cancer tissues, respectively, than that in normal tissues. Cholesterol uptake into isolated nuclei was found to be higher in both non-aggressive and aggressive tumor breast tissue than that in control tissue. There was also corresponding increase in B_max of PBRs in the nucleus of cancer tissues. Furthermore, the nuclear nucleoside triphosphatase (NTPase) activity was found to be higher in aggressive tumor tissues than that in non-aggressive tumor tissues. In conclusion, these data suggest that PBR ligand binding, and PBR-mediated cholesterol transport into the nucleus may be involved in the development of mammary gland adenocarcinoma, thus participating in the advancement of the disease.     

[11C]Olanzapine,radiosynthesis and lipophilicity of a new potential PET 5-HT2 and D2 receptor radioligand

Olanzapine and its precursor desmethyl-Olanzapine were synthesized from malononitrile, propionaldehyde, 1-fluoro-2-nitrobenzene, and substituted piperazine in 4, 4, 5, and 5 steps with 35%, 32%, 26%, and 32% overall chemical yield, respectively. [¹¹C]Olanzapine was prepared from desmethyl-Olanzapine with [¹¹C]CH₃OTf through N-[¹¹C]methylation and isolated by HPLC combined with solid-phase extraction (SPE) in 40–50% radiochemical yield based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB), with 370–740 GBq/μmol specific activity at EOB. The calculated Log P (C Log P) value of [¹¹C]Olanzapine is 3.39.     

Characterization of [125I]AR-M100613, a high-affinity radioligand for delta opioid receptors

AR-M100613 ([I]-Dmt-c[-D-Orn-2-Nal-D-Pro-D-Ala-]) is the iodinated analog of a cyclic casomorphin previously shown to be a potent antagonist at the delta opioid receptor. Specific [125I]AR-M100613 binding to rat whole brain membranes was saturable, reversible, and best fit to a one-site model (Kd = 0.080 +/- 0.008 nM, Bmax = 45.2 +/- 4.4 fmol/mg protein). [125I]AR-M100613 binding was displaced with high affinity by the delta opioid receptor ligands SNC-80, Deltorphin II and DPDPE but not the mu or kappa-selective receptor ligands DAMGO and U69593. Residual non-selective binding of [125I]AR-M 100613 to mu opioid receptors is blocked by the addition of CTOP to the assay buffer. [35S]GTPgammaS binding assays indicate that AR-M100613 is a potent, selective, and reversible antagonist for delta opioid receptors in rat brain membranes. The high-affinity, high specific activity, low nonspecific binding and antagonist profile of [125I]AR-M100613 favor its use as a radiochemical probe for delta opioid receptors.