Human Frontal Cortex Membranes Research Articles

Pharmacological characterisation of the GlyT-1 glycine transporter using two novel radioligands

Inhibitors of the glycine transporter GlyT-1 are being developed as potential treatments for schizophrenia. Here we report on the use of two novel radioligands, [ 3H]-SB-733993 and [ 3H]-GSK931145, for the characterisation of GlyT-1 in both cells and native tissue. Binding was evaluated in membranes either from HEK293 cells expressing recombinant human GlyT-1 (hGlyT-1) or from rat cerebral cortex. Specific binding of both [ 3H]-SB-733993 and [ 3H]-GSK931145 to hGlyT-1 HEK293 cell membranes and rat cerebral cortex membranes was saturable and comprised >90% of total binding. K d and B max values for the two radioligands were fairly similar, with K d values of 1–2 nM and B max values of around 7000 fmol/mg protein in hGlyT-1 membranes and 3000 fmol/mg protein in rat cortex membranes. Association of [ 3H]-SB-733993 was faster, with binding reaching equilibrium within 30 min compared with 90 min for [ 3H]-GSK931145. Dissociation was also much slower for [ 3H]-GSK931145 than for [ 3H]-SB-733993, with 50% of specific binding being dissociated by approximately 40 min and 5 min, respectively. Autoradiography studies with [ 3H]-GSK931145 showed widespread distribution of binding in rat brain, with generally higher binding in caudal compared with rostral areas. Initial studies in human frontal cortex membranes showed clear specific binding of [ 3H]-GSK931145, though with much lower density ( B max 570 fmol/mg protein) and slightly lower affinity (K d 4.5 nM) compared with rat cortex. A human brain autoradiography study showed higher specific binding in cerebellum compared with frontal cortex. All GlyT-1 inhibitors tested, as well as glycine itself, competed fully for the binding of both [ 3H]-SB-733993 and [ 3H]-GSK931145 in both hGlyT-1 and rat cortex membranes. Studies on the effect of varying NaCl concentration showed that [ 3H]-SB-733993 binding was reduced by >90% in the absence of added Na + ions, whilst [ 3H]-GSK931145 binding was unaffected. Glycine produced concentration-dependent decreases in binding affinity of both radioligands without major changes in B max values, suggesting that both [ 3H]-SB-733993 and [ 3H]-GSK931145 bind to sites on GlyT-1 that are orthosteric to the site at which glycine itself binds. Overall, these results show that both [ 3H]-SB-733993 and [ 3H]-GSK931145 are useful radioligands for studies on GlyT-1 in both cell lines and native tissues, with [ 3H]-GSK931145 being the radioligand of choice for further studies on GlyT-1 expression and pharmacology.

Mivazerol, a novel compound with high specificity for α2 adrenergic receptors: Binding studies on different human and rat membrane preparations

Mivazerol, 3-[1(H-imidazol-4-yl)methyl]-2-hydroxybenzamide hydrochloride, a new potential anti-ischemic drug designed by UCB S.A. Pharma Sector, has been studied in binding experiments on adrenergic, dopaminergic, serotoninergic, muscarinic and idazoxan binding sites. Our results indicate that this compound displays high affinity and marked specificity for α 2 adrenoceptors. Mivazerol displaced the binding of the α 2 adrenoceptor antagonist [ 3H]RX 821002 to the α 2A adrenoceptors in human frontal cortex membranes with an apparent K i value of 37 nM. The competition curve was shallow ( n H = 0.55), suggesting that this compound acts as an α 2 adrenergic agonist. Mivazerol was also a potent competitor for [ 3H]RX 821002 binding to human platelet membranes (containing α 2A adrenoceptors) and rat kidney membranes (75 % of the α 2 adrenoceptors of the α 2B subtype), indicating that this compound is not α 2 adrenoceptor subtype selective. Equilibrium dissociation constants for α 1 adrenoceptors (displacement of [ 3H]prazosin) and 5-HT 1A receptors (displacement of [ 3H]rauwolscine) were respectively about 120 times ( K i = 4.4 μM) and 14 times ( K i = 530 nM) higher than that for the α 2 adrenoceptors. Equilibrium dissociation constants were approximately 1000 times higher for all other receptors tested in this study; namely β 1 and β 2 adrenoceptors, D 1- and D 2-dopamine receptors, M 1-, M 2- and M 3-muscarinic receptors, 5-HT 2 receptors and non-adrenergic idazoxan bindings sites.

3H]Rauwolscine behaves as an agonist for the 5-HT a receptors in human frontal cortex membranes

The alpha 2 adrenergic antagonist [3H]rauwolscine binds with comparable nanomolar affinity to alpha 2 adrenoceptors and the nonadrenergic 5-HT1A receptors sites in human frontal cortex membranes. Addition of 0.5 mM GTP into the incubation medium produces a significant decrease in the amount of [3H]rauwolscine binding sites (Bmax = 230 +/- 16 and 115 +/- 11 fmol/mg protein in the absence and presence of GTP, respectively). The affinity for [3H]rauwolscine remains unchanged (i.e. KD = 40 +/- 0.9 nM and 4.1 +/- 1 nM). This effect of GTP can be attributed to decreased binding of the radioligand to the 5-HT1A receptors. GTP decreases binding of [3H]rauwolscine to nearly the same level as the one corresponding to the alpha 2 adrenoceptors in membranes from both the human frontal cortex and hippocampus. The venom of the marine cone snail, Conus tessulatus, preferentially inhibits [3H]rauwolscine binding to 5-HT1A receptors as compared with the alpha 2 adrenoceptors. Following complete masking of the 5-HT1A receptors by this venom. GTP no longer affects the saturation binding characteristics of [3H]rauwolscine for the remaining alpha 2 adrenoceptors. Nucleotides decrease the binding of [3H]rauwolscine to the 5-HT1A receptors with an order of potencies (i.e. GTP gamma S greater than GPP(NH)P much greater than GDP greater than GTP much greater than ATP) that is typical for nucleotide-mediated receptor-G protein dissociation. This suggests that [3H]rauwolscine is a 5-HT1A receptor agonist and this conclusion is compatible with earlier functional studies, indicating that rauwolscine (as well as yohimbine) has agonistic properties at the level of 5-HT autoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Efaroxan (RX 821037) is a potent and selective α2-adrenoceptor antagonist in human frontal cortex membranes

The antagonist [ 3H]idazoxan binds with comparable affinity to α 2 adrenergic receptors and to non-adrenergic “idazoxan sites” in human frontal cortex membranes ( K D = 23 and 28 nM). RX 811033, the 2-ethyl derivative of idazoxan, displays about equal affinity for the α 2 receptors as idazoxan but much lower affinity for the idazoxan sites ( K i = 19 nM and 28 μM). Compared to RX 811033, efaroxan (RX 821037) has a dihydrobenzylfuranyl ring system instead of a benzodioxan ring. Nevertheless, efaroxan and RX 811033 bind with equal high affinity to the α 2 receptors and with equal low affinity to the idazoxan sites. Radioligand binding studies with [ 3H]RX 821002, the 2-methoxy derivative of [ 3H]idazoxan, confirm that efaroxan is an α 2 adrenergic antagonist possessing high affinity for the α 2 receptors and high α 2 receptor/idazoxan site-selectivity in the human frontal cortex.

Identification of α2 adrenergic receptors in human frontal cortex membranes by binding of [3H]RX 821002, the 2-methoxy analog of [3H]idazoxan

The antagonist [(3)H]idazoxan binds with comparable affinity to ?(2) adrenergic receptors and to phentolamine-displaceable non-stereoselective sites in human frontal cortex membranes. In contrast, idazoxan analogs possessing alkyl and alkoxy substituents at the 2-position of the benzodioxan moiety (i.e. RX 821002: 2-methoxy-1,4-[6,7-(3)H]benzodioxan-2-yl-2-imidazolin HCl, 43.8 Ci/mmol) possess 300-1200 times lower affinity for the non-stereoselective sites. Their affinity for the ?(2) receptors is increased as well, resulting in more than a 1000-fold selectivity towards the receptors as compared to the non-stereoselective sites. [(3)H]RX 821002, the 2-methoxy analog of idazoxan possesses an approx. 10-fold higher affinity for the ?(2) receptors (K(D) = 2.8 nM than [(3)H]idazoxan (K(D) = 24 nM) and about equal affinity as [(3)H]rauwolscine (K(D) = 3.6 nM). [(3)H]Rauwolscine binds with comparable affinity to ?(2) receptors and to 5-HT(1A) receptors, and competition studies indicate that the K(i) value of unlabelled RX 821002 for the 5-HT(1A) receptors (30 nM) is about one order in magnitude above its K(i) value for the ?(2) receptors (4.1 nM). Labelling of the 5-HT(1A) receptors by [(3)H]RX 821002 and by [(3)H]rauwolscine can be prevented by selective masking with 8-OH-DPAT (30 nM) or 5-HT (0.3 ?M). Under these conditions, specific binding of [(3)H]RX 821002 to the ?(2) receptors represents 84% of total binding (at its K(D)), as compared to 77% for [(3)H]rauwolscine and 20% for [(3)H]idazoxan. [(3)H]RX 821002 labels the ?(2) receptors as a single class of non-cooperative sites. Association and dissociation kinetics are very fast at 37 degrees C. Antagonist competition curves are steep with Hill coefficients close to one and the agonist curves can be analysed in terms of two affinity sites, confirming the antagonistic properties of [(3)H]RX821002. About 60% of the ?(2) receptors possess high agonist affinity.

σ and PCP receptors in human frontal cortex membranes

σ and PCP receptors in human frontal cortex membranes

Antimigraine drug interactions with serotonin receptor subtypes in human brain.

The interactions of antimigraine agents with serotonin (5-hydroxytryptamine, 5-HT) receptor subtypes were analyzed in human frontal cortex membranes. The drugs studied included 5-HT antagonists, beta-adrenergic antagonists, and calcium channel blockers. At 5-HT1A sites labeled by 3H-8-hydroxy-2-(N,N-dipropylamino)-tetralin, (-)pindolol, alprenolol, (-)propranolol, methysergide, cyproheptadine, and pizotifen are similar in that they display affinities of approximately 100 nM for this receptor. By contrast, only methysergide displays relatively high affinity (120 +/- 60 nM), whereas all other drugs have affinities greater than 1,000 nM for non-5-HT1A sites labeled by 3H-5-HT in human cortex. Finally, at 5-HT2 receptors labeled by 3H-spiperone, cyproheptadine, methysergide, and pizotifen are extremely potent agents (affinity constants of 1 to 10 nM), whereas amitriptyline (23 +/- 4 nM), verapamil (140 +/- 50 nM), and nifedipine (320 +/- 80 nM) are moderately potent. All other drugs are inactive at concentrations below 1,000 nM. These data demonstrate that most antimigraine drugs display high affinity for the 5-HT1A and/or 5-HT2 receptor subtypes in human brain. However, antimigraine efficacy cannot be explained by drug interactions with a single 5-HT receptor subtype.

3H]ketanserin labels 5-HT2 receptors and alpha 1-adrenoceptors in human and pig brain membranes.

The binding characteristics of [3H]ketanserin (a reported selective radioligand for serotonin 5-HT2 receptors) and [125I]BE 2254 (which labels selectively alpha 1-adrenoceptors) were characterized in brain frontal cortex membranes of pig and man. Saturation experiments indicated that both radioligands label apparently a homogeneous class of binding sites in human and pig fontal cortex membranes. Competition experiments with [125I]BE 2254 using 17 agonists and antagonists showed monophasic and steep curves in human and pig frontal cortex membranes. The pharmacological profile of these sites is typical of alpha 1-adrenoceptors. In competition experiments with [3H]ketanserin, most of the tested compounds displayed shallow or biphasic curves. In particular, alpha 1-adrenoceptor-selective antagonists (prazosin, WB 4101, BE 2254...) displaced with nanomolar affinity about 15 and 40% of the specific [3H]ketanserin binding in human and pig frontal cortex membranes, respectively. The minor component of [3H]ketanserin binding correlated highly significantly with [125I]BE 2254 binding in both membrane preparations. The major component of [3H]ketanserin binding to pig and human frontal cortex membranes correlated significantly with [3H]ketanserin binding in rat brain cortex membranes (which is essentially to 5-HT2 receptors). The present data demonstrate that [3H]ketanserin in nanomolar concentrations binds significantly to alpha 1-adrenoceptors in human and pig frontal cortex membranes; this suggests a rather limited degree of selectivity of ketanserin for 5-HT2 receptors in pig and human tissues.