Selective irreversible binding of chloroethylclonidine at alpha 1- and alpha 2-adrenoceptor subtypes

We have determined the alkylating effects and affinity of chloroethylclonidine at alpha 1- and alpha 2-adrenoceptor subtypes in saturation and competition radioligand binding studies. Treatment with chloroethylclonidine (10 microM, for 30 min at 37 degrees, with subsequent washout) abolished [3H]prazosin binding to alpha 1B-adrenoceptors in rat spleen almost completely and reduced specific binding in rat kidney and cerebral cortex by a percentage comparable to the known alpha 1B-adrenoceptor content of these tissues. Chloroethylclonidine treatment also markedly reduced [3H]rauwolscine binding to human platelet and kidney membranes but did not affect [3H]rauwolscine binding to rat kidney. Similar chloroethylclonidine treatment (10 microM, 20 min at 37 degrees) reduced the number of detectable alpha 2-adrenoceptors in cell lines transfected with the alpha 2-C10 or alpha 2-C4 gene but not in those transfected with alpha 2-C2 adrenoceptors. In concentration-response experiments, higher chloroethylclonidine concentrations were required for inactivation of human platelet alpha 2A-adrenoceptors, compared with rat spleen alpha 1B-adrenoceptors, and a smaller maximal inactivation was achieved. The lack of inactivation of rat alpha 1A- and alpha 2B- and human alpha 2-C2-adrenoceptors was not due to a lack of chloroethylclonidine binding, because the affinity of chloroethylclonidine at these subtypes, as determined in competition binding experiments, was at least as high as the apparent affinity at the alkylated subtypes. alpha 2A-Adrenoceptor alkylation by chloroethylclonidine treatment was functionally relevant, because it significantly reduced alpha 2A-adrenoceptor-mediated Ca2+ elevations in HEL cells. We conclude that chloroethylclonidine binds to all major alpha-adrenoceptor subtypes and irreversibly inactivates not only alpha 1B-adrenoceptors but also alpha 2A- and alpha 2C-adrenoceptors, whereas alpha 1A- and alpha 2B-adrenoceptors are relatively resistant to its alkylating action, although they can bind chloroethylclonidine.     

Pharmacological characterization of [3H]idazoxan, [3H]RX821002 and p-[125I]iodoclonidine binding to alpha 2-adrenoceptors in rat cerebral cortical membranes

Binding characteristics of alpha 2-adrenoceptors in rat cerebral cortical membranes were compared using the antagonist radioligands [3H]idazoxan, [3H]2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline ([3H]RX821002), and the partial agonist radioligand [125I]2-[2,6-(dichloro-4-iodophenyl)imino]imidazoline ([125I]iodoclonidine). With [3H]RX821002 and alpha 2-adrenoceptor subtype-selective competitors, both alpha 2A/D- and alpha 2C-adrenoceptor subtypes were detected, suggesting rat cortical membranes contain approximately 90% alpha 2A/D-adrenoceptor subtype and 10% alpha 2C-adrenoceptor subtype. Only alpha 2A/D-adrenoceptors were detected with [3H]idazoxan and [125I]iodoclonidine. All three radioligands bound to a single high affinity site (Kd = 0.3-1.6 nM). However, the densities of sites labeled by [3H]idazoxan and [125I]iodoclonidine were 50% greater than the density labeled by [3H]RX821002, likely representing non-adrenoceptor binding sites. The density of [125I]iodoclonidine binding sites in glycylglycine buffer was similar to that labeled by [3H]RX821002. These results suggest that: (1) alpha 2A/D-adrenoceptors are the predominant subtype in rat cerebral cortex, (2) demonstrate that the small number of alpha 2C-adrenoceptors in this tissue can be detected using prazosin to displace [3H]RX821002 binding, and (3) non-adrenoceptor binding with [125I]iodoclonidine can be minimized with the use of glycylglycine buffer.     

Rat and human hippocampal alpha5 subunit-containing gamma-aminobutyric AcidA receptors have alpha5 beta3 gamma2 pharmacological characteristics

The gamma-aminobutyric acid (GABA)A receptor is a hetero-oligomer consisting of five subunits, the combination of which confers unique pharmacological properties to the receptor. To understand the physiological role of native GABAA receptors, it is critical to determine their subunit compositions. The pharmacological characteristics of human alpha5 beta3 gamma2 and alpha5beta3gamma3 GABAA receptors stably expressed in L(tk-) cells were characterized with the alpha5-selective ligand [3H]L-655,708 and compared with the pharmacological characteristics of [3H]L-655,708 binding sites from rat and human hippocampus. Saturation analyses revealed a 9-fold selective affinity of [3H]L-655,708 for alpha5 beta3 gamma2 receptors (Kd = 1.7 +/- 0.4 nM), compared with alpha5 beta3 gamma3 receptors (Kd = 15 +/- 3 nM). Rat and human hippocampal [3H]L-655,708 binding sites had affinities of 2.2 +/- 0.6 and 1.0 +/- 0.2 nM, respectively, comparable to the affinity of alpha5 beta3 gamma2 receptors. Pharmacological analysis of [3H]L-655,708 binding sites in rat and human hippocampi revealed a strong correlation with the affinities of seven benzodiazepine site ligands for alpha5 beta3 gamma2 but not alpha5 beta3 gamma3 receptors. Immunoprecipitation of [3H]L-655,708 binding sites from rat hippocampus with a gamma2-selective antibody yielded 19 +/- 4% of total benzodiazepine binding sites measured using [3H]Ro15-1788, whereas no specific binding was measured after immunoprecipitation with an anti-gamma3 antibody. Combinatorial immunoprecipitations of [3H]muscimol binding sites with anti-alpha5 and anti-gamma2 or anti-alpha5 and anti-gamma3 antibodies established the preferential expression of alpha5 gamma2 receptors, accounting for 22 +/- 2% of total rat hippocampal GABAA receptors. These observations provide pharmacological and structural evidence for the prevalence of alpha5 beta3 gamma2 GABAA receptors in rat hippocampus, despite the clustering of alpha5 and gamma3 loci on the same chromosome.     

Serotonin modulation of low-affinity ouabain binding in rat brain determined by quantitative autoradiography

Previous results showed that Na+/K+-ATPase may have a functional relationship with the neurotransmitter serotonin which activates the glial sodium pump in the rat brain. Both the reaction rate (V) of Na+/K+-ATPase activity and [3H]ouabain binding were significantly increased in the presence of serotonin. It is not known, however, which alpha isoform is involved in the Na+/K+-ATPase response to serotonin and its regional distribution. Quantitative autoradiography of [3H]ouabain binding to rat brain slices was employed at different [3H]ouabain concentrations in order to gain information on both the distribution and the possible isoform involved. The results showed that 1500 nM [3H]ouabain binding was sensitive to serotonin 10(-3) M and significantly increased in the following brain regions: frontal cortex, areas CA1, CA2, and CA3 of the hippocampus, presubiculum, zona incerta, caudate putamen and the amygdaloid area, confirming and extending previous results. An effect of serotonin on brain but not kidney tissue at high, 1500 nM, and the lack of effect at low, 50 nM [3H]ouabain concentrations, strongly suggests the participation of the alpha2 isoform in the response of the pump to the neurotransmitter. Glial cells showed stimulation of ouabain binding by serotonin at ouabain concentrations above 350 nM. The present results open interesting questions related to the brain regions involved and the K+ handling by the glial alpha2 isoform of the pump.     

Alpha-adrenoceptor interaction of tetrandrine and isotetrandrine in the rat: functional and binding assays

The action of 1S,1'S-tetrandrine, a bisbenzyltetrahydroisoquinoline alkaloid, on alpha1-adrenoceptors has been compared with that of its isomer 1R,1'S-isotetrandrine. The work includes binding assays to analyse the affinity of these products for the [3H]prazosin binding site of rat cerebral cortical membranes and functional studies on rat isolated aorta to examine the effects of both alkaloids on intracellular calcium processes related or not to alpha-adrenoceptor activation. A radioligand receptor-binding study showed that both compounds interacted with the alpha1-adrenoceptors displacing [3H]prazosin from the specific binding site. The Ki values (inhibition constants) were 0.69+/-0.12 and 1.6+/-0.4 microM for tetrandrine and isotetrandrine, respectively. The functional studies showed that both alkaloids concentration-dependently inhibited noradrenaline-induced contraction in Ca2+-free solution (IC50 values, i.e. the concentrations needed to induce 50% inhibition, were 252.8 and 174.9 microM for tetrandrine and isotetrandrine, respectively), the spontaneous contractile response elicited by extracellular calcium after depletion of noradrenaline-sensitive intracellular stores (increase in resting tone; IC50 values 11.6 and 19.6 microM for tetrandrine and isotetrandrine, respectively) and the refilling of intracellular Ca2+ stores sensitive to noradrenaline (IC50 values 7.4 and 14.9 microM for tetrandrine and isotetrandrine, respectively). The results show that tetrandrine and isotetrandrine interact with alpha1-adrenoceptors by displacing the [3H]prazosin binding site and that both compounds inhibit mainly the Ca2+-dependent process and have less action on alpha1-adrenoceptors. Tetrandrine is more potent than isotetrandrine.     

Multiple alpha 2 adrenergic receptor subtypes. I. Comparison of [3H]RX821002-labeled rat R alpha-2A adrenergic receptors in cerebral cortex to human H alpha2A adrenergic receptor and other populations of alpha-2 adrenergic subtypes

In the present study, we examined the binding of the alpha-2 adrenergic receptor (AR) antagonist [3H]-(2-(2-methoxy-1,4-benzodioxan- 2yl)-2-imidazoline ([3H]RX821002) to alpha-2 AR in rat cerebral cortex (CC) and compared the properties of these sites to those of rat alpha-2A (R alpha-2A) AR in submaxillary gland (SMG), human alpha-2A (H alpha-2A) AR in human platelets and alpha-2B AR in neonatal rat lung. In the presence of guanidinium phosphate, [3H]RX821002 bound with high affinity to a large and homogeneous population of sites in CC (Kd = 0.30 +/- 0.03 nM and Bmax = 271 +/- 7 fmol/mg of protein), SMG (Kd = 0.7 and Bmax = 274), human platelets (Kd = 0.6 nM and Bmx = 189) and neonatal rat lung (kd = 0.9 and Bmax = 161). A total of 34 chemically diverse AR ligands monophasically inhibited the binding of [3H]RX821002 from each site with, for the CC, the most potent ligand being atipamezole (Ki = 0.2 nM). For all ligands, and at each site, Hill coefficients did not differ significantly from unity. Although the profiles of inhibition of [3H]RX821002 were virtually identical in rat CC and SMG, these populations revealed several marked differences to human platelets; the alkaloids, rauwolscine and yohimbine, as well as the benzodioxane, [2-(2,6- dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxane] (WB 4101), displayed about 10-fold lower affinity for R alpha-2A as compared to H alpha-2A sites, whereas the benzopyrrolidines, fluparoxan and des-fluorofluparoxan, showed about 10-fold greater affinity for R alpha-2A sites. Further, whereas the calculation of potency ratios for selected pairs of ligands, as well as of correlation coefficients, revealed virtual identity between R alpha-2A AR in CC and SMG, these analyses revealed that each of these populations of R alpha-2A AR clearly differed to H alpha-2A AR in human platelets. In addition, both R alpha-2A AR in rat CC and SMG as well as H alpha-2A AR in human platelets markedly differed to alpha-2B AR in neonatal rat lung; thus, they showed 20-fold higher affinity for [2-(2H-(1-methyl-1,3-dihydroisoindole)methyl)-4,5- dihydroimidazoline] (BRL 44408), oxymetazoline, guanfacine and guanabenz yet 10- to 100-fold lower affinity for [2-(2-4-o- methoxyphenyl)piperazine-1-yl)-ethyl)-4,4-dimethyl-1,3-(2H,4H)- isoquinolinedione] (ARC 239) prazosin, chlorpromazine and corynanthine. Similar differences in R alpha-2A and H alpha-2A sites to alpha-2C sites were apparent upon analysis of literature data.(ABSTRACT TRUNCATED AT 400 WORDS)     

Binding of the radioligand [3H]-SCH 58261, a new non-xanthine A2A adenosine receptor antagonist, to rat striatal membranes

1. The present study describes the binding to rat striatal A2A adenosine receptors of the new potent and selective antagonist radioligand, [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazol o [1,5-c] pyrimidine, [3H]-SCH 58261. 2. [3H]-SCH 58261 specific binding to rat striatal membranes ( > 90%) was saturable, reversible and dependent upon protein concentration. Saturation experiments revealed that [3H]-SCH 58261 labelled a single class of recognition sites with high affinity (Kd = 0.70 nM) and limited capacity (apparent Bmax = 971 fmol mg-1 of protein). The presence of 100 microM GTP in the incubation mixture did not modify [3H]-SCH 58261 binding parameters. 3. Competition experiments showed that [3H]-SCH 58261 binding is consistent with the labelling of A2A striatal receptors. Adenosine receptor agonists competed with the binding of 0.2 nM [3H]-SCH 58261 with the following order of potency: 2-hexynyl-5'-N-ethyl carboxamidoadenosine (2HE-NECA) > 5'-N-ethylcarboxamidoadenosine (NECA) > 2-[4-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosi ne (CGS 21680) > 2-phenylaminoadenosine (CV 1808) > R-N6-phenylisopropyladenosine (R-PIA) > N6-cyclohexyladenosine (CHA) = 2-chloro-N6-cyclopentyladenosine (CCPA) > S-N6-phenylisopropyladenosine (S-PIA). 4. Adenosine antagonists inhibited [3H]-SCH 58261 binding with the following order: 5-amino-9-chloro-2-(2-furyl)-[1,2,4]-triazolo[1,5-c] quinazoline (CGS 15943) > 5-amino-8-(4-fluorobenzyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4-triazolo [1,5-c] pyrimidine (8FB-PTP) = SCH 58261 > xanthine amine congener (XAC) = (E,18%-Z,82%)7-methyl-8-(3,4-dimethoxystyryl)-1,3-dipropylxanthine (KF 17837S) > 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) > or = 8-phenyltheophylline (8-PT). 5. The Ki values for adenosine antagonists were similar to those labelled with the A2A agonist [3H]-CGS 21680. Affinities of agonists were generally lower. The A1-selective agonist, R-PIA, was found to be about 9 fold more potent than its stereoisomer, S-PIA, thus showing the stereoselectivity of [3H]-SCH 58261 binding. Except for 8-PT, the adenosine agonists and antagonists examined inhibited [3H]-SCH 58261 binding with Hill coefficients not significantly different from unity. 6. The present results indicate that [3H]-SCH 58261 is the first non-xanthine adenosine antagonist radioligand which directly labels A2A striatal receptors. High receptor affinity, good selectivity and very low non-specific binding make [3H]-SCH 58261 an excellent probe for studying the A2A adenosine receptor subtype in mammalian brain.     

On the high affinity binding site for [3H]-1,3-dipropyl-8-cyclopentylxanthine in frog brain membranes

1. Radioligand binding properties of the adenosine receptor ligands, [3H]-1,3-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), and [3H]-R-phenylisopropyladenosine ([3H]-R-PIA) were investigated in frog brain membranes. 2. The specific binding of the adenosine antagonist, [3H]-DPCPX to frog brain membranes showed one binding site with Kd and Bmax values of 43.8 nM and 0.238 +/- 0.016 pmol mg-1 protein, respectively. Guanosine 5'-triphosphate (GTP, 100 microM) decreased to 72 +/- 7% and Mg2+ (8 mM) increased to 121 +/- 3% [3H]-DPCPX (40 nM) binding to frog brain membranes. 3. [3H]-DPCPX saturation binding experiments performed in the presence of Mg2+ (8 mM), or in the presence of GTP showed that Mg2+ ions decreased the Kd value of [3H]-DPCPX to 14 nM, and GTP increased this value to 65.6 nM. Bmax values were not significantly (P > 0.05) modified (0.261 +/- 0.018 pmol mg-1 protein, with Mg2+, and 0.266 +/- 0.026 pmol mg-1 protein, in presence of GTP) by the presence of Mg2+ or GTP. 4. The specific binding of [3H]-R-PIA (15 nM) was decreased to 37 +/- 6% by GTP (100 microM) and increased to 123 +/- 4% by Mg2+ (8 mM). [3H]-R-PIA saturation binding experiments performed in the presence of Mg2+ (8 mM) showed one binding site with Kd and Bmax values of 0.9 nM and 0.229 +/- 0.008 pmol mg-1 of protein, respectively. 5. The concentration-inhibition curves of adenosine agonists and antagonists versus [3H]-DPCPX binding showed the following order of potencies: CPA> R-PIA~ NECA> S-PIA> > CGS 21680, for the agonists, and XAC ~-DPCPX> > XCC> PACPX, for the antagonists.6. The present results suggest that the adenosine binding site in the frog brain membranes is G-protein coupled, but that the antagonist affinities and the pharmacological profile is different from the Al or A2 adenosine receptors.     

Solubilized rabbit striatal A2a-adenosine receptors: stability and antagonist binding

The A2a-adenosine binding subunit from rabbit striatal membranes was solubilized using 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and was characterized using the antagonist radioligand [3H]8-[4-[[[[2-aminoethyl)amino]carbonyl]methyl]oxy] phenyl]-1,3-dipropylxanthine (XAC). The solubilized receptor was very stable, with 55% of the specific [3H]XAC binding remaining after storage for 15 days at 4 degrees C. The dissociation constant (Kd) for binding of [3H]XAC to solubilized A2 receptors was determined in saturation studies to be 4.0 nM, with a Bmax of 600 fmol/mg protein. Xanthine inhibitors displaced the specific binding of the adenosine antagonist [3H]XAC (in the presence of 50 nM 8-cyclopentyl-1,3-dipropylxanthine) at 25 degrees C, with Ki values consonant with the expected affinities at A2a receptors. Binding of [3H]XAC (1 nM) or the adenosine agonist [3H]2-(carboxyethylphenylethylamino)adenosine-5'-N-ethyl carboxamide (5 nM) to A2a receptors was diminished in the presence of 0.1 M Na+ in both membranes and solubilized preparations. Agonist binding was increased (by 280% for membranes and 180% for solubilized receptors), and antagonist binding was decreased in the presence of 10 mM Mg2+. Displacement of [3H]XAC by the agonist (R)-N6-phenylisopropyladenosine was biphasic, corresponding to high (IC50 = 188 nM, RH = 30%) and low (IC50 = 9730 nM, RL = 70%) affinity sites. Preincubation with 100 microM GTP (10 mM Mg2+) converted the high affinity binding to low affinity, suggesting that receptor and G-protein are dissociated by the guanine nucleotide. The solubilized receptor was more easily inactivated by exposure to the reducing agent dithiothreitol (IC50 = 3 mM) than in membranes (IC50 = 220 mM), suggesting increased accessibility of structurally essential disulfide bridges.     

The non-peptide neuropeptide Y Y1 receptor antagonist BIBP3226 blocks the [Leu31,Pro34]neuropeptide Y-induced modulation of alpha 2-adrenoceptors in the nucleus tractus solitarii of the rat

The potential blockade of the neuropeptide Y (NPY) Y1 receptor agonist [Leu31,Pro34]NPY-induced modulation of the characteristics of alpha 2-adrenoceptor agonist [3H]p-aminoclonidine binding sites by a selective non-peptide NPY Y1 receptor antagonist BIBP3226, was studied in the nucleus tractus solitarii of the rat by means of quantitative receptor autoradiography. [Leu31,Pro34]NPY at a concentration of 10 nM significantly increased the Kd value of [3H]p-aminoclonidine binding sites in the nucleus tractus solitarii without influencing the Bmax, suggesting the existence of an antagonistic modulation by NPY Y1 receptors of alpha 2-adrenoceptors in the nucleus tractus solitarii. BIBP3226 at 100 nM fully blocked the [Leu31,Pro34]NPY-induced increase in Kd of the [3H]p-aminoclonidine binding sites. The present results therefore provide evidence, by use of a NPY Y1 receptor antagonist, for the existence of a NPY Y1/alpha 2 receptor interaction in the nucleus tractus solitarii.     

Characterization of P2x1 purinoreceptors on rat platelets: effect of clopidogrel

This study aimed to determine the binding characteristics of [3H]alpha,beta-Me-ATP, a specific ligand of the P2x1 receptors to rat platelets, and to investigate the effect of clopidogrel, a thienopyridine compound which has been found to selectively inhibit ADP-induced platelet aggregation and adenylyl cyclase ex vivo. Binding of [3H]alpha,beta-Me-ATP to rat platelets was time-dependent and saturable. Scatchard analysis of the saturation binding data indicated that [3H]alpha,beta-Me-ATP bound to one population of specific binding sites with high affinity (KD = 23.6 +/- 1.6 nM; Bmax = 690 +/- 24 fmole/10[8]cells) (n=3). Unlabelled alpha,beta-Me-ATP as well as 2-MeS-ADP and ADP competitively inhibited the specific binding of [3H]alpha,beta-Me-ATP with IC50 values of 19.0 +/- 6.6, 103 +/- 20 and 1120 +/- 80 nM respectively (n=3). Other nucleotide analogues such as ATP, ATP-gammaS, UTP and GTP also antagonized [3H]alpha,beta-Me-ATP binding. When administered orally (10mg/kg, p.o.), clopidogrel inhibited ADP- or 2-MeS-ADP-induced platelet aggregation but did not affect the binding of [3H]alpha,beta-Me-ATP to rat platelets ex vivo. In vitro, alpha,beta-Me-ATP did not induce the aggregation or shape change of rat platelets and did not interfere with ADP-induced platelet aggregation.     

Metabolic responses of postmenopausal women to supplemental dietary boron and aluminum during usual and low magnesium intake: boron, calcium, and magnesium absorption and retention and blood mineral concentrations

The regulation by neuropeptide Y of alpha2-adrenoceptors in the nucleus tractus solitarii was evaluated in the adult normotensive Wistar Kyoto rat and the adult spontaneously hypertensive rat. The microinjection of a submaximal dose of l-noradrenaline (800 pmol in 50 nl) alone into the nucleus tractus solitarii produced a significant reduction in the mean arterial blood pressure in either strain. The threshold dose (1 pmol in 50 nl) of neuropeptide Y(1-36) for the vasodepressor response in the Wistar Kyoto rat was five times higher than that (0.2 pmol in 50 nl) in the spontaneously hypertensive rat. Furthermore, neuropeptide Y(1-36) at 0.2 pmol in 50 nl could significantly counteract the vasodepressor response to l-noradrenaline (800 pmol in 50 nl) in the spontaneously hypertensive rat, but not in the Wistar Kyoto rat, in which 1 pmol in 50 nl of neuropeptide Y(1-36) must be employed to counteract the vasodepressor response to l-noradrenaline (800 pmol in 50 nl), although the vasodepressor responses are of a similar magnitude. The in situ hybridization and quantitative receptor autoradiographical experiments showed that the alpha2A-adrenoceptor messenger RNA levels and the B(max) value of the alpha2-adrenoceptor agonist [3H]p-aminoclonidine binding sites measured in the nucleus tractus solitarii of the spontaneously hypertensive rat were substantially lower than those in the Wistar Kyoto rat. The quantitative receptor autoradiographical results were consistent with the cardiovascular results and showed that in the spontaneously hypertensive rat, neuropeptide Y(1-36) at 1 nM led to a significant increase in the K(d) value of [3H]p-aminoclonidine binding sites. In the Wistar Kyoto rat, neuropeptide Y(1-36) produced this effect only at 10 nM. The present study provides evidence for an increase of the potency of neuropeptide Y(1-36) to antagonistically modulate alpha2-adrenoceptors in the nucleus tractus solitarii of the spontaneously hypertensive rat. This enhanced antagonistic action may partly be related to a reduction in the number of alpha2A-adrenoceptors in the nucleus tractus solitarii of the spontaneously hypertensive rat, since a decrease has been observed in the alpha2A-adrenoceptor messenger RNA levels and the alpha2-adrenoceptor binding sites in the spontaneously hypertensive rat. This increased potency of neuropeptide Y(1-36) to antagonize alpha2-adrenoceptor function in the nucleus tractus solitarii of the spontaneously hypertensive rat may contribute to the development of high blood pressure in this hypertensive strain.     

Species differences in brain adenosine A1 receptor pharmacology revealed by use of xanthine and pyrazolopyridine based antagonists

1. The pharmacological profile of adenosine A1 receptors in human, guinea-pig, rat and mouse brain membranes was characterized in a radioligand binding assay by use of the receptor selective antagonist, [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX). 2. The affinity of [3H]-DPCPX binding sites in rat cortical and hippocampal membranes was similar. Binding site affinity was higher in rat cortical membranes than in membranes prepared from guinea-pig cortex and hippocampus, mouse cortex and human cortex. pKD values (M) were 9.55, 9.44, 8.85, 8.94, 8.67, 9.39 and 8.67, respectively. The binding site density (Bmax) was lower in rat cortical membranes than in guinea-pig or human cortical membranes. 3. The rank order of potency of seven adenosine receptor agonists was identical in each species. With the exception of 5'-N-ethylcarboxamidoadenosine (NECA), agonist affinity was 3.5-26.2 fold higher in rat cortical membranes than in human and guinea-pig brain membranes; affinity in rat and mouse brain membranes was similar. While NECA exhibited 9.3 fold higher affinity in rat compared to human cortical membranes, affinity in other species was comparable. The stable GTP analogue, Gpp(NH)p (100 microM) reduced 2-chloro-N6-cyclopentyladenosine (CCPA) affinity 7-13.9 fold, whereas the affinity of DPCPX was unaffected. 4. The affinity of six xanthine-based adenosine receptor antagonists was 2.2-15.9 fold higher in rat cortical membranes compared with human or guinea-pig membranes. The rank order of potency was species-independent. In contrast, three pyrazolopyridine derivatives, (R)-1-[(E)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl) acryloyl]-2-piperidine ethanol (FK453), (R)-1-[(E)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl) acryloyl]-piperidin-2-yl acetic acid (FK352) and 6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-pyridazinebutyric acid (FK838) exhibited similar affinity in human, guinea-pig, rat and mouse brain membranes. pKi values (M) for [3H]-DPCPX binding sites in human cortical membranes were 9.31, 7.52 and 7.92, respectively. 5. Drug affinity for adenosine A2A receptors was determined in a [3H]-2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamido ade nosine ([3H]-CGS 21680) binding assay in rat striatal membranes. The pyrazolopyridine derivatives, FK453, FK838 and FK352 exhibited pKi values (M) of 5.90, 5.92 and 4.31, respectively, compared with pKi values of 9.31, 8.18 and 7.57 determined in the [3H]-DPCPX binding assay in rat cortical membranes. These novel pyrazolopyridine derivatives therefore represent high affinity, adenosine A1 receptor selective drugs that, in contrast to xanthine based antagonists, exhibit similar affinity for [3H]-DPCPX binding sites in human, rat, mouse and guinea-pig brain membranes.     

Evidence for direct interaction of ketamine with alpha 1- and beta 2-adrenoceptors

1. Ketamine has a number of effects that suggest that it may interact with alpha- and beta-adrenoceptors. To date, the experimental evidence for this has been indirect and has been based on physiological studies using competitive blocking agents. In the present study we sought to determine from receptor binding studies whether ketamine binds directly to alpha- and beta-adrenoceptors. 2. Membrane preparations of alpha 1- and beta 2-adrenergic binding sites were obtained from urinary bladder and urethrae of sheep. These binding sites were characterized by saturation analyses using [3H]-prazosin for alpha 1-adrenoceptor binding sites and [125I]-cyanopindolol (CYP) for the beta 2-adrenoceptor binding sites. The receptors were further characterized by displacement studies using selective and non-selective antagonists. 3. Studies in which ketamine was used to displace [3H]-prazosin revealed a Kd of 3.40 +/- 1.23 x 10(-3) mol/L for ketamine binding to alpha 1-adrenoceptors. Displacement studies of [125I]-CYP by ketamine showed a Kd of 0.35 +/- 0.03 x 10(-3) mol/L for ketamine binding to beta 2-adrenoceptors. 4. We conclude that ketamine interacts directly with both alpha 1- and beta 2-adrenoceptors and that such interactions probably explain the reported effects of this agent on the vasculature and the bronchial tree.     

Characterization of A2A adenosine receptors in human lymphocyte membranes by [3H]-SCH 58261 binding

1. The present study describes for the first time the characterization of the adenosine A2A receptor in human lymphocyte membranes with the new potent and selective antagonist radioligand, [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4 triazolo [1,5-c] pyrimidine, ([3H]-SCH 58261). In addition, both receptor affinity and potency of reference adenosine receptor agonists and antagonists were determined in binding and adenylyl cyclase studies. 2. Saturation experiments revealed a single class of binding sites with Kd and Bmax values of 0.85 nM and 35 fmol mg-1 protein, respectively. A series of adenosine receptor ligands were found to compete for the binding of 0.8 nM [3H]-SCH 58261 to human lymphocyte membranes with a rank order of potency consistent with that typically found for interactions with the A2A-adenosine receptor. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency similar to that observed in binding experiments. 3. Thermodynamic data indicate that [3H]-SCH 58261 binding to human lymphocytes is entropy and enthalpy-driven, a finding in agreement with the thermodynamic behaviour of antagonists for rat striatal A2A-adenosine receptors. 4. It is concluded that in human lymphocyte membranes [3H]-SCH 58261 directly labels binding sites showing the characteristic properties of the adenosine A2A-receptor. The presence of A2A-receptors in peripheral tissue such as human lymphocytes strongly suggests an important role for adenosine in modulating immune and inflammatory responses.     

3H]-SCH 58261 labelling of functional A2A adenosine receptors in human neutrophil membranes

1. The present study describes the direct labelling of A2A adenosine receptors in human neutrophil membranes with the potent and selective antagonist radioligand, [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4 triazolo[l,5-c]pyrimidine, ([3H]-SCH 58261). In addition, both receptor affinity and potency of a number of adenosine receptor agonists and antagonists were determined in binding, adenylyl cyclase and superoxide anion production assays. 2. Saturation experiments revealed a single class of binding sites with Kd and Bmax values of 1.34 nM and 75 fmol mg(-1) protein, respectively. Adenosine receptor ligands competed for the binding of 1 nM [3H]-SCH 58261 to human neutrophil membranes, with a rank order of potency consistent with that typically found for interactions with the A2A adenosine receptors. In the adenylyl cyclase and in the superoxide anion production assays the same compounds exhibited a rank order of potency identical to that observed in binding experiments. 3. Thermodynamic data indicated that [3H]-SCH 58261 binding to human neutrophils is entropy and enthalpy-driven. This finding is in agreement with the thermodynamic behaviour of antagonists binding to rat striatal A2A adenosine receptors. 4. It was concluded that in human neutrophil membranes, [3H]-SCH 58261 directly labels binding sites with pharmacological properties similar to those of A2A adenosine receptors of other tissues. The receptors labelled by [3H]-SCH 58261 mediated the effects of adenosine and adenosine receptor agonists to stimulate cyclic AMP accumulation and inhibition of superoxide anion production in human neutrophils.     

Autoradiographic visualization of group III metabotropic glutamate receptors using [3H]-L-2-amino-4-phosphonobutyrate

1. In vitro receptor autoradiography using [3H]-L-2-amino-4-phosphonobutyrate ([3H]-L-AP4) binding to sections of rat brain has been characterized and shown to most likely represent labelling of group III metabotropic glutamate receptors. 2. Specific [3H]-L-AP4 binding to rat brain sections was observed at high densities in the molecular layer of the cerebellar cortex and the outer layer of the superior colliculus. Moderate levels were observed throughout the cerebral cortex, in the molecular layer of the hippocampal dentate gyrus, in thalamus, striatum, substantia nigra and in the medial geniculate nucleus. Low levels of [3H]-L-AP4 binding were found in other regions of the hippocampal formation, in the entorhinal cortex and the granule cell layer of cerebellum. 3. Inhibitors of sodium- or calcium/chloride-dependent glutamate uptake did not displace [3H]-L-AP4 binding to rat brain sections indicating that the observed binding does not represent [3H]-L-AP4 uptake via these carriers. Furthermore, in contrast to [3H]-L-AP4 uptake into cerebellar membranes, [3H]-L-AP4 binding to brain sections was sensitive to guanosine-5'-O-(3-thio)trisphosphate-gamma-S. 4. In the molecular layer of the cerebellar cortex, [3H]-L-AP4 binding showed a maximal binding density (Bmax) of 0.52+/-0.06 pmol mg(-1) tissue and an affinity (Kd) of 346 nM. The rank order of affinity for displacement of [3H]-L-AP4 binding to rat brain sections was: L-AP4 > L-serine-O-phosphate > glutamate > (L)-2-aminomethyl-4-phosphonobutanoate > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate which is in agreement with a group III metabotropic glutamate receptor pharmacology.     

Effect of peripherally and centrally administered calcitonin on gallbladder emptying in dogs

The vast molecular heterogeneity of brain gamma-aminobutyric acid type A (GABAA) receptors forms the basis for receptor subtyping. Using autoradiographic techniques, we established the characteristics of cerebellar granule cell GABAA receptors by comparing wild-type mice with those with a targeted disruption of the alpha6 subunit gene. Cerebellar granule cells of alpha6(-/-) animals have severe deficits in high affinity [3H]muscimol and [3H]SR 95531 binding to GABA sites, in agonist-insensitive [3H]Ro 15-4513 binding to benzodiazepine sites, and in furosemide-induced increases in tert-[35S]butylbicyclophosphorothionate binding to picrotoxin-sensitive convulsant sites. These observations agree with the known specific properties of these sites on recombinant alpha6beta2/3gamma2 receptors. In the presence of GABA concentrations that fail to activate alpha1 subunit-containing receptors, methyl-6,7-dimethoxy-4-ethyl-beta-carboline (30 microM), allopregnanolone (100 nM), and Zn2+ (10 microM) are less efficacious in altering tert-[35S]butylbicyclophosphorothionate binding in the granule cell layer of the alpha6(-/-) than alpha6(+/+) animals. These data concur with the deficiency of the cerebellar alpha6 and delta subunit-containing receptors in the alpha6(-/-) animals and could also account for the decreased affinity of [3H]muscimol binding to alpha6(-/-) cerebellar membranes. Predicted additional alterations in the cerebellar receptors of the mutant mice may explain a surplus of methyl-6,7-dimethoxy-4-ethyl-beta-carboline-insensitive receptors in the alpha6(-/-) granule cell layer and an increased diazepam-sensitivity in the molecular layer. These changes may be adaptive consequences of altered GABAA receptor subunit expression patterns in response to the loss of two subunits (alpha and delta) from granule cells.     

Margatoxin increases dopamine release in rat striatum via voltage-gated K+ channels

The distribution of iodinated margatoxin ([125I]margatoxin) binding sites in rat was investigated by autoradiography. Rat striatum expresses a high density of margatoxin binding sites and, therefore, the effects of margatoxin, charybdotoxin and iberiotoxin have been studied on [3H]dopamine release from rat striatal slices in vitro. Margatoxin (0.1-100 nM) and charybdotoxin (10-1000 nM), but not iberiotoxin increased the spontaneous and the electrically evoked [3H]dopamine release. [3H]dopamine release by margatoxin was inhibited by tetrodotoxin and omega-conotoxin GVIA, but not by atropine, naloxone, N(omega)-nitro-L-arginine and neurokinin or neurotensin receptor antagonists. In the buffer solution used for release experiments, [125I]margatoxin labels a maximum of 0.12 pmol of sites/mg protein in rat striatal membranes with a Kd of 5 pM. [125I]margatoxin binding was inhibited by margatoxin (Ki of 4 pM), charybdotoxin (Ki of 162 pM) but not by iberiotoxin. We conclude that inhibition of margatoxin-sensitive voltage-gated K+ channels increases [3H]dopamine release demonstrating their role in repolarization of nigrostriatal projections. In contrast, iberiotoxin-sensitive, high-conductance Ca2+-activated K+ channels are not involved in release of [3H]dopamine.     

3H]SCH 58261, a selective adenosine A2A receptor antagonist, is a useful ligand in autoradiographic studies

We have characterized the new potent and selective nonxanthine adenosine A2A receptor antagonist SCH 58261 as a new radioligand for receptor autoradiography. In autoradiographic studies using agonist radioligands for A2A receptors ([3H]CGS 21680) or A1 receptors (N6-[3H]cyclohexyladenosine), it was found that SCH 58261 is close to 800-fold selective for rat brain A2A versus A1 receptors (Ki values of 1.2 nM versus 0.8 microM). Moreover, receptor autoradiography showed that [3H]SCH 58261, in concentrations below 2 nM, binds only to the dopamine-rich regions of the rat brain, with a K(D) value of 1.4 (0.8-1.8) nM. The maximal number of binding sites was 310 fmol/mg of protein in the striatum. Below concentrations of 3 nM, the nonspecific binding was <15%. Three adenosine analogues displaced all specific binding of [3H] SCH 58261 with the following estimated Ki values (nM): 2-hex-1-ynyl-5'-N-ethylcarboxamidoadenosine, 3.9 (1.8-8.4); CGS 21680, 130 (42-405); N6-cyclohexyladenosine, 9,985 (3,169-31,462). The binding of low concentrations of SCH 58261 was not influenced by either GTP (100 microM) or Mg2+ (10 mM). The present results show that in its tritium-labeled form, SCH 58261 appears to be a good radioligand for autoradiographic studies, because it does not suffer from some of the problems encountered with the currently used agonist radioligand [3H]CGS 21680.