A1 Binding Sites Research Articles

Differential patterns of local cerebral glucose utilization in response to 5-hydroxytryptamine, agonists

Local cerebral glucose utilization was measured in parallel groups of conscious rats following intravenous injection of either 1 mg/kg8-hydroxy-2-(di- N-propylamino)tetralin (a 5-hydroxytryptamine 1A binding site agonist), 3 mg/kg 5-methoxy 3-(1,2,3,6-tetrahydro-4-pyridinyl)1H indole, succinate (a 5-hydroxytryptamine 1B agonist), or saline alone, using the 2-deoxyglucose quantitative autoradiographic techniques ( n = 5 in each of the three groups). Following both drugs, local rates of glucose use in the majority of the 72 brain areas analysed remained unaltered, but in some other regions either increases or decreases were observed. In keeping with the observed behavioural response to8-hydroxy-2-(di- N-propylamino)tetralin there were marked increases in cerebellum (+ 56%) and motor cortex where a columnar arrangement of increased metabolism (+ 34%) contrasted with adjacent columns of decrease (− 26%). Hippocampal areas showed moderate decreases in glucose use (−13 to −21%). All areas which increased following8-hydroxy-2-(di- N-propylamino)tetralin also increased following 5-methoxy 3-(1,2,3,6-tetrahydro-4-pyridinyl)1H indole, succinate, but in the latter case all elements of the basal ganglia were also increased, including globus pallidus (+ 105%) and the striatum where the changes (+ 54%) were limited to a discrete dorsal region of the nucleus. In the hippocampus only dorsal dentate gyrus was decreased (− 24%) whilst a moderate increase (+16%) was observed in dorsal subiculum. The complexity of these results contrasts with previous 2-deoxyglucose investigations where less specific 5-hydroxytryptamine receptor ligands were used, 11,30 and suggests that certain aspects of brain function may be selectively targeted by systemic pharmacological manipulation of endogenous serotonergic systems.

A 5-HT 1-like receptor mediates a sympathetic ganglionic hyperpolarisation

5-HT induced a hyperpolarisation of the rat superior cervical ganglion in vitro which was resistant to both MDL 72222 (10 μM), a 5-HT 3 antagonist, and ketanserin (1 μM), a 5-HT 2 antagonist. The 5-HT 1-selective ligands 5-carboxamidotryptamine and 8-OH-DPAT also hyperpolarised the ganglion. The 5-HT-induced hyperpolarisation was potently antagonised by spiperone. These results suggest that 5-HT hyperpolarises the rat superior cervical ganglion via a 5-HT 1-like receptor which resembles the central 5-HT 1A binding site.

Isapirone is a partial agonist at 5-hydroxytryptamine 1A (5-HT 1A) receptors in the rat hippocampus: electrophysiological evidence

Using iontophoretic techniques we observed that in vivo isapirone (TVX Q 7821), a selective ligand for the 5-HT 1A binding site, at low ejection currents (5–30 nA) antagonised 5-HT and 8-hydroxy-2-(di-n-propylamino) tetralin (DPAT)-induced suppression of hippocampal unit activity, with little effect on baseline firing rate itself. At higher ejection currents (20–100 nA) or following prolonged application, isapirone inhibited unit firing. Responses to GABA were unaffected by isapirone. These data demonstrate that isapirone is a 5-HT 1A receptor antagonist with partial agonist properties on 5-HT sensitive neurones in the rat hippocampus.

Affinity labeling of adenosine A1 binding sites.

The adenosine A1 receptors of sheep brain membranes have been identified by the specific binding of radiolabeled cyclohexyl[3H]adenosine ([3H]CHA). Pretreatment of membranes with periodate-oxidized CHA causes a dose- and time-dependent decrease in the number of binding sites. No decrease occurs when membranes are pretreated with CHA. Binding of [3H]CHA to the remaining sites occurs with the same characteristics as binding to the untreated receptor population.

Apparent affinity of some 8-phenyl-substituted xanthines at adenosine receptors in guinea-pig aorta and atria.

1 Some 8-phenyl-substituted, 1,3 dipropyl xanthines have previously been demonstrated to have a 20-400 fold greater affinity for A1 binding sites in rat CNS membranes than for A2 adenosine receptors in intact CNS cells from guinea-pigs. In the present study these compounds (1,3, dipropyl-8-phenylxanthine: DPPX; 1,3 dipropyl-8-(2 amino-4-chlorophenyl) xanthine: PACPX; 8-(4-(2-amino-ethyl)amino) carbonyl methyl oxyphenyl)-1,3-dipropylxanthine: XAC; and D-Lys-XAC) together with two that have not been reported to exhibit A1-receptor selectively (8-(p-sulphophenyl)theophylline: 8-PST; 8-(4-carboxy methyl oxyphenyl)-1,3-dipropylxanthine: XCC) have been evaluated as antagonists of the effects of 2-chloroadenosine in two isolated cardiovascular tissues. 2 The isolated tissues used were guinea-pig atria (bradycardic response) and aorta (relaxation), which are thought to possess A1 and A2 adenosine receptors, respectively. 3 All the xanthines antagonized responses evoked by 2-chloroadenosine in both tissues but did not affect responses evoked by acetylcholine (atria) or sodium nitrite (aorta). 4 The xanthines, 8-PST, XAC, D-Lys XAC, XCC and DPPX appeared to be competitive antagonists of the effects of 2-chloroadenosine, as Schild plot slopes did not differ significantly from unity. The 1,3-dipropyl substituted compounds had pA2 values from 6.5 to 7.4 and were more potent than the 1,3 dimethyl substituted 8-PST (pA2 4.9 to 5). 5 For individual xanthines, there was no difference between pA2 values obtained in the atria and in the aorta. 6 Responses evoked by 2-chloroadenosine in atria and aorta were antagonized to a similar degree by PACPX (1 microM). The agonist dose-ratio evoked by 10 microM PACPX was no greater than that evoked by 1 microM of the xanthine in both tissues. This was probably a consequence of the limited aqueous solubility of PACPX. 7. These results fail to demonstrate A, receptor selectivity for DPPX, XAC, D-Lys XAC or PACPX in tissues from the guinea-pig. The A, selectivity previously found for these compounds may therefore be due to their high affinity for binding sites in rat CNS cell membranes.

Stimulation of corticosterone and β-endorphin secretion in the rat by selective 5-HT receptor subtype activation

Changes in plasma concentrations of corticosterone and β-endorphin (β-END) were determined in male rats after treatment with the selective 5-HT 1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) or the non-selective 5-HT agonist 6-chloro-2-(1-piperazinyl)pyrazine (MK-212). The administration of either 8-OH-DPAT or MK-212 increased plasma concentrations of both corticosterone and β-END in a dose-related manner. The corticosterone and β-END responses to 8-OH-DPAT were antagonized by spiperone and (−)-pindolol, both of which have been shown to have high affinity for the 5-HT 1A binding site. In contrast, antagonists which are selective for the 5-HT 2 receptor or non-selective 5-HT antagonists were without effect on the hormone responses to 8-OH-DPAT. The MK-212-induced increase in plasma concentrations of corticosterone and β-END were not affected by treatment with the 5-HT 1A antagonists spiperone and (−)-pindolol. However, the corticosterone and β-END responses to MK-212 were attenuated by the selective 5-HT 2 antagonists ketanserin, ritanserin and altanserin, as well as by the non-selective 5-HT antagonist metergoline. It is concluded that stimulation of either 5-HT 1A or 5-HT 2 receptors results in an activation of the hypothalamic-pituitary-adrenal axis in the rat.

GABA and benzodiazepine receptors in the gerbil brain after transient ischemia: demonstration by quantitative receptor autoradiography.

Quantitative receptor autoradiography was used to measure the binding of gamma-aminobutyric acid (GABA) and benzodiazepine receptors after ischemia by means of transient occlusion of bilateral common carotid arteries in the gerbil. [3H]Muscimol was used to label the GABAA receptors and [3H]flunitrazepam to label central type benzodiazepine receptors. In the superolateral convexities of the frontal cortices, [3H]muscimol binding was increased in 60% of the animals killed 3 days after ischemia, and decreased in 67% of the animals killed 27 days after ischemia. Twenty-seven days after ischemia, [3H]flunitrazepam binding in the substantia nigra pars reticulata increased to 252% of the control, though the increase in [3H]muscimol binding was not significant. In the dorsolateral region of the caudate putamen, marked neuronal necrosis and depletion of both [3H]muscimol and [3H]flunitrazepam binding sites were observed 27 days after ischemia, the ventromedial region being left intact. In spite of the depletion of pyramidal cells in the CA1 region of the hippocampus, both [3H]muscimol and [3H]flunitrazepam binding sites were preserved 27 days after ischemia. Since our previous study revealed that adenosine A1 binding sites were depleted in the CA1 subfield of the hippocampus after ischemia correlating with neuronal damage, GABAA and benzodiazepine receptors may not be distributed predominantly on the pyramidal cells in the CA1 region.

Modulation of neuronal activity in the hippocampus by 5-hydroxytryptamine and 5-hydroxytryptamine 1a selective drugs

The interactions between 5-hydroxytryptamine (5-HT), 8-hydroxy-2-( N, N-dipropylamino)tetralin (8-OH-DPAT), buspirone, 2-(4-(4-(2-pyrimidinyl)-1 -piperazinyl)butyl)-1,2-benzisothiazol-3-(2H) one-1,1-dioxide-hydrochloride (TVX Q 7821) and ketanserin, and putative 5-HT receptors were analyzed using both radioligand techniques and an in vitro hippocampal slice preparation. The potencies of the drugs were determined at 5-HT 1A binding sites labelled by [ 3H] 8-OH-DPAT in hippocampal membranes from the rat. The binding site had similar affinity for 5-HT, 8-OH-DPAT, buspirone and TVX Q 7821, whereas ketanserin was essentially inactive. Physiological effects of these drugs were also examined using an in vitro hippocampal slice preparation. With the exception of ketanserin, application of each drug to the bath modulated the amplitude of the field potential recorded in the pyramidal layer of CA1 evoked by stimulation of Schaffer collaterals. Application of micromolar concentrations of 5-HT produced an initial increase in the population spike followed by a return to near baseline levels within 5 min. By contrast, the amplitude of the population spike was reduced in a dose-dependent manner by micromolar concentrations of 8-OH-DPAT, buspirone and TVX Q 7821, beginning 5 min after application of drug. Ketanserin did not affect the amplitude of the population spike and it did not antagonize the effects of 5-HT, buspirone or TVX Q 7821. Neither buspirone nor 8-OH-DPAT altered the initial increase in population spike induced by 5-HT. Therefore, the physiological effects of the novel anxiolytics 8-OH-DPAT, buspirone and TVX Q 7821 in the hippocampus appear to be independent of their interactions with 5-HT 1A receptors.

Ipsapirone depresses neuronal activity in the dorsal raphe nucleus and the hippocampal formation

Ipsapirone, a putative, novel anxiolytic with a high affinity for 5-HT 1A binding sites, suppressed neuronal activity in both the dorsal raphe nucleus and hippocampal formation of urethane-anesthetized rats. In the hippocampus, dentate granule cells matched the responsiveness of dorsal raphe serotonin neurons, the median effective dose for 50% inhibition being 125.0 μg/kg in both areas. In contrast, the responses of CA1 pyramidal cells were related directly to baseline firing rates. Slow-firing neurons, for example, were inhibited by 31.3 μg/kg and fast-firing cells were unresponsive even up to a dose of 500.0 μg/kg. These results indicate a potent effect of ipsapirone on neuronal activity in sites with a high density of 5-HT 1A receptors.

Monoamine receptor sensitivity changes following chronic administration of MDL 72394, a site-directed inhibitor of monoamine oxidase

(E)-β-Fluoromethylene-m-tyrosine (MDL 72394) is not per se an inhibitor of monoamine oxidase (MAO) but is a substrate of aromatic L-amino acid decarboxylase (AADC) which liberates the potent MAO inhibitor (E)-β-fluoromethylene-m-tyramine (MDL 72392). When co-administered to animals with the peripherally selective AADC inhibitor, carbidopa, MDL 72394 inhibited MAO selectively in the brain. Chronic (14 days plus 3 days withdrawal) administration of 0.5 mg/kg per day p.o. MDL 72394 combined with 10 mg/kg per day p.o. carbidopa or 50 mg/kg per day p.o. pargyline produced equivalent inhibition of rat brain MAO and decreased the binding of [ 3H]clonidine and [ 3H]RX 781094 to the α 2-adrenoceptor and of [ 3H]dihydroalprenolol to the β-adrenoceptor without changing the binding of [ 3H]prazosin to the α 1-adrenoceptor. The locomotor depressant effect of clonidine was attenuated without attenuation of the hypotensive effect in rats treated chronically with the MAO inhibitors. Neither the sensitivity of the α 2-autoreceptor nor of the α 2-heteroreceptor was decreased in brain slices. However, the sensitivity of adenylate cyclase to activation by both noradrenaline and isoprenaline was significantly reduced. The number of 5-HT 2 and 5-HT 1A binding sites was decreased; the 5-HT 1B binding sites remained unchanged. The effect of chronic MAO inhibitor treatment on 5-HT 1A receptors was associated with a decreased in the behavioural response to 8-hydroxy-2-(di-n-propylamino) tetralin and the decrease in 5-HT 2 binding was related to a small reduction in the sensitivity of the inositol phosphate system to stimulation by 5-HT. The lack of effect of chronic MAO treatment on the 5-HT autoreceptor measured in cortical slices corresponded to a lack of effect on the 5-HT 1B binding site except that chronic administration of pargyline produced a small but significant decrease in 5-HT autoreceptor sensitivity. Overall, the data show that chronic administration of MDL 72394 has a profile of effects on central monoamine receptor binding and function similar to that seen following chronic administration of a number of clinically effective antidepressants.

Nucleotide interactions with 5-HT 1A binding sites directly labeled by [ 3H]-8-hydroxy-2-(di)-n-propylamino) tetralin ([ 3H]-8-OH-DPAT)

Nucleotide interactions were examined at 5-hydroxytryptamine 1A (5-HT 1A) binding sites labeled by [ 3H]-8-hydroxy-2-(di)-n-propylamino)tetralin (8-OH-DPAT). At a 10 −4M concentration, GTP and GDP decreased specific binding of 0.4 nM [ 3M]-8-OH-DPAT to 47 ± 4 and 61 ± 1% of control values respectively. This nucleotide effect was significantly greater (P < 0.005) than observed at total 5-HT 1 binding sites labeled by 1.5 nM [ 3H]-5-HT. GMP and adenine nucleotides had a minimal effect on [ 3H]-8-OH-DPAT binding at concentrations less than 10 −3 M. Saturation experiments demonstrated that 10 −4 M GTP increased the K D of [ 3H]-8-OH-DPAT for 5-HT 1A binding sites (0.79 to 2.7 nM) without changing the number of binding sites (1.98 to 1.93 pmoles/g tissue). The K i vlaues of classic and novel putative 5-HT agonists were increased 2- to 4-fold in the presence of 10 −4 M GTP. Affinities of 5-HT antagonists for the [ 3H]-8-OH-DPAT site were not affected by the addition of 10 −4 M GTP to the binding assay.

Comparison of the actions of serotoninergic agents on human saphenous veins and platelets

Changes in tension of helical strips from human saphenous veins and reversible aggregation of human platelets were recorded in vitro. Comparison of the activities of 12 serotonin receptor agonists revealed that only 4 of the investigated tryptamine derivatives acted as full agonists on both tissues. 5-Carboxamidotryptamine, a drug with selective affinity for both 5-HT 1A and 5-HT 1B binding sites, though the most potent agonist on veins, failed to produce platelet aggregation but acted as a weak antagonist of the 5-HT-induced reversible aggregation. The tetralin derivative 8-OH-DPAT, a drug with selective affinity for 5-HT 1A binding sites, was a weak partial agonist on veins and completely devoid of any activity on the platelets. The antagonism of 5-HT by spiperone was monophasic on platelets but biphasic on veins. These data are in line with the contention that the 5-HT-induced reversible aggregation of human platelets is initiated by 5-HT 2-like recognition sites while the evidence suggests that the contractile response of human saphenous vein to 5-HT reflects activation of both 5-HT 2- and 5-HT 1-like recognition sites.

Species variations in RU 24969 interactions with non-5-HT 1A binding sites

The interaction of RU 24969 with [ 3H]5-HT) binding to non-5-HT 1A binding sites was analyzed in nine species. Non-5-HT 1A binding was defined as specific [ 3H]5-HT binding observed in the presence of 100 nM 8-OH-DPAT. Computer-assisted iterative curve fitting of RU 24969 competition studies indicate that RU 24969 distinguishes two distinct components of non-5-HT 1A sites in rat and mouse brain. These two binding components display apparent K i values of 0.33–0.39 and 66–130 nM for RU 24969. By contrast, RU 24969 competition for non-5-HT 1A binding in guinea-pig, cow, human, dog, chicken, turtle and frog brain membranes is consistent with a homogeneous population of [ 3H]5-HT binding sites. This population of sites displays apparent K i values of 23–130 nM for RU 24969 and appear to be analogous to the low affinity component of non-5-HT 1A sites identified in rat and mouse.

Inhibition of forskolin-stimulated adenylate cyclase activity by 5-HT receptor agonists

We measured the inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig hippocampal membranes by 5-HT, 5-carboxamidotryptamine (CAT) and 8-hydroxy-2-(di-n-propylamino) ttetralin (PAT). Low concentrations of these agonists inhibited forskolin-stimulated adenylate cyclase activity in a concentration-dependent and saturable manner. The antagonist spiperone shifted the concentration-response curve to CAT to the right in a parallel manner. The EC 50 values of CAT, PAT and 5-HT and the K B of spiperone suggest that this receptor may correspond to the 5-HT 1A binding site.

Selective labeling of 5-HT 1A and 5-HT 1B binding sites in bovine brain

Drug interactions with serotonin(1A) 5-HT 1A and serotonin(1B) (5-HT 1B) binding sites were analyzed in bovine brain membranes. 5-HT 1A binding sites were directly labeled with [ 3H]8-hydroxy-2-(di- n-propylamino)-tetralin (8-OH-DPAT) in bovine hippocampal membranes. 5-HT 1B binding sites were labeled by [ 3H]5-HT in bovine striatal membranes where less than 15% of specific binding sites are sensitive to nanomolar concentrations of 8-OH-DPAT. Each of the 12 agents tested was more potent at the 5-HT 1A than 5-HT 1B binding site. 5-HT, bufotenine, N,N-dimethyltryptamine (DMT) and quipazine were only slightly more potent at the 5-HT 1A binding site. By contrast, 8-OH-DPAT, TVX Q 7821 and buspirone were significantly more potent at [ 3H]8-OH-DPAT binding sites in bovine hippocampus than at [ 3H]5-HT binding sites in bovine striatum. These findings suggest that 5-HT 1A and 5-HT 1B binding sites have distinct pharmacological profiles and can be directly labeled with appropriate [ 3H]ligands in specific brain regions.

CHARACTERISTICS OF HIGH AFFINITY AND LOW AFFINITY ADENOSINE BINDING SITES IN HUMAN CEREBRAL CORTEX: 96

Binding characteristics of human cortical membrane fractions were evaluated to test the hypothesis that there are A1 and A2 adenosine binding sites. Binding of chloroadenosine was time dependent, reversible and concentration dependent. The Kd for chloroadenosine was 280 nM with a Bmax of 1.6 pmoles/mg protein. The apparent Kd was estimated to be 0.74 μM for 5'-N-ethylcarbox-amideadenosine, 1 μM cyclohexyladenosine, 13 μM N6-(L-2-phenylisopropyl)adenosine, 84 μM isobutylmethylxanthine and 105 μM theophylline. Hill slope factors ranged from 0.3 to 0.6. The kob was 0.080 min−1, the K1 7.5 × 104 min−1M−1, and the k2 .074 min−1. The Kd calculated from the rate constants was 990 nM. Cyclohexyladenosine binding was concentration dependent and the Kd was 5 nM with a Bmax of 0.35 pmoles/mg protein. Cyclohexyladenosine binding was displaced by N6-(L-2-phenylisopropyl)adenosine (Kd 4 nM), 2-chloroadenosine (Kd 10 nM), 5'-N-ethylcarboxamideadenosine (Kd 6 nM), isobutylmethylxanthine (4 μM) and theophylline (8 μM). Hill slope factors ranged from 0.5 to 0.8.Our data support the existence of two adenosine binding sites in human cortex compatable with low affinity (A2) and high affinity (A1) adenosine receptors.

Solubilization of adenosine A1 binding sites from sheep cortex

[(3)H]N(6)-cyclohexyladenosine binds with high affinity to sheep brain membranes with a drug specificity indicating an association with A(1) adenosine receptors. The [(3)H]N(6)-cyclohexyladenosine binding site has been solubilized with sodium cholate being the only detergent able to maintain specific binding after solubilization. After solubilization, the kinetics and drug specificity of binding are virtually identical with those obtained in the intact membranes, indicating a conservation of the binding site after removal of the receptor from its lipid environment. Gel filtration experiments indicated an apparent molecular weight of 400,000 for the receptor-detergent complex and a Stokes radius of 6.2 nm.