COMPETITION OF ADENINE NUCLEOTIDES FOR A l,3-[3H]-DIPROPYL-8-CYCLOPENTYLXANTHINE BINDING SITE IN RAT VAS DEFERENS

1. The binding of l,3-[³H]-dipropyl-8-cyclopentylxanthine ([³H]-DPCPX), a specific adenosine Ai receptor antagonist, was examined in rat vas deferens membrane preparations using radioligand binding techniques. 2. l,3-[³H]-Dipropyl-8-cyclopentylxanthine bound to these preparations with a KD of 1.07 ± 0.14 nmol/L (n = 6). The density of [³H]-DPCPX binding sites was 133.38 ± 5.57 fmol/mg protein. 3. Computer analysis indicated that nucleosides competed for [³H]-DPCPX binding at two distinct sites. The rank order of potency at the higher affinity site corresponded to R-phenyliso-propyladenosine (R-PIA) 2-chloroadenosine (2-CI ADO) cyclopentyladenosine (CPA) N-ethylcarboxamidoadenosine (NECA)>s-phenylisopropyladenosine (s-PIA). Kj values were in the low nmol/L range. The rank order of nucleoside potency at the lower affinity site corresponded to R-PIA CPA NECA>=2-ClADO> S-PIA. Ki values were in the low μmol/L range. 4. Nucleotides competed for [³H]-DPCPX binding at a single site only. The rank order of potency at this site corresponded to a,β-methylene ATP βγ-methylene ATP = ATP. Ki values were in the high |xmol/L range. This site seemed to correspond with one of the two binding sites predicted by nucleoside competition binding. 5. The ATP-regenerating compound myokinase did not significantly change the competition curve for ATP, indicating that the competition for [³H]-DPCPX binding observed in the presence of ATP was due to an effect of ATP per se and not to an action of a degradation product. 6. The results demonstrate that in rat vasa deferentia there exist two distinct binding sites for [³H]-DPCPX. One of these sites binds only nucleosides and may represent an adenosine Ai receptor, as usually defined. The other site binds both nucleosides and nucleotides and may represent an atypical adenosine A₁ receptor, an atypical P₂ or a P₃ purinoceptor.     

COMPARATIVE STUDY ON α1-ADRENOCEPTOR ANTAGONIST BINDING IN HUMAN PROSTATE AND AORTA

1. Specific binding of [³H]-prazosin in prostatic and aortic membranes of humans was saturable and of high affinity (prostate: apparent dissociation constant, K_d= 0.35 ± 0.03 nmol/L; aorta: K_d= 0.26 ± 0.03 nmol/L). The density of [³H]-prazosin binding sites (B_max) for prostate and aorta was 546 ± 31 and 61.6 ± 1.6 fmol/mg protein, respectively. 2. Prazosin, YM617, naftopidil and urapidil competed with [³H]-prazosin for the binding sites in a dose-dependent manner in the prostate and aorta of humans. The binding affinities of these antagonists in both tissues were compared, based on the inhibition constant, K_i. Both prazosin and urapidil showed similar affinity to [³H]-prazosin binding sites in human tissue, whereas YM617 and naftopidil showed approximately a 12 and two times higher affinity, respectively, to α₁-adrenoceptor sites of prostate than aorta. 3. The chloroethylclonidine treatment reduced partially the B_max values for specific [³H]-prazosin binding in the prostate and aorta of humans with little effect on the K_d values. 4. These data suggest that YM617 is a relatively selective antagonist of human prostatic α₁-adrenoceptors.     

EFFECTS OF CAPTOPRIL ON [3H]-NOREPINEPHRINE RELEASE IN RAT CENTRAL NERVOUS SYSTEM

1. The present study was performed to investigate the effects of captopril (an angiotensin converting enzyme inhibitor, ACE-I) on noradrenergic transmission in the rat central nervous system. 2. Slices of rat hypothalamus and medulla oblongata were prepared and prelabelled with [³H]-norepinephrine. Slices were continuously superfused with Krebs-Ringer solution, and electrical stimulation (1 Hz) was performed. 3. Captopril significantly inhibited the stimulation-evoked [³H]-norepinephrine release from rat hypothalamic slices in a dose-dependent manner (S2/S1 ratio: control 0.904 ± 0.025, n= 6, captopril l × 10^-5 mol/L 0.617 ± 0.043, n= 6,P<0.05, captopril 5 ×10^-5 mol/L 0.547±0.037, n= 6, P<0.05). However, the basal release of [³H]-norepinephrine was not affected by captopril. 4. Captopril also reduced the stimulation-evoked [³H]-nor-epinephrine release in the medulla oblongata (S2/S1 ratio: control 0.878 ± 0.018, n= 6, captopril 3.3 × 10^-5 mol/L 0.624 ± 0.046, n= 6, P<0.05). 5. These results show that captopril might inhibit the stimulation-evoked norepinephrine release in rat hypothalamus and medulla oblongata. Although the precise mechanisms underlying the neurosuppressive effect of captopril are still uncertain, the finding suggests that the inhibition of noradrenergic transmission might be related to the central action of the ACE-I.     

A1-and A2-PURINOCEPTORS IN THE GUINEA-PIG UTERUS

1. Radioligand binding and functional studies were undertaken to investigate the P₁-purinoceptors present in the separated myometrial layers and the endometrium of the guinea-pig uterus. 2. In preparations of endometrium-denuded circular myometrium, the A₂-selective agonists (2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido-adenosine (CGS 21680, 100 μmol/L) and N-ethylcarboxamido adenosine (NECA, 1–10 μmol/L) inhibited contractile responses to phenylephrine. In preparations of endometrium-intact circular myometrium, NECA (10 μmol/L) enhanced responses to phenylephrine. NECA did not modulate the spontaneous contractions of longitudinal myometrium. 3. Homogenate binding studies with circular myometrium, longitudinal myometrium and endometrium revealed saturable high affinity [³H]-NECA binding sites. The mean maximal densities of binding sites (B_max) were 2.08, 14.7 and 15.5 fmol/mg protein, and pK_D (neg. log dissociation constant) values were 9.82, 9.19 and 7.44, respectively. 4. (R-) and (S-) -N⁶-(2-phenylisopropyl)adenosine (R- and S-PIA) both competed for two [³H]-NECA binding sites in preparations of circular myometrium. CGS 21680 competed for two [³H]-NECA binding sites in preparations of endometrium and longitudinal myometrium. All other agonist competition was for one site only. The rank orders of potency of high affinity binding were S-PIA ≥ R-PIA ≥ CGS 21680 (circular myometrium), R-PIA ≥ CGS 21680 ≥ S-PIA (longitudinal myometrium) and CGS 21680 > > S-PIA ≥ R-PIA (endometrium). 5. In preparations of circular myometrium, longitudinal myometrium and endometrium the selective A₁-purinoceptor antagonist, 1,3-dipropyl-8-(2-amino-4-chloro)-phenylxanthine (PACPX), competed for two [³H]-NECA binding sites, the non-selective antagonist 3,7-dimethyl-1-propargylxanthine (DMPX), competed for one site only. 6. NECA increased cyclic adenosine monophosphate (CAMP) levels in preparations of both circular myometrium and endometrium. 7. These results indicate that P₁-purinoceptors of the A₂-subtype mediate the inhibitory effects of adenosine analogues on the phenylephrine-induced contractions of the circular myometrium of the guinea-pig, this effect is modified by the presence of the endometrium. There is no evidence that the [³H]-NECA binding sites of the longitudinal myometrium correlate with functional P₁-purinoceptors in this tissue.     

High-affinity binding of [3H]doxepin to histamine H1-receptors in rat brain: Possible identification of a subclass of histamine H1-receptors

The binding of the radioactively labeled tricyclic antidepressant, [³H]doxepin, to rat brain tissie was examined. Scatchard plots of specific [³H]doxepin binding indicated the presence of two distinct binding sites. The equilibrium dissociation constant (K_D) of the high-affinity site was 0.020 nM with a maximal binding capacity (B_max) of 13.7 fmol/mg protein. The corresponding values for the low-affinity site were 3.6 nM and 740 fmol/mg protein, respectively. The high-affinity site was sensitive to competition by pharmacologically relevant concentrations of histamine H₁ antagonists such as pyrilamine (K_D = 1.0 nM), diphenhydramine (K_D = 20 nM), d-chlorpheniramine (K_D = 1.7 nM), and 1-chlorpheniramine (K_D = 97 nM). The B_max for [³H]doxepin binding in the high-affinity H₁-receptor, however, was approximately 10% of the B_max obtained using [³H]pyrilamine to label the H₁-receptor. Various tricyclic antidepressants were very potent inhibitors at the high-affinity [³H]doxepin site. Their potencies, however, did not correlated with their potencies previously reported for the H₁-receptor. The regional distribution of [³H]doxepin high-affinity sites correlated with the known distribution of H₁-receptors in the rat brain. These results suggest that [³H]doxepin is binding to a subclass of histamine H₁-receptors.     

EFFECTS OF BRADYKININ ON [3H]-NOREPINEPHRINE RELEASE IN RAT HYPOTHALAMUS

• 1 We examined the regulatory actions of bradykinin on norepinephrine release in the hypo-thalamus of rats.
• 2 Bradykinin increased the stimulation-evoked [³H]-norepinephrine release from hypothalamic slices of Sprague-Dawley rats in a dose-dependent manner (1 Hz: S2/S1 ratio, mean ± s.e.m., control 0.868±0.016, n= 6; bradykinin 1±10^–6mol/L 1.039±0.018, n= 6, P<0.05;bradykinin3.3 ×10^–6 mol/L 1.130 ± 0.064, n= 6, P<0.05). The basal release of [³H]-norepinephrine was not affected by the peptide.
• 3 Bay K 8644, a dihydropyridine-sensitive calcium channel agonist, significantly potentiated the facilitatory effect of bradykinin on norepinephrine release, although Bay K 8644 by itself had no significant effect. By contrast, nicardipine, a dihydropyridine-sensitive calcium channel blocker, reversed the increase in norepinephrine release induced by bradykinin and Bay K 8644.
• 4 These results indicate that bradykinin may increase norepinephrine release in rat hypo-thalamus, partially mediated by interactions with dihydropyridine-sensitive calcium channels.

     

Heterogeneity of Guinea-Pig Lung Muscarinic Receptors Revealed by [3H]4-DAMP-binding

Summary: Muscarinic receptors present in guinea-pig lung were characterized using the M₃ selective radioligand [³H]4-diphenylacetoxy-N-methyl-piperidine methiodide ([³H]4-DAMP). In saturation studies, [³H]4-DAMP identified two populations of binding sites with approximately 4% of the sites displaying high affinity (K_d=0.21 nM and B_max= 10 fmol/mg prot.) while the remaining sites were low affinity ones (K_d=18.11 nM and B_max=269 fmol/mg prot.). In competition studies with [³H]4-DAMP (0.35 nM), methoctramine and hexahydro-siladifenidol (HHSiD) identified 50 and 70% of high affinity binding sites displaying the pharmacological profile of the M₂ and the M₃ receptors, respectively. No evidence was found for high affinity [³H]pirenzepine binding sites in guinea-pig lung. However, pirenzepine/[³H]4-DAMP competition experiments suggested that pirenzepine recognized an equal proportion of [³H]4-DAMP binding sites with intermediate and low affinity binding constants. The intermediate affinity binding constant was inconsistent with the presence of M₁ receptors and reflected more the presence of M₄ or a mixture of M₃ and M₄ receptors. The low affinity pirenzepine binding sites may represent M₂ receptors. These results provide further evidence for the occurrence of M₂ and M₃ receptors and suggest the presence of the M₄ muscarinic receptor subtype in guinea-pig lung.     

Characteristics of high affinity binding of [3H]N-acetylserotonin in rat brain

Evidence that the putative pineal hormone, N-acetylserotonin (NAS), is present in diverse areas of the brain of the rat and that this compound is biologically active, prompted a study of its central binding characteristics. [³H]N-acetylserotonin ([³H]-NAS) exhibits specific, saturable and high affinity binding in synaptosomal fractions from the brain of the rat. In fresh CNS membranes, [³H]N-acetylserotonin appeared to bind to a single population of high affinity sites with a dissociation constant (K_d) of 3–5 nM and a maximal binding site concentration (B_max) of 250–400 fmol mg^?1 protein. A comparison of the regional distribution of the binding of [³H]N-acetylserotonin and [³H]-5-hydroxytryptamine ([³H]-5-HT) indicated that both radioligands exhibited the greatest binding in the striatum and frontal cortex, while relatively more [³H]N-acetylserotonin was bound in the cerebellum and brainstem. The structural specificity of the binding suggests that [³H]N-acetylserotonin labelled serotonergic receptors in the frontal cortex.     

Sodium dependent [3H]cocaine binding associated with dopamine uptake sites in the rat striatum and human putamen decrease after dopaminergic denervation and in Parkinsons disease

Summary The binding of radiolabelled cocaine, an inhibitor of dopamine uptake, to the post-mortem human putamen was studied and compared to that in the rat striatum. Saturation analysis of [³H]cocaine binding to the human putamen revealed the presence of a high affinity component of binding with a K _d of 0.21 mol/l and a B _max of 1.47 pmol/mg protein. In addition a low affinity component (K _d=26.4 mol/l) was demonstrated, having a B _max of 42.2 pmol/mg protein. Also in the rat striatum [³H]cocaine binding was both of high affinity (K _d=0.36 mol/l, B _max=5.56 pmol/mg protein) and low affinity (K _d=25.9 mol/l, B _max=35.6 pmol/mg protein). A pharmacological characterisation of high affinity [³H]cocaine binding to rat striatal membranes clearly indicates an association with the neuronal dopamine transporter. The IC₅₀ values of 8 selected drugs for inhibition of [³H]cocaine binding in the rat striatum were highly significantly correlated with their potency to inhibit [³H]dopamine uptake into slices of the rat striatum. [³H]Cocaine binding was stereospecifically inhibited by (+)nomifensine and (+)diclofensine which were 50–80-fold more active than their respective (-)isomers. Drugs with dopamine releasing activity were more potent at inhibiting [³H]dopamine uptake than at competing for the high affinity site of [³H]cocaine binding. A highly significant correlation was found between IC₅₀ values for [³H]cocaine binding in the rat striatum and the human putamen. Further evidence in support of an association of [³H]cocaine binding in the rat striatum with the dopamine transporter was obtained from lesion studies. Thus, intranigral 6-hydroxydopamine administration produced a marked (67%) decrease in striatal [³H]cocaine binding. Also in the human putamen high affinity [³H]cocaine binding sites appear localized on dopaminergic nerve terminals as evidenced by a prominent decrease in binding in the putamen obtained from subjects with Parkinsons disease. It is concluded that [³H]cocaine may be a useful ligand to examine the dopamine transporter in the rat striatum and the human putamen. Therefore it offers a new and valuable approach in the study of drug effects and neuropsychiatric diseases.Preliminary results on some parts of this study have appeared previously in abstract form (Langer et al. 1984a) or as a rapid communication (Pimoule et al. 1983)     

赛庚啶对大鼠心肌细胞膜[3H]-d-cis-硫氮卓酮结合部位的影响

苯并噻嗪类钙通道阻滞剂[³H]-d-cis-硫氮酮能以一种特异和可饱和的方式与离体大鼠心肌细胞膜结合,其K_D值和B_max分别为84nmol·L^-1和0.279pmol·mgprotein^-1。非标记的硫氮酮和赛庚啶均能完全抑制这种结合,其Ki值分别为102nmolL^-1和5.5umol·L^-1。上述结果证实在大鼠心肌细胞膜上也存有[³H]-硫氮酮受体,同时还提示赛马庚啶对心肌细胞膜的钙通道阻滞作用可能与作用于心肌细胞膜[³H]硫氮酮受体有关。     

Characteristics and regulation of high affinity [3H]imipramine binding to rat hippocampal membranes

The imipramine binding sites located in the membrane of the rat hippocampus have been characterized in order to obtain a better understanding of their role in the treatment of mental depression. The binding of [³H]imipramine to hippocampal membranes has a K_d of 8.3 ± 1.5 nM, B_max of 800 ± 60 fmol/mg protein and a Hill coefficient of 1.0. The number of specific binding sites for [³H]imipramine was rapidly and irreversibly reduced when the incubation temperature was raised from 0 to 37°C. Experiments using leupeptin and EGTA suggest that this temperature-dependent inactivation is due to a destruction of the binding sites by Ca²⁺-dependent proteases released upon disruption of the cells. The binding of [³H]imipramine to the hippocampal membrane is also sensitive to phospholipase A₂ as well as pronase, suggesting the complexity of the regulation of this binding. The [³H]imipramine binding sites appear to have been solubilized from membranes by treatment with Triton X-100. However, these solubilized binding sites also display a high level of nonspecific binding of this ligand. Studies of tissue distribution and subcellular localization of the imipramine binding sites suggest that these sites are located in neurons. This possibility is strengthened by the finding that the high affinity imipramine binding sites are also detected in the membranes of neuroblastoma N4TG1 cells.     

[3H]2-(2-Benzofuranyl)-2-imidazoline,a highly selective radioligand for I2-imidazoline receptor binding sites Studies in rabbit kidney membranes

2-(2-Benzofuranyl)-2-imidazoline (2-BFI) has recently been characterised as a selective ligand for the I₂-type of imidazoline-receptor binding site(s) (I₂-RBS). The present studies determined the relative levels of specific [³H]2-BFI binding to membrane homogenates of brain and kidney from rat, guinea pig and rabbit and identified the pharmacological characteristics of [³H]2-BFI binding sites in rabbit kidney membranes. Rabbit kidney membranes had the highest relative density of specific [³H]2-BFI binding of all tissues studied (2000?fmol/mg protein). Rabbit brain and guinea pig kidney had moderate levels of specific [³H]2-BFI binding (350–500?fmol/mg protein), while rat kidney and guinea pig and rat brain displayed much lower densities of binding (40–65?fmol/mg protein). Studies of [³H]2-BFI binding kinetics in rabbit kidney homogenates revealed binding to two distinct sites with K _d values of 0.10?±?0.01?nmol/l and 1.00?±?0.36?nmol/l respectively. Equilibrium saturation studies were also consistent with the presence of two binding sites – [³H]2-BFI (0.01–20?nmol/l) bound to sites with affinities of 0.10?± 0.01?nmol/l and 0.92?±?0.13?nmol/l and binding densities of 470?±?80 and 840?±?60?fmol/mg protein (n=3), representing 36 and 64% respectively. Drug inhibition studies revealed that l-adrenaline; α₁-adrenoceptor drugs (prazosin, l-phenylephrine) and α₂-adrenoceptor drugs (rauwolscine, methoxyidazoxan, 2-(2,4-(O-methoxyphenyl)-piperazin-1-yl)-ethyl-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione (ARC-239) had extremely low affinities for [³H]2-BFI binding sites (IC₅₀?≥?10?μmol/l). Putative I₁-RBS compounds, p-aminoclonidine, moxonidine, imidazole-4-acetic acid and cimetidine, inhibited [³H]2-BFI binding to rabbit renal membranes with low to very low affinities (K _i values 3 to ≥100?μmol/l), suggesting [³H]2-BFI does not label I₁-RBS in rabbit kidney membranes. I₂-RBS compounds – 2-(4,5-dihydroimidaz-2-yl)-quinoline (BU224), 2-(4,5-dihydroimidaz-2-yl)-quinoxaline (BU239), idazoxan and cirazoline – potently inhibited [³H]2-BFI binding (K _i values 0.37–1.6?nmol/l), confirming the labelling of I₂-RBS. Inhibition of [³H]2-BFI binding by certain compounds was consistent with their interaction with two binding site populations – for example (drug, K _i values) guanabenz, 0.65?nmol/l and 0.17?μmol/l; naphazoline, 0.94?nmol/l and 2.8?μmol/l; amiloride, 76?nmol/l and 26?μmol/l rilmenidine, 150?nmol/l and 50?μmol/l; and clonidine, 230?nmol/l and 70?μmol/l. The high affinity of amiloride for a high proportion (85%) of the binding is consistent with the presence of the I_2A-subtype of I-RBS in rabbit kidney. These results demonstrate that [³H]2-BFI is a highly selective and high affinity radioligand for I₂-RBS which should be useful for the further characterisation of these sites in mammalian tissues.     

粉防己碱,小檗胺等四氢异喹啉类生物碱对大鼠脑内M-胆碱受体的作用

用放射受体结合方法,研究了36种四氢异喹啉类生物碱及其半合成衍生物对大鼠脑内M-胆碱受体的结合特性。实验发现,粉防己碱对M-胆碱受体的亲和力最高,其K_i值为7.3×10^-8mol/L。小檗胺、四氢黄连碱和半合成原小檗碱衍生物B₁亦具有较高亲和力,K_i值分别为1.9×10^-7,6.8×10^-7和8.1×10^-7mol/L。四氢小檗碱半合成原小檗碱衍生物B₂,B₃,B₄,B₅,B₆和半合成小檗胺衍生物E₁及半合成蝙蝠葛碱衍生物D₁对M-胆碱受体的亲和力为中等强度,K_i值在1～2×10^-6mol/L之间。实验结果提示,某些四氢异喹啉类生物碱的药理作用可能与M-胆碱受体有关。     

Effect of synthetic purines and purine nucleosides on [3H]diazepam binding in brain

We have compared fifteen synthetic purines and purine nucleosides on their ability to displace [³H]diazepam binding to rat brain membranes. Among these analogs, 6-methylthioguanine was found to be most potent, inhibiting competitively the specific binding of [³H]diazepam with a K_i value of 16 μM. At a concentration of 50 μM, 6-methyl-thioguanine increased the apparent K_D of specific diazepam binding from 4.3 nM to 13.3 nM without affecting the B_max, nor had it any effect on the non-specific binding. Binding with membrane preparations from developing rat brain was slightly less sensitive to 6-methylthioguanine inhibition than that with membranes prepared from mature brain.     

Ca2+-DEPENDENT AND -INDEPENDENT MECHANISMS OF ISCHAEMIA-EVOKED RELEASE OF [3H]-DOPAMINE FROM RAT STRIATAL SLICES

1. Ischaemia was induced by 5 min of deprivation of glucose and an additional 5 min of deprivation of glucose and oxygen from Mg²⁺-free artificial cerebrospinal fluid in vitro. 2. During the ischaemic period, 11 ± 1.5% of the total [³H]-dopamine ([³H]-D A) was released into the incubation medium. 3. Ischaemia-evoked release of [³H]-DA from striatal slices was attenuated by tetrodotoxin (TTX), MgSO₄, dizocilpine, ketamine, 6,7-dinitroquinoxaline-2,3-dione (DNQX), or carbetapentane. 4. Release of [³H]-DA was attenuated by verapamil, ω-conotoxin GVIA and dantrolene. 5. Nomifensin inhibited the ischaemia-induced release of [³H]-DA. 6. Omission of Ca²⁺ from incubation media potentiated ischaemia-evoked [³H]-DA release. The inhibitory effect of nomifensin was potentiated in Ca²⁺-free incubation media. 7. These results suggest that ischaemia induces release of [³H]-DA by dual mechanisms; one is Ca²⁺-dependent exocytosis and the other is reversal of transporter.     

[3H] sertraline binding to rat brain membranes

Tritiated sertraline, a radiolabeled form of a potent and selective inhibitor of serotonin uptake, was found to bind with high affinity to rat whole brain membranes. Characterization studies showed that [³H] sertraline binding occurred at a single site with the following parameters:K _d 0.57 nM,B _max 821 fmol/mg protein,n _h 1.06. This binding was reversible; the dissociation constant calculated from kinetic measurements (K _d 0.81 nM) agreed with that determined by saturation binding experiments. [³H] Sertraline binding in the presence of serotonin, paroxetine, fluoxetine or imipramine suggested competitive inhibition of binding (large increase inK _d with little change inB _max). The rank order of potency of inhibition of [³H] sertraline binding was similar to that of inhibition of serotonin uptake for known uptake inhibitors and the 1-amino-4-phenyltetralin uptake blockers. A marked decrease in ex vivo [³H] sertraline binding in the brain of rats 7 days after treatment withp-chloroamphetamine was consistent with the loss of serotonin uptake sites induced by this agent. The results of our study indicated that [³H] sertraline labels serotonin uptake sites in rat brain.     

蝙蝠葛碱和粉防己碱对[3H]WEB 2086与体外牛脑前动脉平滑肌细胞结合的影响(英文)

用标记的血小板活化因子拮抗剂[³H]WEB 2086,在培养的牛脑前动脉平滑肌细胞上鉴定了血小板活化因子受体。结果表明在25℃时该细胞上存在两种与配基具有不同亲和力的受体结合位点,其中K_d-1=22.8±5.0 nmol·L^-1,K_d-2=186+20.5 nmol·L^-1;B_max-1=2.1±0.3 pmol/10⁴细胞,B_max-2=12.1±1-5 pmol/10⁶细胞。蝙蝠葛碱和粉防己碱均能抑制[³H]WEB2086与上述细胞的结合。     

CHARACTERIZATION AND LOCALIZATION OF [3H]-CLONIDINE BINDING IN MEMBRANES PREPARED FROM GUINEA-PIG SPLEEN

1. [3H]-Clonidine binds to membranes prepared from guinea-pig spleen with high affinity. 2. Kinetic experiments indicated that [3H]-clonidine associates rapidly to the binding site and that the binding is reversible. A study of the dissociation of [3H]-clonidine from splenic membranes revealed two components. The slowly dissociating component corresponded to a high affinity process (Kd = 2.1 nmol/l) in good agreement to that obtained by saturation analysis. 3. Over the concentration range used, saturation experiments revealed only a single population of sites with a dissociation constant (Kd) of 2.4 nmol/l and a density of 5.1 pmol/g wet weight tissue. 4. Examination of the relatively potency of a series of alpha-adrenoceptor agonists and antagonists indicates that [3H]-clonidine binding is to alpha 2-adrenoceptors. 5. High levels of binding were obtained to lymphocytes prepared from guinea-pig spleen and to membranes from the splenic capsule. Pretreatment of animals with 6-hydroxydopamine produced changes in apparent affinity of binding with little change in the number of receptor sites. 6. It is concluded that [3H]-clonidine labels is a site resembling the alpha 2-adrenoceptor in guinea-pig spleen. Few if any of these sites are located prejunctionally and a significant fraction are associated with lymphocytes.     

Binding of adrenergic ligands ([3H]clonidine and [3H]WB-4101) to multiple sites in human brain

In order to identify alpha-adrenoceptors in post-mortem human brain and to detect the possible existence of multiple types of binding sites for adrenergic [³H]ligands, we studied the binding of [³H]clonidine and [³H]WB-4101 to human brain cerebral cortex, hippocampus, hypothalamus and striatum. Frontal cortex revealed two binding sites for [³H]clonidine (with K_D values of approximately 1 and 8 nM), as indicated by the biphasic Scatchard plot, the biphasic pattern of dissociation kinetics, and the biphasic inhibition by phentolamine on the binding of [³H]clonidine; the high-affinity site was heat-labile. Two high-affinity binding sites for [³H]WB-4101 were also detected in the human frontal cortex (with K_D values of about 0.09 and 1.5 nM), as revealed by a biphasic pattern of dissociation. A third site with low affinity binding for [³H]WB-4101 was detected by the biphasic inhibition by phentolamine (as well as by WB-4101 and prazosin) on the binding of [³H]WB-4101. The three other brain regions revealed very similar patterns exhibited by the frontal cortex, except that the density of the [³H]clonidine sites (of either high or low affinity) was highest in the hypothalamus, whereas the density of [³H]WB-4101 sites was highest in the hippocampus.     

Characterization of peripheral type benzodiazepine binding sites in human and rat platelets by using [3H]PK 11195. Studies in hypertensive patients

Peripheral type benzodiazepine binding sites have been studied in human and rat platelets and platelet membranes by using PK 11195 (1-(2-chlorophenyl)-N-methyl-N-(1-methyl propyl)-3-isoquinolinecarboxamide) as a ligand. [³H]PK 11195 binding to the intact cells and membranes is saturable, with high affinity and presents the pharmacological specificity corresponding to the peripheral binding sites (PK 11195 > RO5–4864 > diazepam > clonazepam). [³H]PK 11195 affinity is not affected by cell lysis, but there is a loss of binding capacity, contrarily to RO5–4864 whose affinity is greatly diminished. For this reason [³H]RO5–4864 binding can only be demonstrated in intact cells. Furthermore opposite to RO5–4864, PK 11195 affinity is not decreased by increasing temperatures. No difference was found between binding parameters (K_D and B_max) for [³H]PK 11195 between normotensive and hypertensive subjects. The very high binding capacity of human and rat platelets (B_max > pmole/10⁸ cells) makes them a good biological model for studying the physiological significance of “peripheral type” benzodiazepine binding sites.