甲基黄酮醇胺盐酸盐对β受体的阻断作用

The β-receptor blocking action of methylflavonolamine hydrochloride(MFA) was studied and compared with those of propranolol. The doseresponse curves of isoproterenol were shifted to the right by MFA on isolated rabbit atrium in this experiment. The pA₂ value and the slope of the regression line of MFA calculated from Schild plot were 5.53 and -0.84 respectively. The effects of MFA and propranolol on duck erythrocyte membranes were studied by the radioligand binding method. Both MFA and propranolol inhibited the binding of [³H] dihydroalprenolol to β-receptors. Their apparent equilibrium dissociation constants were 1.12×10^-5 mol/L and 5.50×10^-9 mol/L respectively. The affinity of propranolol to β-receptors of duck erythrocyte membranes was 2039-fold higher than that of MFA. These results demonstrates that MFA is a weak competitive β-receptor blocking agent.     

一叶萩碱对5种神经递质受体和蛙脊髓膜电位的影响

Using radioligand binding assay method, securinine was shown to be specifically bound to GABA receptors of rat brain with IC₅₀ of 10^(-4)～10^-5mol/L. However, securinine did not exhibit affinity for a₁-, a₂- adrenoceptors and M-cholinergic receptors of rat brain and the β-adrenoceptors of duck erythrocyte membrane. In isolated and perfused bullfrog spinal cord experiment, the depolarization induced by GABA was slightly depressed by securinine (1 mmol/L), but no antagonistic effect on glycine or taurine evoked depolarization was observed with securinine. These results indicate that securinine is a weak GABA antagonist.     

Interaction of the diuretics torasemide and U-37883A with the KATP channel in rat isolated aorta

In this study we have investigated the interaction of the loop diuretics torasemide and furosemide and of the eukalemic diuretic U-37883A (4-morpholinocarboximidine-N–1-adamantyl-N’-cyclohexylhydrochloride) with the ATP-sensitive K⁺ channel (K_ATP channel) in rat aortic rings. Torasemide contains a sulphonylurea group which might enable the compound to interfere with K_ATP channels; this group is lacking in furosemide. U-37883A blocks several types of K_ATP channels. The interaction with the vascular K_ATP channel was probed in binding studies, ⁸⁶Rb⁺ efflux experiments and vasorelaxation assays. Torasemide inhibited the binding of the K_ATP channel inhibitor [³H]glibenclamide and of the opener [³H]P1075 with IC₅₀ values of 19 and 45 μM, respectively; furosemide and U-37883A were inactive or interfered with binding in a nonspecific way. In ⁸⁶Rb⁺ efflux experiments, the loop diuretics, at μM concentrations, inhibited basal tracer efflux to 50% whereas U-37883A had no effect. P1075-stimulated ⁸⁶Rb⁺ efflux, a qualitative measure of K_ATP channel opening, was inhibited by U-37883A and torasemide with IC₅₀ values of 0.06 and 130 μM, respectively; furosemide induced only a small (23%) inhibition. In experiments measuring isometric force, torasemide and furosemide partially relaxed endothelium-denuded aortic rings precontracted with noradrenaline or KCl with EC₅₀ values between 6 and 10 μM. The vasorelaxant effect of P1075 was inhibited in a noncompetitive manner by torasemide (300 μM) but unaffected by furosemide. U-37883A increased noradrenaline-induced force and inhibited the vasorelaxant effect of P1075 in an apparently competitive manner with an inhibition constant of 0.4 μM. The data show that torasemide interferes specifically with the binding of the K_ATP channel modulators [³H]glibenclamide and [³H]P1075 and with the K_ATP channel opening and vasorelaxant effects of P1075 whereas furosemide is inactive. This suggests that the interaction of torasemide with the vascular K_ATP channel is due to the sulphonylurea group present in torasemide. U-37883A, which does not inhibit P1075 binding, is one of the most potent blockers of P1075-induced ⁸⁶Rb⁺ efflux yet described but is relatively weak as an inhibitor of P1075-mediated vasorelaxation. The opposite vascular actions of torasemide and U-37883A are expected to contribute to the renal effects of these drugs. Received: 28 January 1998 / Accepted: 20 April 1998     

Methoclocinnamox: time course of changes in alfentanil-reinforced responding in rhesus monkeys

Rationale: Methoclocinnamox (MC-CAM) possesses initial partial μ-opioid agonist activity with subsequent long-lasting μ-antagonist effects. This profile of activity is similar to that of buprenorphine, a compound with proposed use in the treatment of opioid abuse, suggesting a possible therapeutic use for MC-CAM as well. Objective: The current study assessed the time course of the ability of MC-CAM and buprenorphine to antagonize the reinforcing effects of alfentanil and compared this with that of buprenorphine. Methods: Rhesus monkeys self-administered a range of doses of alfentanil (0.03–1 μg/kg per injection) under a fixed-ratio 30, time-out 45 s schedule of i.v. drug delivery. MC-CAM was substituted for alfentanil on occasion, and a dose of 1.0 mg/kg MC-CAM or buprenorphine was given prior to sessions in which alfentanil was available. In the pretreatment studies, a wider range of alfentanil doses was utilized (0.03–30 μg/kg per injection). Results: MC-CAM maintained self-administration behavior and was nearly equipotent with buprenorphine as a reinforcer in this para digm. Both drugs, when given prior to a session in which alfentanil was available, produced a decrease in the reinforcing potency of alfentanil. The antagonist effects of the pretreatments were largest 30 min following administration and decreased over the next several days. The duration of MC-CAM’s antagonism of alfentanil was approximately 4 days; the duration of buprenorphine as an antagonist was approximately 2 days. Conclusion: These data suggest that MC-CAM has a longer duration of antagonist effects than buprenorphine and it may therefore have an advantage in the treatment of opioid abuse. Received: 27 April 1999 / Final version: 29 September 1999     

Picrotoxin in the medial prefrontal cortex impairs sensorimotor gating in rats: reversal by haloperidol

  Rationale: Neuropathological data indicate a GABAergic dysfunction in the prefrontal cortex and hippocampus of schizophrenics. On this basis, the construct validity of an animal model of schizophrenia was tested. Objective: This study assessed prepulse inhibition (PPI) of startle in rats after injections of the GABA antagonist picrotoxin into the prefrontal cortex and the ventral hippocampus. It was also tested if reductions in PPI are reversed by the dopamine antagonist haloperidol. PPI is a measure of sensorimotor gating and is impaired in schizophrenia patients. The hypothesis underlying this study was that blockade of prefrontocortical and hippocampal GABA receptors disrupts PPI in a dopamine- dependent way. This hypothesis was based on neuropathological data from schizophrenics indicating a loss of GABAergic neurons in the prefrontal cortex and hippocampus and on the observation that PPI is reduced in schizophrenics. Methods: Picrotoxin (0, 5, 10 ng/0.5 μl) was infused through chronically indwelling cannulae into the medial prefrontal cortex (mPFC), into the lateral prefrontal cortex and into the ventral hippocampus. The effect on PPI was measured directly after picrotoxin infusion. The neuroleptic compound haloperidol (0.1 mg/kg) was administered intraperitoneally 30 min before testing. Results: Picrotoxin in the mPFC dose-dependently reduced PPI and this effect was antagonized by systemic pretreatment with the dopamine antagonist haloperidol. No significant effects on PPI were observed after picrotoxin infusions into the lateral prefrontal cortex or into the ventral hippocampus. Conclusions: These findings indicate that acute blockade of GABA receptors in the mPFC impairs sensorimotor gating in a dopamine-dependent manner. Since PPI in rats has been shown to possess face, predictive, and construct validity as an animal model for some psychotic symptoms, we discuss the potential relevance of our findings for the pathophysiology of schizophrenia. Received: 16 August 1998 / Final version: 25 January 1999     

Dissociation of multiple effects of acute LSD on exploratory behavior in rats by ritanserin and propranolol

Rats tested for 1 h in the Behavioral Pattern Monitor (BPM) after injection of the mixed serotonergic agonistd-lysergic acid diethylamide (LSD) exhibit a behavioral profile similar to that produced by various hallucinogenic 5HT-2 agonists. The characteristic effects of the hallucinogens include suppression of locomotor and exploratory behavior and a preferential decrease in entries into the center of the BPM during the initial half of the test session. After LSD, the initial suppression of responding is followed by a subsequent increase in locomotor activity that is not observed with other serotonergic agonists. In the present studies, the 5HT-1 andβ-adrenergic antagonistd,1-propranolol and the 5HT-2 antagonist ritanserin were administered individually or in combination prior to the acute administration of LSD to test for the involvement of these receptor subtypes in the mediation of the effects of LSD in the BPM paradigm. Propranolol (20 mg/kg) abolished the initial suppression of activity induced by 60 μg/kg LSD without affecting the subsequent increase in locomotion. Conversely, 2.0 mg/kg ritanserin failed to block the initial suppressive effects of 60 or 120 μg/kg LSD, but attenuated the LSD-induced increases in activity during the second half of the session. The combination of propranolol and ritanserin prevented both these effects of LSD. By contrast, the more selective 5HT-2 agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) (0.27 mg/kg) produced an initial suppression of activity in the BPM that was blocked by 2.0 mg/kg ritanserin and was not followed by a subsequent increase in activity. These findings suggest that the initial suppressive effects of LSD in the BPM paradigm are dissociable from the subsequent increases in locomotion and that the two effects are mediated via different serotonergic orβ-adrenergic receptors.     

蝙蝠葛中的新生物碱——N-去甲基蝙蝠葛碱

A new phenolic dauricine-type alkaloid together with the know dauricine, were iso-lated from the rhizoma of Menispermum dauricum DC cultivated in Xianning district, Hubei province.Dauricine was obtained as the major alkaloid and was confirmed by comparison with authentic sample.The new alkaloid is an unstable white powder. Based on spectrometric analysis (UV, IR, FAB--MSand ¹HNMR) and N-methylation which offered clauricine dimethiodide(Ⅴ), the structure was elucidat-ed as RR, N-desmethyldauricine(Ⅱ), which was isolated for the first time from nature.     

Histaprodifen, methylhistaprodifen, and dimethylhistaprodifen are potent H1-receptor agonists in the pithed and in the anaesthetized rat

Selective H₂- and H₃-receptor agonists, exhibiting an at least tenfold higher potency than histamine itself at the respective receptors, have been known for several years. Selective H₁-receptor agonists with a potency exceeding that of histamine have become available only recently; the most potent are methylhistaprodifen and dimethylhistaprodifen [N ^α-methyl- and N ^α,N ^α-dimethyl-2-(3,3-diphenylpropyl)histamine, respectively] with 3.4- and 2.4-fold higher potencies than histamine in vitro (in the guinea-pig ileum). The aim of the present study was to examine whether these compounds and the parent compound histaprodifen are potent H₁-receptor agonists in the pithed and in the anaesthetized rat. In pithed, vagotomized rats diastolic blood pressure was decreased by 2-(2-thiazolyl)ethanamine i.v. (which was used as a reference H₁-receptor agonist) and by histaprodifen, methylhistaprodifen, and dimethylhistaprodifen; the maximum decrease was about 45 mmHg for each compound, and the potencies, expressed as pED₅₀, the negative logarithm of the dose (in mole per kilogram body weight) eliciting a half-maximal response, were 7.23, 7.55, 8.43 and 8.12, respectively. The dose/response curves of the four compounds were shifted to the right to about the same extent by the H₁-receptor antagonist dimetindene (1 μmol/kg i.v.). The vasodepressor response was not affected by combined i.v. administration of the H₂- and H₃-receptor antagonists ranitidine and thioperamide, by combined i.v. administration of the α₁- and α₂-adrenoceptor antagonists prazosin and rauwolscine, and by the β-adrenoceptor antagonist propranolol i.v. but was attenuated by the inhibitor of NO synthase, N ^ω-nitro-l-arginine methyl ester i.v. In anaesthetized rats 2-(2-thiazolyl)ethanamine, histaprodifen, methylhistaprodifen and dimethylhistaprodifen i.v. also decreased diastolic blood pressure in a manner sensitive to dimetindene i.v. Our data show that histaprodifen and, in particular, methyl- and dimethylhistaprodifen are highly potent H₁-receptor agonists in vivo. Received: 3 September 1998 / Accepted: 23 October 1998     

Dopamine transporter binding without cocaine-like behavioral effects: synthesis and evaluation of benztropine analogs alone and in combination with cocaine in rodents

Rationale: Previous SAR studies demonstrated that small halogen substitutions on the diphenylether system of benztropine (BZT), such as a para-Cl group, retained high affinity at the cocaine binding site on the dopamine transporter. Despite this high affinity, the compounds generally had behavioral effects different from those of cocaine. However, compounds with meta-Cl substitutions had effects more similar to those of cocaine. Objectives: A series of phenyl-ring analogs of benztropine (BZT) substituted with 3′-, 4′-, 3′,4′′- and 4′,4′′-position Cl-groups were synthesized and their pharmacology was evaluated in order to assess more fully the contributions to pharmacological activity of substituents in these positions. Methods: Compounds were synthesized and their pharmacological activity was assessed by examining radioligand binding and behavioral techniques. Results: All of the compounds displaced [³H]WIN 35,428 binding with affinities ranging from 20 to 32.5 nM. Affinities at norepinephrine ([³H]nisoxetine) and serotonin ([³H]citalopram) transporters, respectively, ranged from 259 to 5120 and 451 to 2980 nM. Each of the compounds also inhibited [³H]pirenzepine binding to muscarinic M₁ receptors, with affinities ranging from 0.98 to 47.9 nM. Cocaine and the BZT analogs produced dose-related increases in locomotor activity in mice. However, maximal effects of the BZT analogs were uniformly less than those produced by cocaine, and were obtained 2–3 h after injection compared to the relatively rapid onset (within 30 min) of cocaine effects. In rats trained to discriminate IP saline from 29 μmol/kg cocaine (10 mg/kg), cocaine produced a dose-related increase in responding on the cocaine lever, reaching 100% at the training dose; however, none of the BZT analogs fully substituted for cocaine, with maximum cocaine responding from 20 to 69%. Despite their reduced efficacy compared to cocaine in cocaine discrimination, none of the analogs antagonized the effects of cocaine. As has been reported previously for 4′-Cl-BZT, the cocaine discriminative-stimulus effects were shifted leftward by co-administration of the present BZT analogs. Conclusions: The present results indicate that although the BZT analogs bind with relatively high affinity and selectivity at the dopamine transporter, their behavioral profile is distinct from that of cocaine. The present results suggest that analogs of BZT may be useful as treatments for cocaine abuse in situations in which an agonist treatment is indicated. These compounds possess features such as reduced efficacy compared to cocaine and a long duration of action that may render them particularly useful leads for the development of therapeutics for cocaine abusers. Electronic Publication     

Ontogeny of adenosine receptors in the longitudinal muscle and muscularis mucosae of the rat distal colon

The development of adenosine A₁ and A_2B receptors on the longitudinal muscle and muscularis mucosae of the neonatal rat distal colon has been investigated using homogenate binding, quantitative autoradiography and functional studies. In homogenate binding studies 1,3-[³H]-dipropyl-8-cyclopentylxanthine ([³H]DPCPX) bound with high affinity to A₁ receptors in the muscularis mucosae and intact colon from rats aged 10, 15, 20, 25 and 30 days. The affinity of [³H]DPCPX was similar to that in the adult at all ages, but the density of binding sites was higher in the neonatal tissues. Quantitative autoradiography showed a higher density of [³H]DPCPX binding sites in the longitudinal muscle than in the muscularis mucosae at all ages studied (day 10 to adult), and this binding was concentration-dependently displaced by N ⁶-cyclopentyladenosine (CPA). In functional studies the longitudinal muscle relaxed in response to 5’-N-ethylcarboxamidoadenosine (NECA) and CPA at all ages studied (15–30 days), NECA being more potent than CPA. The potency of NECA remained constant and it was antagonised by 1 μM DPCPX at all ages with pA ₂-values consistent with activation of A₂ receptors. The inactivity of 2-[p-(carboxyethyl)-phenylethylamino]-5’-N-ethylcarbox-amidoadenosine (CGS 21680) indicated that the A₂ receptors were of the A_2B subtype. The muscularis mucosae contracted in response to CPA at all ages studied (day 15 to adult) and the antagonism by DPCPX (10 nM) were consistent with activation of A₁ receptors. In conclusion, binding, autoradiographic and functional studies identified A₁ receptors on the rat colon muscularis mucosae at all ages studied. However, while binding and autoradiographic localisation showed the presence of A₁ receptors in the longitudinal muscle at all ages studied, functional data only revealed the presence of A_2B receptors. Received: 3 July 1998 / Accepted: 25 November 1998     

Effects of cell isolation procedures and radioligand selection on the characterization of human leukocyte beta-adrenergic receptors

Radioligand binding techniques are commonly used in the characterization of beta-adrenergic receptors on human peripheral leukocytes. Accurate interpretation of receptor binding parameters necessitates appropriate radioligand selection. In addition, cell isolation techniques should have minimal effect on the binding parameters of receptors. Our observation of curvilinear Scatchard plots with (-)-[125I]iodocyanopindolol (ICYP) resulted in a re-evaluation of this radioligand and the influence of cell isolation techniques on leukocyte beta-adrenergic receptor binding parameters. Membranes from mononuclear (MN) and polymorphonuclear (PMN) cells isolated by a standard procedure (Ficoll-Hypaque) resulted in biphasic Scatchard plots with ICYP in three of four subjects. In contrast, linear Scatchard plots were observed for ICYP binding to membranes from MN and PMN cells isolated from the same four subjects with an alternative procedure utilizing plasma Percoll. Competition and saturation binding assays with ICYP identified a high degree of nonspecific binding. Decreased stereoselectivity with (-)- and (+)-propranolol was observed with membranes from Ficoll-Hypaque cells as compared to plasma Percoll cells. Kinetic analysis with ICYP demonstrated apparent irreversible binding whether displacement was initiated with a beta-adrenergic receptor antagonist or agonist. These problems with ICYP prompted evaluation of an alternative radioligand, (-)-[125I]iodopindolol (IPIN); this radioligand demonstrated rapid and completely reversible binding, improved stereoselectivity, and low nonspecific binding. Using IPIN, Scatchard plots from three additional subjects were linear for both cell isolation procedures. Based on these observations, the preferred method of human leukocyte beta-adrenergic receptor analysis incorporates the plasma Percoll cell isolation technique and the radioligand IPIN.     

The change of beta-adrenergic system after cessation of lead exposure

For understanding a reversible or irreversible harm of beta-adrenergic system in lead induced cardiovascular disease (hypertension), We set up animal model to estimate the change of blood pressure and sympathetic nervous system after lead exposure withdrawn in the study. We address three topics in this study: (a) the relationship between withdrawal time of lead exposure and beta-adrenergic receptor, plasma catecholamine level, blood pressure, and lead level in heart, aorta, and kidney in lead-induced hypertensive rats after lead exposure stopped; (b) the relationship between blood pressure and beta-adrenergic receptor in heart, aorta, and kidney; (c) the estimation of relationship between lead withdrawn and the variation of beta-adrenergic system. Wistar rats were chronically fed with 2% lead acetate (experimental group) and water (control group) for 2 months. The rats were divided into 8 groups by withdrawal time of lead exposure stopped. Plasma catecholamine level was measured by high-performance liquid chromatography. Radioligand binding assay was measured by a method that fulfilled strict criteria of beta-adrenergic receptor using the ligand [125I]iodocyanopindolol. The levels of lead were determined by electrothermal atomic absorption spectrometry. The results showed that a close relation between reduced lead level and the plasma catecholamine level decreased, aorta beta-adrenergic receptor increased, kidney beta-adrenergic receptor diminished, heart beta-adrenergic receptor increased, and blood pressure dropped after lead exposure withdrawn. The study on the regulation of beta-adrenergic system in lead-induced hypertension after lead withdrawn might also provide insight about the nature of this disease state.     

Comparison of two putatively selective radioligands for labeling central nervous system beta-adrenergic receptors: inadequacy of [3H]dihydroalprenolol

[3H]Dihydroalprenolol ([3H]DHA) has been used extensively in receptor binding studies to measure beta-adrenergic receptors in the central nervous system. Usually, nonspecific binding has been defined by high concentrations of the beta-adrenergic receptor agonist isoproterenol or antagonists such as alprenolol or propranolol. Scatchard plots of such "specific" [3H]DHA saturation data in rat cerebral cortex membranes are linear. However, computer analysis demonstrated that the competition curves of these drugs for 2.0 nM [3H]DHA binding are biphasic, with a continuous inhibition of [3H]DHA binding in the concentration range usually used to determine nonspecific binding. These data indicate that another saturable high affinity site was being labeled by the radioligand and that the definition of nonspecific binding with any of these unlabeled drugs is not satisfactory. We used the nonlinear, least squares, curve-fitting program LIGAND to analyze total [3H]DHA binding, allowing the program to mathematically define nonspecific binding as a function of 3H-ligand concentration. Significantly lower Bmax (-44%) and Kd (-58%) values for beta-adrenergic receptors were found, indicating that under normal experimental procedures (defining [3H]DHA non-specific binding with these nonradioactive drugs) a second binding site was being labeled. We found that [3H]DHA binding to this site could be inhibited by drugs such as RU24969, a 5-hydroxytryptamine1A (5HT1A) and 5HT1B receptor subtype-selective agonist, and CGS12066B, a 5HT1B receptor subtype-selective agonist, which were able to compete for 15-20% of [3H]DHA binding in the nanomolar concentration range, whereas drugs that are selective for other serotonin receptor subtypes inhibited [3H]DHA binding only at much higher concentrations. Another beta-adrenergic receptor antagonist radioligand, [3H]CGP-12177, was found to be more selective for beta-adrenergic receptors. Alprenolol competition curves for [3H]CGP-12177 binding were monophasic and saturation curves, with nonspecific binding defined either by 10 microM alprenolol or by LIGAND, yielded Bmax values close to those obtained with [3H]DHA when its nonspecific binding was defined by LIGAND. [3H]DHA cannot be considered a suitable radioligand to quantify central nervous system beta-adrenergic receptors in the manner in which it has been typically used.     

Structure and biological activity of (-)-[3H]dihydroalprenolol, a radioligand for studies of beta-adrenergic receptors.

(-)-Alprenolol is a potent competitive beta-adrenergic antagonist. "(-)-[3H]Alprenolol", a radioactive form of this agent produced by catalytic reduction with tritium, has recently been used successfully as a radioligand for direct studies of beta-adrenergic receptors. In this communication it is documented that the compound formed by catalytic reduction of (-)-alprenolol with tritium gas is the saturated product (-)-[3H]dihydroalprenolol in which tritium is added across the double bond and exchanged into the adjacent benzylic position. No exchange into the aromatic ring was observed. These conclusions were substantiated by results obtained on hydrogenation and deuteration of (-)-alprenolol. The biological activity of (-)-[3H]dihydroalprenolol, dihydroalprenolol, and alprenolol was also shown to be identical as assessed by direct ligand binding and inhibition of catecholamine-stimulated adenylate cyclase.     

Expression of beta-adrenergic receptors in synchronous and asynchronous S49 lymphoma cells. I. Receptor metabolism after irreversible blockade of receptors and in cells traversing the cell division cycle

We have examined the metabolism of beta-adrenergic receptors in intact S49 lymphoma cells. Centrifugal elutriation was used to prepare synchronized cells enriched in particular phases of the cell division cycle. In these synchronized cells, the rate of appearance of beta-adrenergic receptors (i.e., [125I]iodocyanopindolol binding sites) was continuous, approximately 75 sites/cell/hr, and receptor number per cell increased in proportion to the increase in cell size. Thus, receptor number, normalized for cell size, remains constant throughout the cell cycle. Receptors on cells in G1, S, and G2/M phases of the cell cycle displayed similar affinities for the radiolabeled antagonist [125I]iodocyanopindolol and apparent affinities for the agonist isoproterenol. We examined rates of receptor turnover in asynchronous cells by following receptor recovery after inactivation of beta-adrenergic receptors with bromoacetylalprenololmenthane (BAAM), an irreversible beta-adrenergic antagonist. The beta-adrenergic receptors on S49 cells demonstrated an average "half-life" of 28-30 hr. Since the population doubling time for S49 cells is 16-17 hr, this would indicate that receptor protein is conserved through successive cell generations. Moreover, receptor reappearance after blockade by BAAM was a function of newly appearing receptors during the S49 cell cycle and not loss of BAAM from receptors. The rate of receptor metabolism indicates that, under basal conditions (i.e., in the absence of agonist), beta-adrenergic receptors on S49 cells are metabolized more slowly than are other classes of receptors that bind peptides and cholinergic agonists in several other cell types.     

Investigation of cardiac beta-adrenoceptors using 125I-labelled 1-(4-iodophenoxy)-3-isopropylaminopropan-2-ol.

1-(4-iodophenoxy)-3-isopropylaminopropan-2-ol (IIP) is a potent beta-adrenergic antagonist which has been labelled to high specific activity with 125I and used to bind to rat myocardial membranes. The characteristics of binding were consistent with the known properties of beta-receptors. Thus, binding was highly stereospecific for the L-stereoisomer since L-propranolol was two orders of magnitude more potent than the D-isomer in competing for these sites. The beta-adrenergic agonists isoproterenol, epinephrine and norepinephrine competed for binding with potencies paralleling their pharmacological potencies as beta-adrenergic effectors. The dissociation constant for binding of IIP was 4--5 nM as measured either by direct binding studies or by its inhibition of isoproterenol stimulated adenylate cyclase. Binding was saturable with 0.06 pmoles of IIP per mg of membrane protein binding at saturation. 125IIP is a high affinity, high specific activity ligand suitable for use as a selective probe for the detection and quantitation of cardiac beta-receptors. Its introduction should help solve the problems involved in the investigation of myocardial beta-adrenergic receptors.     

Interaction of human serum albumin with furosemide glucuronide: a role of albumin in isomerization, hydrolysis, reversible binding and irreversible binding of a 1-O-acyl glucuronide metabolite

Furosemide 1-O-acyl glucuronide (Fgnd) was reversibly bound to a single class of binding sites on human serum albumin (HSA), and the binding of Fgnd decreased with increasing F concentrations, suggesting that Fgnd binds to the same warfarin binding sites on HSA as F binds. The rate of Fgnd degradation (hydrolysis and acyl migration) decreased in the presence of HSA. Although the formation of acyl migration isomers of Fgnd was slower in the presence of HSA than in its absence, hydrolysis of Fgnd to F was faster in the presence of HSA. Rapid minor irreversible binding of Fgnd to HSA within 30 min was followed by slow major irreversible binding. Slow irreversible binding of Fgnd to HSA was decreased by F, though not significantly. This suggests that major irreversible binding may proceed via reversible binding. It has been reported that acyl migration is a prerequisite for irreversible binding. Therefore, these results indicate that HSA decreases irreversible binding of Fgnd to protein by suppressing acyl migration. Furthermore, these results suggest that HSA may prevent irreversible binding of Fgnd to other proteins in the body by decreasing the concentration of reactive Fgnd in the unbound form. HSA eliminates reactive Fgnd by hydrolysis to F. Therefore, it is concluded that HSA works as a scavenger to decrease reactive compounds by reversible binding or eliminates reactive compounds by irreversible binding.     

Alpha 2-adrenergic receptor turnover in adipose tissue and kidney: irreversible blockade of alpha 2-adrenergic receptors by benextramine

The recovery of post- and extrasynaptic alpha 2-adrenergic receptor-binding sites was studied in vivo in male golden hamsters after treatment with an irreversible alpha-adrenoceptor antagonist benextramine, a tetramine disulfide that possesses a high affinity for alpha 2-binding sites. The kidney alpha 2-adrenergic receptor number was measured with [3H]yohimbine, whereas [3H]clonidine was used for fat cell and brain membrane alpha 2-binding site identification. Benextramine treatment of fat cell, kidney, and brain membranes reduced or completely suppressed, in an irreversible manner, [3H] clonidine and [3H]yohimbine binding without modifying adenosine (A1-receptor) and beta-adrenergic receptor sites. This irreversible binding was also found 1 and 2 hr after intraperitoneal administration of benextramine to the hamsters. Although it bound irreversibly to peripheral and central alpha 2-adrenergic receptors on isolated membranes, benextramine was unable to cross the blood-brain barrier of the hamster at the concentrations used (10-20 mg/kg). After the irreversible blockade, alpha 2-binding sites reappeared in kidney and adipose tissue following a monoexponential time course. Recovery of binding sites was more rapid in kidney than in adipose tissue; the half-lives of the receptor were 31 and 46 hr, respectively in the tissues. The rates of receptor production were 1.5 and 1.8 fmol/mg of protein/hr in kidney and adipose tissue. Reappearance of alpha 2-binding sites was associated with a rapid recovery of function (antilipolytic potencies of alpha 2-agonists) in fat cells inasmuch as occupancy of 15% of [3H]clonidine-binding sites was sufficient to promote 40% inhibition of lipolysis. Benextramine is a useful tool to estimate turnover of alpha 2-adrenergic receptors under normal and pathological situations using the approach described in the present paper.     

Characterization and autoradiographic distribution of the beta-adrenergic receptor in the rat lung

An unexpected distribution of the beta-adrenergic receptor in the rat lung was revealed by an autoradiographic technique. [3H]-Dihydroalprenolol binding was stereoselective. L-propranolol was 300 times more potent than D-propranolol in competition experiments. Scatchard analysis revealed a Kd of 1.1 nM and Bmax of 14 fmol/mg wet weight. Relative potencies of beta-adrenergic ligands were: propranolol greater than alprenolol greater than timolol greater than pindolol greater than isoproterenol greater than epinephrine greater than soterenol greater than metoprolol greater than terbutaline greater than norepinephrine. The autoradiograms generated revealed a diffuse pattern with binding always associated with tissue structures. We conclude that beta-adrenergic receptors are extensively distributed in the lung, being present in large and small airways and blood vessels.     

Down-regulation of central serotonin S2 receptors after repeated treatment with quinupramine in rats

The present studies were undertaken to determine whether the repeated administration of quinupramine caused down- or up-regulation of beta-, alpha 2-adrenergic, serotonin S2, imipramine and muscarinic cholinergic receptors, as had been demonstrated for tricyclic and atypical antidepressant drugs. Quinupramine administered at 10 mg/kg (p.o.) twice daily for 10 days caused a down-regulation of serotonin S2 receptors in the frontal cortex of the rat as determined by [3H]ketanserin binding. However, quinupramine did not alter the binding populations of beta-adrenergic, muscarinic cholinergic and alpha 2-adrenergic receptors in the rat brain as determined by the Scatchard analysis of the [3H]ligand binding data. Differing from quinupramine, imipramine caused down-regulation of beta-adrenergic and serotonin S2 receptor bindings, and it caused slight but significant up-regulation of muscarinic cholinergic receptor bindings. These results show that the antidepressant activity of quinupramine is associated with the central serotonin system, but not with the beta-adrenergic system. Accordingly, quinupramine, chemically one of the typical tricyclic antidepressant drugs, seems to be pharmacologically one of the atypical antidepressant drugs, and it was suggested that the central serotonin system plays an important role in the antidepressant activity of quinupramine.