去甲乌药碱对火鸡红细胞膜β受体及腺苷环化酶活性的影响

本文研究去甲乌药碱(DMC)和异丙肾上腺素(ISO)对火鸡红细胞膜的腺苷环化酶(AC)活性的影响,并用放射配基结合测定法研究了DMC与ISO对β受体的作用。结果表明DMC能激活火鸡红细胞膜上的AC,此作用能被GTP所加强,被心得安(PRO)所阻断。DMC和不同浓度ISO合用,能加强低浓度ISO激活AC活性的作用,而减弱高浓度ISO的效力。DMC和ISO对β受体有相似的亲和力,而DMC的内在活性较小。这些结果直接证明DMC是β受体部分激动剂。     

去甲乌药碱对实验性心力衰竭的治疗作用

去甲乌药碱(DMC)是中药附子的有效成分之一。静脉滴注DMC2μg/kg/min共5min,使豚鼠正常心脏的收缩力明显加强,LVSP和LV dP/dt_max分别增加58±7和25±7%;心衰后,LVSP和LV dP/dt_max分别下降到心衰前的40±5和30.5±2.8%。DMC可使之恢复到79±14和75±9.9%,DMC也能加强离体豚鼠衰竭心脏的收缩力。DMC的强心作用与ISO相似,但前者作用较弱,作用维持时间较长,这可能与他们的作用机制不同有关。     

去甲乌药碱衍生物及其开环类似物的合成

去甲乌药碱具有强心作用,为了寻找更有效的化合物,设计合成了一系列去甲乌药碱类似物,其中包括B环开环类似物;双-四氢异喹啉类及其开环类似物;氨基侧链取代C环等。在所合成的化合物中,D₂呈明显活性,优于去甲乌药碱。     

去甲乌药碱衍生物及其开环类似物的合成

去甲乌药碱具有强心作用,为了寻找更有效的化合物,设计合成了一系列去甲乌药碱类似物,其中包括B环开环类似物;双-四氢异喹啉类及其开环类似物;氨基侧链取代C环等。在所合成的化合物中,D_2呈明显活性,优于去甲乌药碱。     

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.     

甲状腺激素对小鼠脑β-肾上腺素能受体的影响

甲状腺激素可通过调节外周组织β受体的数量,从而影响交感神经的活动。为证实甲状腺激素对中枢神经系统的影响是否也与β受体的变化有关,我们用注射T_3的方法使小鼠体内甲状腺激素增高(甲高),用放射配基结合分析法测定甲高小鼠脑β受体最大结合容量(B_(max))和亲和力(KD)的变化,并以滋阴药进行实验性治疗。结果表明:甲高小鼠脑组织β受体B_(max)较对照组显著增加(P<0.001),KD无明显变化。滋阴药生地可使甲高时升高的β受体结合位点数下降(P<0.001)。     

维拉帕米对去甲肾上腺素诱导的培养大鼠心肌细胞β受体下调的影响

目的：研究维拉帕米（Ｖｅｒ）是否能抑制去甲能上腺素（ＮＥ）诱导的培养大鼠心肌细胞β受体下调配可能机制。方法：β受体密度用［＾３Ｈ］－ＤＮＡ放射配基标记法，细胞内游离钙用钙离子荧光探针Ｆｕｒａ２－ＡＭ法测定。结果：Ｖｅｒ以明显降低培养大鼠心肌细胞内游离钙水平，增加β受体密度；ＮＥ增加细胞内游离钙水平，降低β受体密度；Ｖｅｒ能明显抑制ＮＥ引起的细胞内游离钙增加和β受体密度的降低。结论：Ｖｅｒ能增加正常     

北京鸭红细胞膜β肾上腺素受体放射配基结合测定法

本文以[³H]dihydroalprenolol([³H]DHA)为放射配基。对北京鸭红细胞(RBC)膜β受体进行了系统研究。结果表明,[³H]DHA与北京鸭RBC结合具有饱和性,肾上腺素能激动剂与[³H]DHA竞争结合的强弱顺序与它们的生物活性一致,提示结合有结构特异性和立体特异性,[³H]DHA与鸭RBC结合迅速、可逆。可见北京鸭RBC膜存在β受体,用于放射配基结合测定有取材方便,受体较丰富,膜蛋白得率高,[³H]DHA非特异结合低和易于保存等优点,可以代替火鸡RBC。     

去甲乌药碱的药理作用与心脏β－AR关系的初步研究

附子有效成分去甲乌药碱（ＤＭＣ）的药理作用以及与肾上腺素能β受体（β－ＡＲ）的关系的研究表明，０．５ｍｇ·ｋｇ－１的ＤＭＣ可使正常小鼠心肌β－ＡＲ轻度上调，与异丙肾上腺素（ＩＳＯ）相比，ＤＭＣ能轻度激动ｃＡＭＰ，使其血浆含量升高，升高的峰值时间在１０ｍｉｎ左右。ＤＭＣ的最小激动量为０．５ｍｇ·ｋｇ－１，最大激动量为５ｍｇ·ｋｇ－１。ＤＭＣ对１２５Ｉ－ＰＩＮ的竞争抑制实验表明，在１×１０－６～１×１０－５ｍｏｌ·Ｌ－１时，ＤＭＣ可抑制１２５Ｉ－ＰＩＮ与β－ＡＲ结合，与心得安比较抑制浓度大４～５个数量级。与ＩＳＯ合并用药观察血浆ｃＡＭＰ浓度变化提示，０．５ｍｇ·ｋｇ－１ＤＭＣ与小剂量（０．１ｍｇ·ｋｇ－１）和较大剂量（１ｍｇ·ｋｇ－１）的ＩＳＯ合用时，既无协同作用，也未见拮抗作用。     

去甲乌药碱的药理作用与心脏β－AR关系的初步研究

附子有效成分去甲乌药碱（ＤＭＣ）的药理作用以及与肾上腺素能β受体（β－ＡＲ）的关系的研究表明，０．５ｍｇ·ｋｇ－１的ＤＭＣ可使正常小鼠心肌β－ＡＲ轻度上调，与异丙肾上腺素（ＩＳＯ）相比，ＤＭＣ能轻度激动ｃＡＭＰ，使其血浆含量升高，升高的峰值时间在１０ｍｉｎ左右。ＤＭＣ的最小激动量为０．５ｍｇ·ｋｇ－１，最大激动量为５ｍｇ·ｋｇ－１。ＤＭＣ对１２５Ｉ－ＰＩＮ的竞争抑制实验表明，在１×１０－６～１×１０－５ｍｏｌ·Ｌ－１时，ＤＭＣ可抑制１２５Ｉ－ＰＩＮ与β－ＡＲ结合，与心得安比较抑制浓度大４～５个数量级。与ＩＳＯ合并用药观察血浆ｃＡＭＰ浓度变化提示，０．５ｍｇ·ｋｇ－１ＤＭＣ与小剂量（０．１ｍｇ·ｋｇ－１）和较大剂量（１ｍｇ·ｋｇ－１）的ＩＳＯ合用时，既无协同作用，也未见拮抗作用。     

Positive coupling of cholinergic muscarinic receptors to adenylate cyclase activity in membranes of rat olfactory bulb

Summary In membranes of rat olfactory bulb acetylcholine stimulated adenylate cyclase activity in a concentration-dependent manner. The maximal stimulation corresponded to 53% increase of basal enzyme activity and was obtained with 100 M acetylcholine. The concentration of the cholinergic agonist eliciting a half-maximal effect was 0.4 M. The stimulatory effect of acetylcholine was antagonized by 0.1 M atropine but not by 10 M (+)-tubocurarine. Moreover, the addition of micromolar concentrations of GTP was absolutely required for the enzyme stimulation by acetylcholine. The results demonstrate the presence in rat olfactory bulb of muscarinic receptors coupled to stimulation of adenylate cyclase probably via a GTP regulatory protein and provide evidence for a novel signal transduction mechanism of central muscarinic receptors. Send offprint requests to P. Onali at the above address     

A novel cognition enhancer NS-105 modulates adenylate cyclase activity through metabotropic glutamate receptors in primary neuronal culture

The effect of (+)-5-oxo-D-prolinepiperidinamide monohydrate (NS-105), a novel cognition enhancer, on adenylate cyclase activity was investigated in cultured neurons of the mouse cerebral cortex. NS-105 (10^–7 and 10^–6 M) inhibited forskolin-stimulated cyclic AMP formation, an action that was dependent on pertussis toxin-sensitive G proteins. Conversely, in pertussis toxin-pretreated neurons, NS-105 (10^–7 –10^–5 M) significantly enhanced the forskolin-stimulated cyclic AMP formation, and this action was completely reversed by cholera toxin. A metabotropic glutamate receptor agonist (1S, 3R)-1-aminocyclopentane-1, 3-dicarboxylic acid (1S, 3R-ACPD) produced similar bi-directional actions on the cyclic AMP formation. Both of these inhibitory and facilitatory actions of NS-105 and 1S, 3R-ACPD were blocked by L(+)-2-amino-3-phosphopropinoic acid (L-AP3). NS-105 (10^–6 M) and 1S, 3R-ACPD (10^–4 M) significantly enhanced isoproterenol- and adenosine-stimulated cyclic AMP formation. The enhancement of such Gs-coupled receptor agonists-stimulated cyclic AMP formation was also produced by quisqualate but not by L(+)-2-amino-4-phosphonobutanoate (L-AP4). The phosphoinositides hydrolysis was enhanced by 1S, 3R-ACPD (10^–4 M) but not by NS-105 (10^–6 M), however, 1S, 3R-ACPD-induced increase in phosphoinositides turnover was attenuated by NS-105. These findings suggest that NS-105 stimulates metabotropic glutamate receptor subclasses that are coupled both negatively and positively to adenylate cyclase, but it acts as an antagonist at the receptor subclasses that are linked to phosphoinositides hydrolysis. Received: 3 February 1997 / Accepted: 25 April 1997     

Effects of short-chain alcohols on adenylate cyclase in plasma membranes of rat adipocytes

Summary In rat adipocyte plasma membranes the effects of methanol, ethanol, propanol-1 and butanol-1 on adenylate cyclase activity were studied in the absence (basal) and presence of maximally activating concentrations of 5-guanylylimidodiphosphate [Gpp(NH)p; 10 M] and NaF (3 mM).Basal adenylate cyclase activity increased maximally 2-fold at 0.03–0.3 M propanol or 0.01–0.1 M butanol, with increasedV _max and essentially unchangedK _m for ATP. In most experiments, methanol and ethanol had no measurable activating effect. Above appr. 0.3 M of all alcohols, basal adenylate, cyclase activity declined sharply below control values.Adenylate cyclase activity stimulated by Gpp-(NH)p was further increased severalfold by all alcohols in a concentration-dependent manner, with increasedV _max and unchanged apparentK _m for the guanyl nucleotide. The potency of the alcohols increased with chain length (1 through 4 carbons). The effect of the alcohols on Gpp(NH)p-stimulated adenylate cyclase activity was still found at concentrations where basal activity was already reduced. Adenylate cyclase activity stimulated by NaF was affected similarly but definitely less marked than Gpp(NH)p-stimulated activity.Hormone-stimulated adenylate cyclase activity expressed relative to basal enzyme activity remained essentially unaffected by the alcohols. Increase in absolute enzyme activity apparently reflected corresponding changes in basal adenylate cyclase activity.Above alcohol concentrations of 0.3 M both basal and hormone-stimulated enzyme activities were inhibited.Part of the results have been presented at the Spring Meeting of the Deutsche Pharmakologische Gesellschaft (Stock, 1977)     

Comparative effects of alpha-methyldopa, propranolol and hydralazine therapy on cardiac adenylate cyclase activity in normal and spontaneously hypertensive rats

Normotensive (WKY) and spontaneously hypertensive (SHR) male rats were treated orally, one week after weaning and for 9 weeks, with alpha-methyldopa (100 mg/kg per day), propranolol (30 mg/kg per day) or hydralazine (10 mg/kg per day). Untreated WKY and SHR rats served as controls. The development of hypertension in SHR rats were attenuated by treatment but none of the drugs was able to restore the impairment in isoproterenol, secretin and glucagon responsiveness of cardiac adenylate cyclase activity which is characteristic of these animals. In heart membranes from both WKY and SHR rats, alpha-methyldopa treatment increased the number of beta-adrenoceptors by 20-32% and the maximal response of adenylate cyclase activity to isoproterenol and glucagon by 20-34%. By contrast, the beta-blocker propranolol was ineffective on these parameters. The results obtained are consistent with the hypothesis that the change in adenylate cyclase seen in SHR rats is genetic in origin and is not a consequence of hypertension.     

Effect of pretreatment with bronchodilator drugs on in vitro responsiveness of guinea pig lung adenylate cyclase.

Pretreatment of guinea pigs with 5 microgram/kg isoprenaline or 10 microgram/kg salbutamol s.c. thrice daily for 7 days reduced the responsiveness of lung slice and tracheal ring adenylate cyclase to isoprenaline, but not to prostaglandin E1. Pretreatment of guinea pigs with isoprenaline also reduced the sensitivity of tracheal smooth muscle strip adenylate cyclase to isoprenaline. Cross-tolerance developed to noradrenaline in lung slices obtained from guinea pigs pretreated with isoprenaline. Propranolol blocked the response of lung slice adenylate cyclase of control and isoprenaline-pretreated animals to approximately the same degree. The presence of phentolamine in the incubation medium did not affect the reduced sensitivity to isoprenaline. Possible mechanisms of development of tolerance to sympathomimetic bronchodilator drugs are discussed.     

Separation of solubilized A2 adenosine receptors of human platelets from non-receptor [3H]NECA binding sites by gel filtration

Summary Human platelet membranes were solubilized with the zwitterionic detergent CHAPS (3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate) and the solubilized extract subjected to gel filtration. Binding of the adenosine receptor agonist [3H]NECA (5-N-ethylcarboxamidoadeno-sine) was measured to the eluted fractions. Two [³H]NECA binding peaks were eluted, the first of them with the void volume. This first peak represented between 10% and 25% of the [³H]NECA binding activity eluted from the column. It bound [³H]NECA in a reversible, saturable and GTP-dependent manner with an affinity of 46 nmol/1 and a binding capacity of 510 fmol/mg protein. Various adenosine receptor ligands competed for the binding of [³H]NECA to the first peak with a pharmacological profile characteristic for the A₂ adenosine receptor as determined from adenylate cyclase experiments. In contrast, most adenosine receptor ligands did not compete for [³H]NECA binding to the second, major peak. These results suggest that a solubilized A₂ receptor-GS protein complex of human platelets can be separated from other [³H]NECA binding sites by gel filtration. This allows reliable radioligand binding studies of the A₂ adenosine receptor of human platelets.Abbreviations CHAPS 3-[3-(cholamidopropyl)dimethylammoniol-l-propanesulfonate - CIA 2-chloroadenosine - CPA N⁶-cyclopentyladenosine - DPX 1,3-diethyl-8-phenylxanthine - NECA 5-N-ethylcarboxamidoadenosine - PAA 2-phenylaminoadenosine - PIA N⁶-phenyhsopropyladenosine - XAC 8-{4-[([{(2-aminoethyl)amino}carbonyl}methyl)oxy]phenyl]-1,3-dipropylxanthine Send offprint requests to M. J. Lohse     

5-Hydroxytryptamine 5-HT1B and 5-HT1D receptors mediating inhibition of adenylate cyclase activity

Summary 5-Hydroxytryptamine_1B (5-HT_1B) receptor mediated-inhibition of forskolin-stimulated adenylate cyclase activity in rat substantia nigra was characterized pharmacologically and compared to 5-HT_1D receptor mediated-inhibition of forskolin-stimulated adenylate cyclase activity in calf substantia nigra. Special attention was paid to the effects of drugs known to bind with high affinity to 5-HT_1B (pindolol, propranolol, cyanopindolol, SDZ 21-009, isamoltane) or 5-HT_1D recognition sites (yohimbine, rauwolscine).PEC₅₀ or pK _B values of a variety of 5-HT-receptor ligands (6 agonists including 5-HT, and 12 antagonists) for the inhibition of adenylate cyclase activity in rat substantia nigra, correlated significantly to the corresponding pK _D values at 5-HT_1B binding sites (r = 0.90, P = 0.0001). Amongst the ₂- and -adrenoceptor antagonists tested, none of the drugs expressed more than 35% of the intrinsic activity of 5-HT at 5-HT_1B receptors. When tested as antagonists, their pK _B values were in good agreement with their pK _D values for 5-HT_1B sites. By contrast, these drugs displayed marked intrinsic activity at 5-HT_1D receptors: their pEC₅₀ values were close to their pK _D values for 5-HT_1D sites and their effects could be potently antagonized by methiothepin. The rank orders of potency of the tested compounds at 5-HT_1B and 5-HT_1D were markedly different.The results strengthen the identity between 5-HT receptors mediating inhibition of adenylate cyclase activity in rat and calf substantia nigra and 5-HT_1B and 5-HT_1D binding sites, respectively. They underline the differences between these receptors in terms of intrinsic activities and potencies of drugs. Send offprint requests to: D. Hoyer at the above address     

Hormone metabolism and response of adenylate cyclase to parathyroid hormone in kidney

1. Incubation of parathyroid hormone with plasma membranes from rat kidney cortex resulted in rapid loss of all hormal activity. 2. Chick kidney membranes showed no ability to inactivate parathyroid hormone even with prolonged incubation. 3. Biologically active, labelled parathyroid hormone was degraded to fragments by rat kidney membranes, but not by chick kidney. 4. Hormone-responsive adenylate cyclase activity in a mixture of rat and chick kidney membranes was additive. 5. Parathyroid hormone bound specifically to chick kidney palsma membranes. 6. It is concluded that hormone in activation during incubation has little relevance to the effectiveness of parathyroid hormone in stimulating adenylate cyclase activity in kidney, and furthermore that failure of chick kidney to metabolize the hormone is not the explanation for the greater sensitivity of this species to the hormone.     

Effect of thyroid status on beta-adrenergic receptor, adenylate cyclase activity and guanine, nucleotide regulatory unit in rat cardiac and erythrocyte membranes

The effect of thyroid hormone on the β-receptor coupled adenylate cyclase in rat crude cardiac membranes was analysed by measuring the number of DHA-binding sites, adenylate cyclase activity and the amount of cholera toxin catalysed ADP-ribosylation of a protein with a molecular weight of 42,000 in cardiac and erythrocyte membranes. In crude rat cardiac membranes, the number of DHA-binding sites (78 ± 15 fmole/mg protein in the euthyroid state) is increased to 158 ± 20 fmole/mg protein in the hyperthyroid state and decreased to 51 ± 6 fmole/mg protein in the hypothyroid state; the affinity of the binding sites remained unchanged (K_D 2.9?4.3 nM). l-Isoprenaline (10^?4 M)-stimulated adenylate cyclase activity varied in parallel to the number of DHA-binding sites in hyper- and euthyroidism. Thyroid hormone, however, did not influence GppNHp (10^?4 M)-stimulated adenylate cyclase activity. Cholera toxin catalysed ADP-ribosylation of normal crude cardiac membranes resulted in a 1.8 fold increase in adenylate cyclase activity in the presence of GTP (10^?4) and l-isoprenaline (10^?4M), presumably as a result of inhibition of GTPase. In crude cardiac membranes cholera toxin catalyses the ADP-ribosylation of one major protein, which comigrates on sodium dodecylsulfate-polyacrylamide gel electrophoresis with the putative regulatory component of adenylate cyclase (mol. wt 42,000). In different thyroid states the amount of the regulatory component (as determined by cholera toxin dependent labelling) was equal (112 fmole/mg protein in euthyroid crude cardiac membranes). Basal activity of adenylate cyclase showed a significant difference between activity in euthyroid (3.7 ± 0.2 pmole cAMP/mg protein/min) and hypothyroid (5.4 ± 0.2 pmole cAMP/mg protein/min), but not in hyperthyroid crude cardiac membranes (3.4 ± 0.2 pmole cAMP/mg protein/min). Our results indicate, that thyroid hormone regulates the number of DHA-binding sites and basal activity (in hypothyroidism) in crude cardiac membranes and thereby causes different results in l-isoprenaline-induced adenylate cyclase activity.