Adrenergic and serotonergic receptor-blocking potencies of terazosin, a new antihypertensive agent, as assessed by radioligand binding assay.
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Adrenergic alpha-Antagonists↗
Biomedical subjects
H Tsuchihashi
Publications and source records attributed to H Tsuchihashi.
Biphasic binding of 125I-iodocyanopindolol to beta-adrenergic receptors in rat cerebral cortical membranes. I. Assessment by the use of agonists.
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Adrenergic beta-Agonists↗
Characterization of 3H-dihydroalprenolol binding to beta-adrenergic receptors of rat brain: two binding sites of racemic propranolol in displacement experiments.
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Alprenolol↗
Binding characteristics of 125I-iodocyanopindolol to beta-adrenergic receptors: biphasic Scatchard plots. II. Effects of selective antagonists.
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Adrenergic beta-Antagonists↗
Effects of bunitrolol on adrenergic and serotonergic receptors.
To assess the importance of anti-adrenergic and anti-serotonergic activities of bunitrolol for its efficacy as an antihypertensive and antianginal agent, effects of this substance on the binding of adrenergic and serotonergic agents to the respective receptors of the rat brain, rat heart, dog brain, and/or dog aorta were examined using the radioligand binding assay methods. In addition, the pA2 values of bunitrolol as an antagonist against the positive chronotropic and inotropic actions (beta 1-adrenoceptor) of isoproterenol were also determined by pharmacological methods using the isolated guinea pig atria. To assess the specificity, pA2 values were also obtained in the isolated trachea (beta 2-adrenoceptor) using isoproterenol as an agonist and in the isolated aorta from the guinea pig and the rat using phenylephrine as an agonist (alpha 1-adrenoceptor). A strong inhibition by bunitrolol of 3H-dihydroalprenolol (3H-DHA) binding to beta-adrenoceptors was observed, while the inhibition of 3H-prazosin binding to alpha 1-adrenoceptors, 3H-serotonin binding to 5HT1-receptors. 3H-p-aminoclonidine binding to alpha 2-adrenoceptors, and 3H-ketanserin binding to 5HT2-receptors were found to be very weak. The rank order of antagonistic potencies of bunitrolol against the adrenergic receptors as assessed with pA2 values were beta 1 greater than beta 2 much greater than alpha 1. From these two different types of experiments, it is clear that the antihypertensive and antianginal effects of bunitrolol are mainly due to its beta-blocking actions, with the alpha 1-blocking action of this drug playing a minor role.
Animals↗
Carbohydrate composition of sarcolemma of rat myocardium.
The carbohydrate composition of SL prepared from rat ventricular muscle was examined and compared with those of Mw and FSR prepared from the same species. The total carbohydrate content of SL was 308.58 micrograms/mg of protein, which was greater than that of Mw (74.49 micrograms/mg of protein) and FSR (76.50 micrograms/mg of protein). The carbohydrate of SL was composed of hexose (35.0%), hexuronic acid (7.3%), sialic acid (6.1%), methyl pentose (7.4%), and hexosamine (44.2%). The peculiar PAS-positive glycoproteins of SL were observed as five bands on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and the molecular weight of the main band of these was 85.4 kDa. Thus, each of the cell fractions obviously shows a distinct carbohydrate composition and the results obtained in the present study may suggest that the presence of the PAS-positive glycoprotein (85.4 kDa) can be used for routine monitoring of the purity of the SL fraction.
Animals↗
Effects of deoxyribonuclease I and neuraminidase treatments on the specific binding of 3H-prazosin and 3H-quinuclidinyl benzilate (3H-QNB) to alpha-adrenergic and muscarinic receptors in rat myocardial membranes.
The effects of neuraminidase and deoxyribonuclease I (DNase) treatments on the specific binding of 3H-prazosin and 3H-quinuclidinyl benzilate (3H-QNB) to alpha-adrenoceptors and muscarinic cholinergic receptors in the membrane of the rat myocardium was examined, and the dissociation constant (Kd) and the maximum number of binding sites (Bmax) was analyzed using the method of Scatchard analysis. Although no changes in Kd values were observed when DNA or sialic acid was removed from cardiac muscles by treatments with DNase or neuraminidase, the Bmax values of alpha-adrenergic and muscarinic receptors were markedly decreased after treatment with DNase, while neuraminidase treatment induced an increase in the Bmax values of the alpha-adrenoceptors. The possibility that these results provide the basis for elucidation of the characteristics of these receptors in the rat myocardium was discussed.
Animals↗
Binding characteristics of [3H]dihydroalprenolol to beta-adrenergic receptors of rat brain: comparison with those of rat heart treated with neuraminidase.
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Alprenolol↗
Displacement by alpha-adrenergic agonists and antagonists of 3H-prazosin bound to the alpha-adrenoceptors of the dog aorta and the rat brain.
To characterize the alpha 1-adrenoceptors in the dog aorta and the rat brain and to assess the antagonistic potencies of various chemicals inclusive of newly synthesized ones, radioligand binding assays were performed, and the potencies thus obtained were compared with those obtained from pharmacological observations. Reproducible binding to and displacement from the dog aorta of 3H-prazosin were observed. The rank orders of the inhibition of 3H-prazosin binding to the membrane preparations of the aorta expressed as IC50 (the concentration of drugs inhibiting 50% of maximal specific binding of 3H-prazosin) were: prazosin greater than YM09538 greater than phentolamine greater than yohimbine greater than phenoxybenzamine greater than labetalol greater than S-596 greater than dibenamine greater than K-351 greater than propranolol greater than hydralazine greater than N-696 for alpha-blockers and I-epinephrine greater than clonidine greater than I-norepinephrine greater than phenylephrine greater than I-isoproterenol for agonists. IC50 values of phenoxybenzamine, labetalol, dibenamine and K-351 obtained in the brain preparations were higher than those obtained in the preparation of the aorta. There was a good correlation (r = 0.90) between the IC50 values obtained in the dog aorta and in the rat brain, suggesting that the alpha 1-adrenergic receptors in the dog aorta and the rat brain resemble each other. Good correlations (aorta, r=0.97 and brain, r=0.94) were also observed between IC50 values derived from the binding assay in the aorta or the brain and the pA2 values obtained as regards to the contractile response of the rat aorta to phenylephrine. Thus, this method could be useful for the assessment of newly synthesized chemicals as alpha-adrenergic antagonists.
Adrenergic alpha-Agonists↗
Binding characteristics of 3H-dihydroalprenolol to beta-adrenergic receptors of rat brain: influence of lectins.
The significance of the carbohydrate moieties of the beta-adrenergic receptor molecule in the rat brain was examined using the radioligand binding assay method. Thus, this experiment was designed to assess the effects of lectins, concanavalin A (Con A), Phaseolus vulgaris agglutinin (PHA), and wheat germ agglutinin (WGA) on the affinity of the beta-adrenoceptor. The rat brain was used and the beta-adrenoceptor binding assay was carried out using 3H-dihydroalprenolol as a ligand. Con A and PHA significantly caused an increase in the values of the density of beta-adrenoceptor (Bmax) and a reduction in the values of the dissociation constant (Kd), but significant changes were not observed with WGA. These results strongly suggest that the carbohydrate moieties of the cell surface containing the beta-adrenoceptor molecule may have a crucial role in the drug-receptor interaction, and they imply that the beta-adrenoceptor molecule is a glycoprotein which contains N-linked carbohydrate chains.
Alprenolol↗
Influence of polymeric effectors on binding of 3H-dihydroalprenolol to beta-adrenergic receptor of rat brain.
The significance of anionic and cationic charges of glycocalyx, phospholipid or protein, etc. on the cell surface of the rat brain was examined for beta-adrenoceptors using the radioligand binding assay method. Thus, this experiment was designed to assess the effects of polymeric effectors, DNA, heparin, polymyxin B, histone, gelatin, colominic acid and bovine serum albumin (BSA), on the affinity of beta-adrenoceptors. The rat brain was used and the beta-adrenoceptor binding assay was carried out using 3H-dihydroalprenolol as a radioligand. Polymyxin B, DNA and heparin significantly caused a reduction in the maximum number of beta-adrenoceptors (Bmax), but only small changes were observed with histone, gelatin, BSA and colominic acid. Only DNA induced a decrease in the value of the dissociation constant (Kd) of beta-adrenoceptors. These results suggest that anionic or cationic charges in the environment of the receptor sites could have a crucial role in drug-receptor interaction.
Alprenolol↗
Binding characteristics of 3H-dihydroalprenolol to beta-adrenergic receptors of rat brain: influence of exo- and endo-glycosidases and glycopeptidase.
The significance of carbohydrate moieties containing the beta-adrenoceptor molecule in the rat brain was examined using radioligand binding assay methods. Thus, this experiment was designed to assess the effects of exoglycosidase (alpha-D-mannosidase and neuraminidase), endoglycosidase (endoglycosidase D and endoglycosidase H), and glycopeptidase A on the affinity of beta-adrenoceptor. The main reason why five kinds of enzymes were used in the present study is that they can hydrolyze different carbohydrate molecules from cell membranes. Rat brain was used and beta-adrenoceptor binding assay was carried out using 3H-dihydroalprenolol (3H-DHA) as a ligand. 3H-DHA binding to beta-adrenoceptors was sensitive to very low concentration of endoglycosidase H and glycopeptidase A, thus indicating that the treatments with these enzymes of rat brain membrane appear to decrease the number of beta-receptor binding sites. On the other hand, the treatment with neuraminidase, endoglycosidase H, and glycopeptidase A of the membrane induced lower values of the dissociation constant (Kd) than those of the control. alpha-D-mannosidase and endoglycosidase D are without effect in spite of the removal of hexose contents and total carbohydrate contents with these treatments, respectively. These results imply that complex type N-linked acidic carbohydrate chains containing neuraminic acid and high mannose type N-linked carbohydrate chains, which are hydrolyzed with endoglycosidase H and glycopeptidase A, of the rat brain membrane containing beta-adrenoceptor molecules play a crucial role in the drug-receptor interaction.
Alprenolol↗
Beneficial effects of diltiazem on the ischemic derangements of the myocardial metabolism assessed by 31P-NMR in the isolated perfused rat heart.
Using the isolated perfused heart preparations of the rat, effects of diltiazem, a calcium antagonist, on the ischemic derangements of the mechanical function and the energy metabolism of the ventricular myocardium were studied. The myocardial tissue levels of creatine phosphate (CP), ATP, inorganic phosphate (Pi) and pH were determined with 31P-NMR. Global ischemia was induced by cross-clamping of the aortic inflow line for 15 min, which resulted in a fall of CP, ATP and pH and a rise of Pi. The test hearts were perfused with diltiazem-containing solution (10(-7), 10(-6) and 10(-5) M) for 12 min prior to the induction of the global ischemia. A significant dose-related decline of the myocardial mechanical function expressed as (left ventricular pressure) X (heart rate) was observed in diltiazem-treated hearts. In doses above 10(-6) M, diltiazem delayed the onset of the fall of the myocardial CP and pH levels and the rise of Pi induced by ischemia, and there was an excellent correlation between the suppression of the myocardial mechanical function observed before induction of ischemia and the level of the myocardial CP and pH at the initial phase of ischemia, indicating that the improvement of the myocardial energy metabolism was due to the cardiodepressant effects of the compound.
Animals↗
Exo-beta-D-glucuronidase from the fungus Kobayasia nipponica.
The occurrence of three forms, I, II, and III, of exo-beta-D-glucuronidase of the fungus Kobayasia nipponica was demonstrated. These three forms were purified 1,905-fold, 857-fold, and 357-fold, respectively. Forms I, II, and III of exo-beta-D-glucuronidase behaved differently on heparin-Sepharose chromatography, and differed in optimum pH (3.5, 3.2, and 2.6, respectively), pH-stability, Km (0.22, 0.16, and 0.13 mM, respectively), and Vmax (V) values. Their molecular weights, as estimated by gel filtration through Sephacryl S-200, were all 70,000; polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gave a value of 72,000. These three forms were very active towards 1-4 linked beta-D-glucuronans, (Formula: see text) and (Formula: see text), but weak or inactive towards protuberic acid and several glycosaminoglycans.
Basidiomycota↗
Isolation and characterization of an endo-beta-D-glucuronidase from the fungus Kobayasia nipponica.
An endo-beta-D-glucuronidase was isolated and characterized from Kobayasia nipponica. The enzyme was purified by ammonium sulfate fractionation, CM-Sephadex chromatography, gel filtration with Sephacryl S-200, and heparin-Sepharose chromatography. The enzyme shows the following properties: optimum pH 5.0, thermal stability below 37 degrees C, pH stability 5-6, optimum temperature 45-55 degrees C, and Km 0.12% for L-idurono-D-glucuronan (protuberic acid (PA), L-IdUA:D-GlcUA = 1:2) from Kobayasia nipponica, 0.19% for PF (L-IdUA:D-GlcUA = 1:3) from Pseudocolus fusiformis, and 0.23% for (1-4)-beta-D-glucuronan(mucoric acid) from Mucor mucedo as determined from Hofstee plots. The molecular weight values estimated by gel filtration through Sephacryl S-200 and Sephadex G-50 and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate were 10,500 and 10,200, respectively. The endo-beta-D-glucuronidase was inactive towards several glycosaminoglycans.
Basidiomycota↗
Beta-receptor blocking potencies of the three newly synthesized beta-adrenergic antagonists (S-596, K-351, N-696) as assessed with the radioligand binding assay method in rat cardiac muscle membrane treated with neuraminidase.
To assess the beta-blocking potencies of three newly synthesized beta-blockers, i.e., S-596: dl-2-(3'-t-butylamino-2'-hydroxypropylthio)-4-(5'- carbamoyl-2'-thienyl)-thiazole hydrochloride, K-351: 3,4-dihydro-8-(2-hydroxy-3-isopropyl-aminopropoxy)-3-nitroxy -2H-1-benzopyran, and N-696: 4,3-(tert-butylamino)-2-hydroxypropoxy)-N-methylisocarbostyril hydrochloride, values of Ki (inhibition constant) were obtained by the radioligand binding assay method using rat heart membrane treated with neuraminidase. The pA2 values of these blockers as antagonists against the positive chronotropic and inotropic actions of isoproterenol were also determined by pharmacological methods using guinea pig atria. To assess the selectivity of these beta-blockers, pA2 values were also obtained in the isolated trachea using isoproterenol as an agonist. The order of potencies as assessed by Ki and pA2 values were S-596 greater than K-351 greater than N-696. When the Ki values were plotted against the pA2 values together with the Ki and pA2 values of our previous study (Horii et al., 1974; Nakazawa et al., 1978; Nakagawa et al., 1980; Nakagawa et al., 1983; Nagatomo et al., 1983), straight lines were obtained with r values of 0.93 (P less than 0.001) (positive chronotropic action) and 0.91 (P less than 0.001) (positive inotropic action), respectively. Thus, the validity of using the beta-adrenoceptor binding assay method developed in this laboratory using cardiac membrane treated with neuraminidase for assessment of the potencies of beta-adrenoceptor blockers in cardiac muscle was further substantiated.
Adrenergic beta-Antagonists↗
Protuberic acid-hydrolytic enzyme in Kobayasia nipponica and characterization of the hydrolytic products.
When the cold water-extract of Kobayasia nipponica containing a glycuronan, protuberic acid (PA), was allowed to stand at room temperature, PA was hydrolyzed. The optimum conditions for this PA-hydrolysis were 37 degrees C and pH 4-5 in 0.1 M acetate buffer. Characterization of the hydrolytic products was performed by chemical analysis, and by 1H- and 13C-NMR spectroscopy. They were identified as D-GlcUA and O-(alpha-L-IdUAp)-(1-4)-D-GlcUA. These results suggest that PA-hydrolytic enzyme(s) include at least endo-beta-D-glucuronidase.
Basidiomycota↗
Binding characteristics of 3H-dihydroalprenolol to beta-adrenoceptors of rat heart treated with neuraminidase.
Binding characteristics of the beta-blockers and beta-agonists with the beta-adrenoceptors were investigated in 3H-dihydroalprenolol (3H-DHA) binding to rat heart membranes treated with neuraminidase. When 60% of the total sialic acid content in the membranes was removed, reproducibility of the binding assay became much better than was attainable without neuraminidase treatment, and the maximum density of beta-adrenoceptors was increased. These data suggest that the binding of the test compounds with the beta-adrenergic receptors in cardiac muscle was under the influence of the sialic acid of the glycocalyx of the membrane. The 3H-DHA binding sites in membranes treated with neuraminidase showed a strict stereo-specificity when tested with propranolol. The ranking order of inhibition of beta-antagonists or agonists is: dl-propranolol greater than oxprenolol greater than alprenolol greater than pindolol greater than YM-09538 greater than labetalol greater than acebutolol greater than atenolol greater than metoprolol greater than sotalol greater than butoxamine greater than practolol as antagonists or l-isoproterenol greater than l-epinephrine greater than l-norepinephrine as agonists. A good correlation (r = 0.91, P less than 0.001) was observed between the Ki values observed by the present binding assay and the pA2 observed in the guinea-pig atria relative to the positive inotropic effect by Bieth et al. (Br. J. Pharmacol. 68, 563-569, 1980), indicating that the present method will be useful for screening new beta-adrenergic receptor antagonists or agonists.
Alprenolol↗