昭 医 会 誌 第44巻 第3号 〔317-321頁 〕

Original

INHIBITORY EFFECTS OF SEVERAL 13-ADRENERGIC BLOCKING DRUGS ON HUMAN PLATELET MONOAMINE OXIDASE

Koji WATANABE, Sakae WADA, Shinichi KOBAYASHI and Katsuji OGUCHI Department of Pharmacology, School of Medicine, Showa University (Director: Prof. Hajime YASUHARA)

Abstract: Effects of six β-adrenergic blocking drugs (β-blockers) on human platelet mono-

amine oxidase (MAO) were studied. Platelet rich plasma (PRP) was used as enzyme material. MAO activities were determined by the radiometric assay using 14C-tyramine as substrate. Indenolol, propranolol, pindolol, oxprenolol, atenolol and carteolol, at concentration of lmM inhibited human platelet MAO. Theseβ-blockers inhibited human platelet MAO activity from ll to 47% of control activity. The order of potency of β-blockers was indenolol>propranolol

>pindolol>oxprenolol>atenolol>carteolol. Inhibitions of MAO activity by propranolol, indenolol, carteolol and atenolol were reversible, while inhibitions of MAO activity by oxpre-

nolol and pindolol were irreversible. Propranolol, indenolol and pindolol inhibited human

platelet MAO competitively, while oxpernolol, carteolol and atenonol inhibited noncompetit- ively. Ki values were 1,1.9,1,1.6,4.4 and 2.7 mM using propranolol, oxprenolol, inden- olol, pindolol, carteolol and atenolol, respectively. These results suggest that inhibitions of

human platelet MAO activity by β-blockers are weak and propranolol, indenolol and pindolol

inhibit human platelet MAO activity in active site or at least near active site.

Recently, ƒÀ-adrenergic blocking drugs (ƒÀ- SHR was significantly lower than that in the

blockers) have been widely used in cardiovas- untreated SHR concluding the changes in

cular diseases, especially, as antianginal, anti- MAO activity may be related in part to the

arrythmic and antihypertensive drugs1,2). onset of hypertension.

Wada et al3). demonstrated that propra- MAO activity in human platelets has some

nolol, pindolol and oxprenolol inhibited mo- similarities in substrate and inhibitor chara-

noamine oxidase (MAO, monoamine: O2 cteristics to MAO activity in brain and other

oxidoreductase (deaminating) EC 1, 4, 3, 4) in tissues5-8).

rat brain homogenate and in liver mitochond- In this experiment, the inhibitory effects of ƒÀ -blockers on human platelet MAO were stu- ria. Inhibition pattern of propranolol was

competitive and pindolol, oxprenolol were died.

non-competitive. And it was concluded that Materials and Methods 19•blockers preferentially inhibited type A MAO rather than type B MAO. Enzyme mate rial Furthermore, Yasuhara et al4), reported that Venous blood (3-4 ml) was collected in the the the effects of propranolol on spontaneously bottles containing one drop of 10% EDTA-

hypertensive rats (SHR), and MAO activity 2K solution. The blood was left for two

in the brain stem of the propranolol-treated hours at a glass tube of 5 mm in diameter.

317 318 昭和医学会雑誌 第44巻 第3号

The supernatant, platelet rich plasma (PRP), was collected and used as enzyme material of human platelet MAO. Assay of MAO activity MAO activity was determined by the ra- diometric assay of Watanabe et al91. PRP was pretreated at concentration of 1 mM semi- carbazide.

PRP(20μl)in 0.05 M phosphate buffer, pH

7.4(20μl)were mixed with l mM semicarba- zide(20,ul)for 20 min at room temperature, Fig. 1 Lineweaver-Burk's plots for inhibi- and this mixture was preincubated with β-blo- tion of tyramine deamination by pr- ckers for 30min at room temperature. At opranolol. the start of the reaction, radiolabelled tyra- Various concentrations of tyramine mine(50μM)was added in a mixture. were incubated in the presence and After incubation for 20min in air at 37℃, absence of different concentrations the reaction was terminated by adding of 20μl of propranolol with human platelet of 3N HC1, then l ml of toluene-ethylacetate MAO.

(1:1)was added and shaken for 20 sec and

centrifuged at 3000 rpm for l min.0.5 ml of

the supernatant fluid was carefully extracted

and poured into the counting vials containing

6ml of scintillator. Product formation was

assayed by Packard Tri-Garb Scintillation

Spectrometer. MAO activities were express-

ed in disintegrations per min(DPM).

Results

Effects of β-blockers on human platelet

MAO activity Fig. 2 Lineweaver-Burk's plots for inhibi- The effects of propranolol, indenolol, pin- tion of tyramine deamination by dolol, carteolol, oxprenolol and atenolol on oxprenolol. human platelet MAO activity were investi- Conditions are the same as Fig. 1.

gated using tyramine as substrate. The inhi- bitory effects on platelet MAO activity by concentrations of propranolol intersected at 1mM β-blockers were summarized in Table the same point on the ordinate, indicating

1. The order of potency was indenolol>pro- that the pattern of inhibition by propranolol

pranolol>pindolol> oxprenolol>atenolol> was competitive (Fig, 1). On the other hand, carteolol and percent inhibitions of human in case of oxprenolol, the lines intersected at

platelet MAO activity were 47,45,30,27,20 the same point on the abscissa, indicating that and 11%, respectively. the pattern of inhibition by oxprenolol was

Inhibition pattern and Ki values of β-blo- non-competitive (Fig. 2). Furthermore, the

ckers pattern of inhibition by indenolol and pindol-

Inhibition pattern of propranolol, oxpre- ol were competitive, while the pattern of in-

nolol, indenolol, pindolol, carteolol and ate- hibition by carteolol and atenolol were non- nolol were determined from Lineweaver- competitive (Table 2). Burk's plots. In case of propranolol, the lines The Ki values were determined by the re- in the absence and presence of three different plot of the reciprocal slopes versus the cor-

318 昭 和59年6月28日 319

Table 1 The inhibitory rate of human Table 3 Reversibility of effects several ƒÀ-blockers platelet MAO activity by ƒÀ- blockers

Percent inhibition of human platelet MAO Effects of dialysis on human platelet MAO activity by several 13-blockers was determi- activities inhibited by several ƒÀ-blockers. ned from PI curves using 14C-tyramine as MAO activities were assayed using tyra- substrate. mine as substrate.

3. The MAO activities which had been in- Table 2 The inhibition patterns of human hibited by propranolol, indenolol, carteolol platelet MAO activity by ƒÀ-blo- and atenolol returned to that of the control ckers by dialysis. So, it was indicated that inhibi- tion of MAO activities by propranolol, in- denolol, carteolol and atenolol were reversi- ble. But, the MAO activities which had been inhibited by oxprenolol and pindolol did not return to that of the control by dialysis. So, it was indicated that inhibition of MAO acti- vities by oxprenolol and pindolol were irre- versible. The inhibition pattern of human platelet MAO by several 19-blockers was determined Discussion from Linweaver-Burk's plots and Ki values were detarmined by a plot of the recipro- MAO is responsible for the oxidative dea- cal slopes versus the corresponding conce- mination of a variety of biogenic amines ntration of inhibitors. in many mammalian tissues. Data obtained from studies of enzyme inhibitor sensitivities responding concentration of inhibitors (Table have suggested that MAO exists in two froms, 2). The Ki values of propranolol, oxprenol- termed type A and type B MAO10) . 5-Hy- ol, indenolol, pindolol, carteolol and atenolol droxytryptamine (5-HT) and norepinephrine were 1, 1.9, 1, 1.6, 4.4, 2.7 mM, respectively. are deaminated by type A MAO. Benzyla- Reversibility of effects of fl-blockers on hu- mine and 1S-phenylethylamine (PEA) are dea- man platelet MAO minated by type B MAO and tyramine is dea- The reversibility of effects of ƒÀ-blockers minated by both types of MAO11-13). were tested using 50 ƒÊM tyramine as sub- According to the difinition of the classifi- strate. The preparations of PRP were mixed cation of MAO, only type B MAO is present with 1 mM n-blockers or with 50 mM pho- in human platelets14). Human platelet MAO sphate buffer as control. Both mixture were metabolizes only type B substrates15), while dialized overnight 1 mM phosphate buffer (pH brain MAO metabolizes both types of sub- 7.4) at 10•Ž, after which their activities were strates7). compared. The results are shown in Table W ada et al3). showed that propranolol and

319 320 昭和医学会雑誌 第44巻 第3号

pindolol inhibited 5-HT oxidation much more it was observed that pindolol, oxprenolol, in- than PEA oxidation and concluded that in denolol and propranolol inhibited more than the concept of multiplicity of MAO, propra- carteolol and atenolol on human platelet nolol and pindolol were considered to inhibit MAO activity. These results suggested that type A MAO preferentially. degree of inhibition of platelet MAO activity

Our results show that jS-blockers inhibit hu- is proportional to quinidine-like activity of ƒÀ- man platelet MAO activity at highest con- blockers. centration of 1 mM. They inhibited human In the concept of multiplicity of MAO, platelet MAO activity from 11 to 47 %. The both types MAO (type A and type B) exist in inhibitory effects of ƒÀ-blockers were conside- the brain7). Since ƒÀ-blockers were conside- red to be weak since only type B MAO are red to inhibit type A MAO preferentially, present in human platelets. it is suggested that $-blockers possess only

Kinetic studies of ƒÀ-blockers on human pla- weak inhibitory effect on platelet MAO com- telet MAO activity revealed that inhibitions pared to that on brain MAO. by propranolol, indenolol and pindolol are References competitive, while inhibitions by oxprenolol, carteolol and atenolol are non-competitive. 1) Waal-Manning, H. J.: Drugs, 12: 412, 1976. These results suggested that propranolol, in- 2) Prichard, B. N. C.: Br. J. Clin. Pharmacol., denolol and pindolol inhibit human platelet 5: 379, 1978.

MAO activity in active site or at least near 3) Wada, I., et al.: J. Showa Med. Assoc., 41: active site. 129, 1981. Yasuhara et al4). reported that changes in 4) Yasuhara, H., et al.: Jpn. J. Pharmacol., 33 : 1057, 1983. MAO activity may be related in part to the 5) Robinson, D. S., et al.: Biochem.PharmacoL, onset of hypertension. 17: 109,1968. Therapeutic concentration of propranolol is 6) Collins, G. G. S., et al.: Nature, 225 : 817, from 50 to 100 ng/ml (1.5-3.0•~10-7 M). Ki 1970. values of propranolol in human platelet MAO 7) Collins, G.G.S. and Sandier, M.: Biochem. and rat brain MAO were 1 mM and 0.5 mM, Pharmacol. 20 : 289, 1971. respectively. These Ki values are about 3000 8) Murphy, D. L. and W eiss, R.: Am. J. Psychi- times higher for platelet and 1500 times high- atry, 128 : 1351, 1972. er for brain than therapeutic concentration. 9) Watanabe, K., et al.: J. Showa Med. Assoc.,

Volume of distribution (Vd) of propranolol 44: 205, 1984 is 31/kg16) and lipid soluble drug, so that, 10) Johnston, J. P.: Biochem. Pharmaco1.,17 : 1285, 1968. propranolol passes easily from plasma to peri- 11) Hall, D. W. R., et al.: Biochem. Pharmacol., pheral tissues and accumulate in peripheral 18 : 1447, 1969. tissues, especially brain. So, it is suggested 12) Goridis, C. and Neff, N. H.: Br. J. Pharma- that ƒÀ-blockers inhibit brain MAO much more col., 43 : 814, 1971. than human platelet MAO. 13) Yang, H.-Y. T. and Neff, N. H.: J. Pharma- Wada et al3), reported that inhibition of col. Exp. Ther.,187: 365, 1973. MAO activity by a-blockers were well cor- 14) Donelly, C. H. and Murphy, D. L.: Biochem. relation to their surface anesthetic effects and Pharmacol., 26: 853, 1977. membrane stabilizing effects of ,8-blockers 15) Fowler, C. L, et al.: Biochem. Pharmacol., 28 : 3063, 1979. seem to be important to inhibit MAO acti- 16) Bochner, F., et al.: Handbook of clinical vity. Pindolol, oxprenolol, indenolol and Pharmacology., p. 110, Little, Brown and propranolol possess local anesthetic or quini- Company, Boston, 1981. dine-like activity and carteolol, atenolol do not possess its activity. In this experiment,

320 昭和59年6月28日 321

人血小板MAO活 性 に対 する 種 々 β-blockersの 影響

昭 和 大 学 医学 部 第 二 薬 理 学 教 室(主 任:安 原 一 教 授) 渡 辺 浩 次 和 田 栄 小 林 真 一 小 口 勝 司

人 血 小 板MAO活 性 に 対 す る 種 々 β-blockersの 影 響 を 検 討 し た.酵 素 材 料 はplatelet rich plasma (PRP)を 使 用 し た.MAO活 性 の 測 定 はRI法 に よ つ た. Indenolol, propranolol, pindolol, Oxprenolol. atenolol, carteololは1mMの 濃 度 で 血 小 板MAOを 阻 害 した.こ れ ら の β-blockersは11～47%血 小 板 MAO括 性 を 阻 害 し た.β-blockersの 阻 害 の 強 さ はindenolol>propranolol>pindolol>oxprenolol> atenolol>carteololの 順 で あ っ た. propranelol, indenolol, carteolol, atenololに よ る 阻 害 は 可 逆 的 で, oxprenolol, pindololに よ る 阻 害 は 非 可 逆 的 で あ っ た.ま たpropranolol, indenolol, pindololは 血 小 板 MAOを 競 合 的 に 阻 害 し,oxprenolol, atenololは 非 競 合 的 に 阻 害 し た.更 にpropranolol, Oxprenolol, indenolol, pindolol, carteolol, atenololのKi値 は そ れ ぞ れ1,1.9,1,1.6,4.4,2.7mMで あ っ た. こ れ ら の 結 果 よ り,β-blockersに よ る 人 血 小 板MAO活 性 へ の 阻 害 は 弱 く, propranolol, indenolol,pi- ndololはactive siteあ る い は 少 く と も そ の 近 く で 人 小 血 板 活 性 を 阻 害 す る こ と が 示 唆 さ れ た.

〔受 付:1日30月,1984〕

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