The effect of evodiamine on catecholamine secretion from bovine adrenal medulla was investigated. Evodiamine, a bioactive component isolated from dry unripened fruit of Evodia rutaecarpa Bentham, was found to stimulate the secretion of catecholamine from perfused bovine adrenal medulla at a concentration of 10 μM and its effect persisted for at least 30 min. This stimulatory effect of evodiamine was abolished by omission of Ca2+ from the perfusion fluid. Evodiamine (0.1-10 μM) markedly enhanced the secretion of catecholamine from the adrenal medulla induced by acetylcholine (100 μM) or high K+(56 mM). The secretion of catecholamine was promptly enhanced by acetylcholine or high K+, but returned to the control level on treatment for 20 min. However, when evodiamine was added to the perfusion fluid after acetylcholine or high K+ stimulation for 10 min, the secretion of catecholamine again increased greatly. These results indicate that evodiamine not only stimulated the secretion of catecholamine from bovine adrenal medulla but also reversed insensitivity of these cells to acetylcholine or high K+ stimulation

Hirose, Masao

Houchi, Hitoshi

Ishimura, Yasuko

Kitagawa, Tetsuya

Minakuchi, Kazuo

Tamaki, Toshiaki

Tsuchiya, Koichiro

Yoshizumi, Masanori

Tokushima University Institutional Repository

INTRODUCTIONEvodiamine is a bioactive component isolated from dryunripened fruit of Evodia rutaecarpa Bentham(1). Thesefruit have long been used in Chinese medical practice forthe treatments of headache, abdominal pain, dysentery,postpartum hemorrhage and amenorrhea(1). Pharma-cological studies demonstrated that evodiamine has apositive inotropic action on isolated left atria of the guineapig(1), an antianoxic action in anoxia induced by KCN inmice(2)and an effect of retaining the body temperature ofrats treated with chlorpromazine(3).Adrenal medulla contains neural crest-derived chromaf-fin cells. Therefore, the adrenal medulla is useful forstudying the mechanism of stimulus-secretion couplingand is regarded as a model for catecholamine containingneurons. Physiological stimulations of the adrenal medullasuch as acetylcholine cause an increase in the levels ofintracellular free Ca2+([Ca2+]i). The increase in [Ca2+]ileads to the stimulation of catecholamine secretion(4).However, the effects of evodiamine on the secretions ofhormones and neurotransmitters have not yet beenreported. In the present study, we examined the effect ofevodiamine on catecholamine secretion from bovineadrenal medulla.MATERIALS AND METHODSFresh bovine adrenal glands were used throughout.The glands were perfused retrogradly(5)with a mediumconsisting of (in mM : NaCl 154, KCl 5.6, CaCl2 2.2, glu-cose 10 and Tris-HCl buffer 20 pH 7.4)saturated with 100%O2 and maintained at 37℃. The glands were perfused at arate of 4 ml/min for 40 min and then stimulated bychanging to a medium containing high K+(56 mM K+ withequimolar reduction of Na+), acetylcholine or evodiamine.Samples of 8 ml of perfusion fluid were collected at 2 minintervals. Catecholamine secretion was determined byHPLC with electrochemical detection (Yanaco model L-2000).Evodiamine was obtained from Wako Pure ChemicalIndustries (Osaka, Japan). Other chemicals used werecommercial products of reagent grade.RESULTSTable 1 shows the effect of evodiamine on secretion ofcatecholamine from perfused bovine adrenal medulla.Evodiamine at a concentration of 10 μM slightly, butsignificantly, stimulated the secretion of catecholaminefrom the adrenal medulla. This stimulatory effect ofevodiamine was observed at concentrations of more thanEffect of evodiamine on catecholamine secretion from bovineadrenal medullaMasanori Yoshizumi＊, Hitoshi Houchi＊, Yasuko Ishimura＊, Masao Hirose†,Tetsuya Kitagawa‡, Koichiro Tsuchiya, Kazuo Minakuchi, and Toshiaki Tamaki＊＊Department of Pharmacology ; †Division of Transfusion Medicine ; ‡Department of Cardiovascular Surgery ;andDepartment of Pharmacy, The University of Tokushima School of Medicine, Tokushima, JapanAbstract : The effect of evodiamine on catecholamine secretion from bovine adrenal medulla wasinvestigated. Evodiamine, a bioactive component isolated from dry unripened fruit of Evodiarutaecarpa Bentham, was found to stimulate the secretion of catecholamine from perfused bovineadrenal medulla at a concentration of 10 μM and its effect persisted for at least 30 min. Thisstimulatory effect of evodiamine was abolished by omission of Ca2+ from the perfusion fluid.Evodiamine (0.1-10 μM) markedly enhanced the secretion of catecholamine from the adrenal medullainduced by acetylcholine (100 μM) or high K+(56 mM). The secretion of catecholamine was promptlyenhanced by acetylcholine or high K+, but returned to the control level on treatment for 20 min.However, when evodiamine was added to the perfusion fluid after acetylcholine or high K+stimulation for 10 min, the secretion of catecholamine again increased greatly. These results indicatethat evodiamine not only stimulated the secretion of catecholamine from bovine adrenal medulla butalso reversed insensitivity of these cells to acetylcholine or high K+ stimulation. J. Med. Invest. 44 : 79-82, 1997Key Words : evodiamine, catecholamine secretion, adrenal medullaReceived for publication July 31, 1997 ; accepted August 5, 1997.１ Address correspondence and reprint requests to Masanori Yoshizumi,M.D., Ph.D., Department of Pharmacology, The University of TokushimaSchool of Medicine, 3-18-15 Kuramoto-cho, Tokushima, Japan.The Journal of Medical Investigation Vol.44 1997７９1 μM (control, 24.5±2.8 μg/2 min ; 1 μM evodiamine,27.6±2.9 μg/2 min, N=6) and continued for at least 30min. To determine whether the stimulatory effect ofevodiamine on catecholamine secretion depends on Ca2+in the perfusion fluid, the adrenal medulla was perfusedwith Ca2+-free fluid. As shown in Table 1, the slight effectof evodiamine on catecholamine secretion was notobserved in Ca2+-free fluid. Therefore, evodiamine maystimulate the secretion of catecholamine through increasein Ca2+ uptake into the cells of the adrenal medulla.Catecholamine secretion from the adrenal medulla isknown to be induced by stimulation of acetylcholinereceptors or depolarization of the cell membrane by highK+(6). As shown in Fig.1, the effect of evodiamine onacetylcholine-induced catecholamine secretion from per-fused adrenal medulla was examined. Acetylcholinetransiently increased the secretion of catecholamine fromthe adrenal medulla. This catecholamine secretion waspotentiated by concentrations between 0.1 to 10 μMevodiamine. To exclude effects of evodiamine on acetyl-choline receptors, we examined its effects on high K+-induced catecholamine secretion from perfused adrenalmedulla(Fig.2). High K+ also transiently increased thesecretion of catecholamine from the adrenal medulla.Evodiamine potentiated both the amount and the durationof catecholamine secretion induced by high K+ stimula-tion.There are reports that desensitization, in other words,insensitive to catecholamine secretion produced byfrequent same stimulations, of stimulus secretion couplingin the bovine adrenal medulla is induced by physiologicalstimulations such as acetylcholine as well as high K＋(7-10). To determine whether evodiamine influences theprocess of desensitization of catecholamine secretion, weinvestigated the effects of evodiamine on catecholaminesecretion after acetylcholine- or high K+-induced desensiti-zation (Fig.3 and 4). The secretion of catecholamine waspromptly enhanced by acetylcholine or high K+, butreturned to the control level on treatment for 20 min.When evodiamine was added to the perfusion fluid afteracetylcholine or high K+ stimulation for 10 min, thesecretion of catecholamine again increased greatly (Fig.3and 4). Nifedipine, a typical voltage-dependent calciumchannel blocker, did not change the effect of evodiamineon acetylcholine- or high K+-stimulated catecholaminesecretion in the same experimental protocol (Table 2).Moreover, 12-O -tetradecanoyl phorbol 13-acetate (TPA),Table 1. Effect of evodiamine on catecholamine secretion fromperfused bovine adrenal medullaAdrenal medulla was stimulated by 10 μM evodiamine with orwithout Ca2+ in the perfusion fluid. Catecholamine secretion wasdetermined as described in Materials and Methods. Data aremeans±SE for 4-6 separate experiments.*Significantly greater than control (p<0.05).#Significantly less than with Ca2+ (p<0.01).Fig.1. Effect of evodiamine on acetylcholine-induced catecholaminesecretion from perfused bovine adrenal medulla.Adrenal medulla was stimulated by 100 μM acetylcholine for 10min without evodiamine, and then stimulated again by 100 μMacetylcholine with (hatched columns) or without (open columns)0.1-10 μM evodiamine. Catecholamine secretion was significantlygreater with 1 and 10 μM evodiamine than without evodiamine (p<0.01).Catecholamine secretion was determined as described in Materialsand Methods. Data are means±SE for 4-6 separate experiments.Fig. 2. Effect of evodiamine on high K+-induced catecholaminesecretion from perfused bovine adrenal medulla.Adrenal medulla was stimulated by 56 mM K+ for 10 min withoutevodiamine, and then stimulated again by 56 mM K+ with (hatchedcolumns) or without (open columns) 0.1-10 μM evodiamine.Catecholamine secretion was significantly greater with 1 and 10 μMevodiamine than without evodiamine (p<0.01).Catecholamine secretion was determined as described in Materialsand Methods. Data are means±SE for 4-6 separate experiments.Catecholamine Secretion(μg/2 min)2.2 mM Ca2+ 0 mM Ca2+Control 24.5±2.8Evodiamine(10 μM)(0-2 min) 32.3±3.5*(8-10 min) 37.5±4.1*(28-30 min) 38.4±4.3*24.3±2.225.2±2.925.1±2.6#24.8±2.7#M. Yoshizumi et al. Evodiamine Enhances Catecholamine Secretion８０an activator of protein kinase C, did not mimic evodiaminewhich enhances the secretion of catecholamine from thebovine adrenal medulla, after stimulation by acetylcholineor high K+ (Table 3). These results suggest that evodia-mine may reverse the insensitivity of the bovine adrenalmedulla to induction of catecholamine secretion thatdevelops during acetylcholine or high K+ stimulation.DISCUSSIONLittle is known about the effects of evodiamine orrelated compounds on stimulus-secretion coupling inseveral hormonal organs or nervous systems. In thepresent study, we demonstrated that evodiamine greatlyincreased acetylcholine- or high K+-stimulated catechola-mine secretion from the bovine adrenal medulla even afterdesensitization of the response (Fig. 3 and 4), althoughevodiamine alone had only a slight, but significant, effecton the spontaneous secretion (Table 1). Therefore, it isdifficult to conclude that the effects of evodiamine andacetylcholine or high K+ on catecholamine secretion weresimply additive. In the experimental conditions for Fig.3and 4, we found that the stimulatory effect of evodiamineon acetylcholine- or high K+-induced catecholamine secre-tion was not affected by nifedipine, a typical voltage-dependent calcium channel blocker (Table 2). Therefore,the stimulatory effect of evodiamine on catecholaminesecretion after acetylcholine or high K+ stimulation doesnot seem to be regulated by activation of voltage-dependent calcium channels on the cell membrane.Fig. 4. Effect of evodiamine on catecholamine secretion afterstimulation of high K+ from perfused bovine adrenal medulla.Adrenal medulla was stimulated by 56 mM K+, and 1-10 μMevodiamine (hatched columns) was added after this stimulation for10 min. Catecholamine secretion was significantly greater with 0.1,1 and 10 μM evodiamine than without evodiamine (p<0.01).Catecholamine secretion was determined as described in Materialsand Methods. Data are means±SE for 4-6 separate experiments.Fig. 3. Effect of evodiamine on catecholamine secretion afterstimulation of acetylcholine from perfused bovine adrenal medulla.Adrenal medulla was stimulated by 100 μM acetylcholine, and 1-10 μM evodiamine (hatched columns) was added after thisstimulation for 10 min. Catecholamine secretion was significantlygreater with 0.1, 1 and 10 μM evodiamine than without evodiamine(p<0.01).Catecholamine secretion was determined as described in Materialsand Methods. Data are means±SE for 4-6 separate experiments.Table 2. Effects of nifedipine on evodiamine-induced catechola-mine secretion treated with acetylcholine or high K+ from perfusedbovine adrenal medullaAdrenal medulla was stimulated by 100 μM acetylcholine or 56mM high K+, and then 10 μM evodiamine and/or 100 μMnifedipine were added after this stimulation for 10 min. Catechola-mine secretion was determined as described in Materials andMethods. Data are means±SE for 4-6 separate experiments.Table 3. Effects of TPA on catecholamine secretion treated withacetylcholine or high K+ from perfused bovine adrenal medullaAdrenal medulla was stimulated by 100 μM acetylcholine or 56 mMhigh K+, and then 1 μM 12-O -tetradecanoyl phorbol 13-acetate(TPA) was added after this stimulation for 10 min. Catecholaminesecretion was determined as described in Materials and Methods.Data are means±SE for 4-6 separate experiments.Catecholamine Secretion (18 to 20 min)(μg/2 min)Acetylcholine (100 μM)+ Evodiamine (10 μM) 130±15Acetylcholine (100 μM) NS+ Evodiamine (10 μM)+ Nifedipine (100 μM) 125±13High K+ (56 mM)+ Evodiamine (10 μM) 100±13High K+ (56 mM) NS+ Evodiamine (10 μM)+ Nifedipine (100 μM) 110±13Catecholamine Secretion (18 to 20 min)(μg/2 min)Acetylcholine (100 μM) ３０±４Acetylcholine (100 μM) NS+ TPA (1 μM) ３１±５High K+ (56 mM) ３６±４High K+ (56 mM) NS+ TPA (1 μM) ３６±５The Journal of Medical Investigation Vol.44 1997 ８１Acetylcholine is known to stimulate nicotinic andmuscarinic receptors in adrenal medullary cells (11).Activation of the nicotinic receptor induces Ca2+ influxthrough the cell membrane(11,12). However, activation ofthe muscarinic receptor stimulates the formation ofinositol 1,4,5-trisphosphate (IP3) and diacylglycerol(13).Diacylglycerol is produced concurrently with IP3 onbreakdown of phosphatidyl inositol 4,5-bisphosphate(PIP2) by phospholipase C and is thought to activateprotein kinase C by increasing the affinity of the enzymefor calcium(14,15). Activation of protein kinase C usingphorbol ester TPA increases secretion of catecholaminefrom leaking adrenal medullary cells(16). However, TPAdid not mimic evodiamine which enhances the secretionof catecholamine from the bovine adrenal medulla, afterstimulation by acetylcholine or high K+ (Table 3).Therefore, the stimulatory effect of evodiamine oncatecholamine secretion after acetylcholine or high K+stimulation may not be mediated by the activation ofprotein kinase C.We are now studying the effects of evodiamine on theintracellular mechanisms of the catecholamine secretoryresponse in bovine adrenal chromaffin cells in culture.ACKNOWLEDGMENTSThis work was supported by a grant from TheYamamura Yuichi Memorial WAKAN-YAKU ResearchFoundation, Japan.REFERENCES1. Shoji N, Umeyama A, Takemoto T, Kajiwara A,Ohizumi Y : Isolation of evodiamine, a powerfulcardiotonic principle, from Evodia rutaecarpa Ben-tham (rutaceae). J Pharm Sci 75 : 612-613, 19862. Yamahara J, Yamada T, Kitani T, Naitoh Y, FujimuraH : Antianoxic action of evodiamine, an alkaloid inEvodia rutaecarpa fruit. J Ethnopharmacol 27 : 185-192, 19893. Kano Y, Zong QN, Komatsu K : Pharmacologicalproperties of galenical preparation. XIV. Body tem-perature retaining effect of the Chinese traditionalmedicine,“goshuyu-to" and component crude drugs.Chem Pharm Bull Tokyo 39 : 690-692, 19914. Masserano JM, Vulliet PR, Tank AW, Weiner N : Therole of tyrosine hydroxylase in the regulationof catecholamine synthesis. In ; Trendelenburg U,Weiner N, eds. : Handbook of Experimental Pharma-cology. Catecholamines. Springer, Berlin, 1990, pp.427-4695. Banks P : The release of adenosine triphosphatecatabolites during the secretion of catecholamines bybovine adrenal medulla. Biochem J 101 : 536-541,19666. Oka M, Isosaki M, Yanagihara N : Isolated bovineadrenal medullary cells : studies on regulation ofcatecholamine synthesis and release. In : Usdin E,Kopin I J, Barchas J, eds. Catecholamines : Basic andClinical Frontiers. Pergamon Press, Oxford, 1979, pp70-727. Douglas WW, Rubin RP : The role of calcium in thesecretory response of the adrenal medulla toacetylcholine. J Physiol (Lond)159 : 40-57, 19618. Baker PF, Rink TJ : Catecholamine release frombovine adrenal medulla in response to maintaineddepolarization. J Physiol (Lond)253 : 593-620, 19759. Bevington A, Radda GK : Declining catecholaminesecretion in adrenal medulla on prolonged stimula-tion with acetylcholine. Biochem Pharmacol 34 :1497-1500, 198510. Marley PD, Livett BG : Effects of opioid compoundson desensitization of the nicotinic response ofisolated bovine adrenal chromaffin cells. BiochemPharmacol 36 : 2937-2944, 198711. Oka M, Isosaki M, Watanabe J : Calcium flux andcatecholamine release in isolated bovine adrenalmedullary cells : Effects of nicotinic and muscarinicstimulation. In : Izumi F, Oka M, Kumakura K, eds.Advances in the biosciences. Synthesis, storage andsecretion of adrenal catecholamines. PergamonPress, Oxford, 1981, pp 29-3612. Wada A, Yanagihara N, Izumi F, Sakurai S,Kobayashi H : Trifluoperazine inhibits 45Ca2+ uptakeand catecholamine secretion and synthesis in adrenalmedullary cells. J Neurochem 40 : 481-486, 198313. Plevin R, Boarder MR : Stimulation of formation ofinositol phosphates in primary cultures of bovineadrenal chromaffin cells by angiotensin II, histamine,bradykinin and carbachol. J Neurochem 51 : 634-641,198814. Nishizuka Y : The role of protein kinase C in cellsurface signal transduction and tumor promotion.Nature (Lond)312 : 315-321, 198415. Houchi H, Nakanishi A, Uddin MM, Ohuchi T, OkaM : Phorbol ester stimulates catecholamine synthe-sis in isolated bovine adrenal medullary cells. FEBSLett 188 : 205-2０8, 198516. Knight DE, Baker PF : The phorbol ester TPAincreases the affinity of exocytosis for calcium inleaky adrenal medullary cells. FEBS Lett 160 : 98-100, 1983M. Yoshizumi et al. Evodiamine Enhances Catecholamine Secretion８２

Effect of evodiamine on catecholamine secretion from bovine adrenal medulla

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Effect of evodiamine on catecholamine secretion from bovine adrenal medulla

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