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ORIGINAL ARTICLE
Stimulatory effect of nobiletin, a citrus polymethoxyflavone, on catecholamine synthesis through Ser19 and Ser40
phosphorylation of tyrosine hydroxylase in cultured bovineadrenal medullary cells
Han Zhang & Nobuyuki Yanagihara & Yumiko Toyohira & Keita Takahashi &Hirohide Inagaki & Noriaki Satoh & Xiaoja Li & Xiumei Goa & Masato Tsutsui &Kojiro Takahaishi
Received: 13 April 2013 /Accepted: 4 September 2013 /Published online: 17 September 2013# Springer-Verlag Berlin Heidelberg 2013
Abstract We previously reported the dual effects of nobiletin,a compound of polymethoxy flavones found in citrus fruits, oncatecholamine secretion in cultured bovine adrenal medullarycells. Here, we report the effects of nobiletin on catecholaminesynthesis in the cells. Nobiletin increased the synthesis of 14C-catecholamines from [14C]tyrosine in a time (20–30 min)- andconcentration (1.0–100 μM)-dependent manner. Nobiletin(10–100 μM) also activated tyrosine hydroxylase activity.The stimulatory effect of nobiletin on 14C-catecholamine syn-thesis was not observed when extracellular Ca2+ was notpresent in the incubation medium. Protein kinase inhibitorsincluding H-89, an inhibitor of cyclic AMP-dependent proteinkinase, andKN-93, an inhibitor of Ca2+/calmodulin-dependent
protein kinase II, suppressed the stimulatory effects ofnobiletin on catecholamine synthesis as well as tyrosine hy-droxylase activity. Nobiletin also induced the phosphorylationof tyrosine hydroxylase at Ser19 and Ser40. Nobiletin (1.0–100 μM) inhibited 14C-catecholamine synthesis induced byacetylcholine. The present findings suggest that nobiletin, byitself, stimulates catecholamine synthesis through tyrosine hy-droxylase phosphorylation at Ser19 and Ser40, whereas it in-hibits catecholamine synthesis induced by acetylcholine inbovine adrenal medulla.
Keywords Adrenal medulla . Catecholamine synthesis .
Citrus flavonoid . Nobiletin . Phosphorylation . Tyrosinehydroxylase
Introduction
Nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone) is a major compo-nent of polymethoxylated flavones found in the peel of citrusfruits (Bocco et al. 1998) which is used in a Chinese traditionalherbal medicine to treat a poor appetite, cough, vomiting, andhypertension. Nobiletin exhibits many favorite pharmacologicalactivities, including antiatherogenic (Whitman et al. 2005), car-diovascular protective effects (Middleton et al. 2000), neuronaleffects such as neurotrophic effects in vitro (Nagase et al.2005a), and antidementia activities in vivo (Nakajima et al.2007; Onozuka et al. 2008). The precise mechanisms underly-ing nobiletin’s effects, however, remain to be determined.
Adrenal medullary cells derived from the embryonic neuralcrest are functionally homologous to the sympathetic postgan-glionic cells. In cultured bovine adrenal medullary cells, theNa+ influx induced by acetylcholine (ACh) via nicotinic ACh
H. Zhang :X. GoaResearch Center of Traditional Chinese Medicine, TianjinUniversity of Traditional Chinese Medicine, Tianjin, China
N. Yanagihara (*) :Y. Toyohira :K. Takahashi :H. Inagaki :X. LiDepartment of Pharmacology, School of Medicine, University ofOccupational and Environmental Health, 1-1, Iseigaoka,Yahatanishi-ku, Kitakyushu 807-8555, Japane-mail: [email protected]
N. SatohShared-Use Research Center, University of Occupational& Environmental Health, University of Occupational& Environmental Health, Japan
M. TsutsuiDepartment of Pharmacology, Graduate School of Medicine,University of the Ryukyus, Okinawa 903-0215, Japan
K. TakahaishiDepartment of Hospital Pharmacy, University of Occupationaland Environmental Health, 1-1, Iseigaoka, Yahatanishi-ku,Kitakyushu 807-8555, Japan
Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:15–22DOI 10.1007/s00210-013-0916-6
receptor (nAChR)-ion channels is a prerequisite for Ca2+ influxvia the activation of voltage-dependent Ca2+ channels andsubsequent catecholamine synthesis and secretion. Stimulationof catecholamine synthesis induced by ACh is associated withan activation of tyrosine hydroxylase (Yanagihara et al. 1987),which catalyzes the conversion of tyrosine to L -3,4-dihydroxyphenylalanine (DOPA), the rate-limiting step of cat-echolamine biosynthesis (Nagatsu et al. 1964). It has beenestablished that the activity of tyrosine hydroxylase is regulatedby protein kinases. Indeed, tyrosine hydroxylase can be phos-phorylated and activated by several protein kinases such ascyclic AMP-dependent protein kinase (protein kinase A), ex-tracellular signal regulated-protein kinase (ERK), and calcium/calmodulin-dependent protein kinase II (CaM kinase II) (for areview, see Zigmond et al. 1989), although activation of theenzyme phosphorylated by CaM kinase II needs an activatorprotein (14-3-3 protein) (Yamauchi and Fujisawa 1981).
Our recent study revealed that nobiletin stimulates cate-cholamine secretion via activation of voltage-dependent Ca2+
channels and/or Na+/Ca2+ exchangers, but it inhibits catechol-amine secretion induced by ACh in cultured bovine adrenalmedullary cells (Zhang et al. 2010). In the present study, weinvestigated the effects of nobiletin on catecholamine synthe-sis and tyrosine hydroxylase activity in these cells. We foundthat nobiletin, by itself, stimulates the activity of tyrosinehydroxylase and subsequently catecholamine synthesisthrough the phosphorylation of Ser19 and Ser40 of tyrosinehydroxylase, whereas it inhibits catecholamine synthesisinduced by ACh.
Materials and methods
Materials
Oxygenated Krebs–Ringer phosphate (KRP) buffer wasused throughout. Its composition is as follows (in millimo-lar): 154 NaCl, 5.6 KCl, 1.1 MgSO4, 2.2 CaCl2, 0.85NaH2PO4, 2.15 Na2HPO4 and 10 glucose, adjusted pH to7.4. Reagents were obtained from the following sources:Eagle’s minimum essential medium (Eagle’s MEM) wasfrom Nissui Pharmaceutical (Tokyo, Japan); calf serumwas from Nacalai Tesque (Kyoto, Japan); collagenase wasfrom Nitta Zerachin (Osaka, Japan); nobiletin was fromWako Pure Chemical Industries, Ltd (Osaka, Japan). AChwas from Sigma (St. Louis, MO, USA); L-[U-14C]tyrosine(17.9 GBq/mmol) and L-[3-14C]DOPA (0.25 GBq/mmol)from GE Healthcare UK Ltd. (Little Chalfont, Bucks., UK);L -[1-14C]tyrosine (2 GBq/mmol) from Perkin-Elmer LifeSciences (Boston, MA USA). Nobiletin was dissolved in100 % dimethyl sulfoxide (DMSO) and then diluted in areaction medium before use at a final DMSO concentrationnot exceeding 0.5 %, unless otherwise specified.
Isolation and primary culture of bovine adrenal medullarycells
Bovine adrenal medullary cells were isolated by collagenasedigestion of adrenal medullary slices according to the methodas reported previously (Yanagihara et al. 1979, 1996). Cellswere suspended in Eagle’s MEM containing 10 % calf serum,3 μM cytosine arabinoside, and several antibiotics, andmaintained in monolayer culture at a density of 4×106 or 2×106 cells per dish (35 mm dish; Falcon, Becton DickinsonLabware, Franklin Lakes, NJ) or 106 cells per well (24-wellplate; Corning Life Sciences, Lowell, MA) at 37 °C under ahumidified atmosphere of 5 % CO2 and 95 % air. The cellswere used for experiments between 2 and 5 days of culture.
14C-Catecholamine synthesis from [14C]tyrosineor [14C]DOPA in the cells
Cells (4 × 106/dish) were incubated with 20 μML-[U-14C]tyrosine (1 μCi) in KRP buffer in the presence orabsence of various concentrations of nobiletin and/or 0.3 mMACh at 37 °C for 20 min (for [14C]tyrosine ) or 15 min (for[14C]DOPA). In some experiments with Ca2+-free medium,2.2 mM CaCl2 was omitted from and 0.1 mM EGTA wasadded to the KRP buffer. After removing the incubation me-dium by aspiration, cells were harvested in 0.4 M perchloricacid (PCA) and centrifuged at 1,600×g for 10 min. 14C-Cate-cholamines were separated further by ion exchange chroma-tography on Duolite C-25 columns (H+-type, 0.4×7.0 cm) andcounted for radioactivity (Yanagihara et al. 1987). 14C-Cate-cholamine synthesis was expressed as the sum of the 14C-catecholamines (adrenaline, noradrenaline, and dopamine).
Tyrosine hydroxylase activity in situ
The cells (106/well; 24 well, Falcon) were exposed to 200 μlof the KRP buffer with or without nobiletin (1.0–100 μM),supplemented with 18 μML-[1-14C] tyrosine (0.2 μCi) for10 min at 37 °C. Upon addition of the labeled tyrosine, eachwell was sealed immediately with an acrylic tube capped witha rubber stopper and fitted with a small plastic cup containing200 μl of NCS-II tissue solubilizer (GE Healthcare UK Ltd.)to absorb the 14CO2 released by the cells, and counted for theradioactivity (Bobrovskaya et al. 1998).
Phosphorylation of tyrosine hydroxylase in the cells
Cells (2×106/dish) were incubated with or without nobiletinfor 10 min in KRP buffer. After incubation, cells wereharvested with a buffer containing 1 mMEGTA, 1 mMEDTA,1 mM dithiothreitol, phosphatase inhibitors (80 mM ammoni-um molybdate, 2 mM tetrasodium pyrophosphate, 1 mM so-dium vanadate and 1 mM β-glycerophosphate) and protease
16 Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:15–22
inhibitor cocktail (2 mM 4-(2-aminoethyl)benzenesulfonylfluoride hydrochroride, 1.6 μM aprotinin, 30 μM E-64,40 μM leupeptin, 100 μM bestatin, 20 μM pepstatin) (NacalaiTesque, Kyoto, Japan). Cell lysates were centrifuged at20,000×g for 30 min, and an aliquot of each sample was usedfor SDS–polyacrylamide gel electrophoresis (SDS-PAGE) andWestern blotting was as follows. The tyrosine hydroxylaseproteins were separated by SDS-PAGE (10 %), and wereelectroblotted onto polyvinylidene difluoride (PVDF) mem-brane (Immobilon-P) with transfer buffer (39 mM glycine,48 mM Tris, 0.0375 % SDS, 20 % methanol, pH 8.5). Afterblocking with a blocking buffer (PVDF Blocking Reagent forCan Get Signal, Toyobo, Osaka, Japan) for 1 h at roomtemperature, the membranes were incubated with a primaryantibody against phospho-Ser19, -Ser31, and -Ser40 of tyrosinehydroxylase (1:2,000; Millipore, Billerica, MA, USA) in CanGet Signal Solution-1 (Toyobo, Osaka, Japan) for 1 h at roomtemperature and then washed with Tris-buffered saline-Tween(10 mM Tris–HCl, pH 7.4, 150 mM NaCl and 0.1 % Tween20) (TBS-T). The immunoreactive bands were reacted in asolution (Can Get Signal Solution-2, Toyobo, Osaka, Japan)with a polyclonal goat anti-rabbit antibody conjugated tohorseradish peroxidase (1:10,000; Cell Signaling Technology,Beverly, MA, USA) for 1 h at room temperature, and washedrepeatedly as above. The immunoreactive bands were visual-ized by Immobilon Western (Millipore Corporation, Billerica,MA, USA), and quantified by Light-Capture with CSAnalyzer(ATTO corporation, Tokyo, Japan).
Statistical analysis
All experiments were performed in duplicate or triplicate,and each experiment was repeated at least three times. Allvalues are given as means±SEM. The significance of dif-ferences between means was evaluated using one-way anal-ysis of variance (ANOVA). When a significant F value wasfound by ANOVA, Dunnett’s or Scheffe’s test for multiplecomparisons was used to identify differences among thegroups. Values were considered statistically different whenp was less than 0.05. Statistical analyses were performedusing StatView for Macintosh version 5.0 J software (SanDiego, CA) and PRISM for Windows version 5.0 J soft-ware (Abacus Concept, Berkeley, CA, USA).
Results
Stimulatory effects of nobiletin on 14C-catecholaminesynthesis from [14C]tyrosine in cultured bovine adrenalmedullary cells
The cells were incubated with or without nobiletin (100 μM)and 20 μM [14C]tyrosine (1 μCi) at 37 °C for the indicated
lengths of time (Fig. 1a). The basal synthesis of 14C-catechol-amines (the sum of [14C]adrenaline, [14C]noradrenaline, and[14C]dopamine production) from [14C]tyrosine was linear up to30min, as reported (Yanagihara et al. 1987). Nobiletin caused asmall (about 15–25 % over the control) but significant (p <0.05) increase in 14C-catecholamine synthesis during the incu-bation for 20–30 min. On the basis of this result, we performedsubsequent measurements of 14C-catecholamine synthesis witha 20-min incubation. Nobiletin increased the 14C-catechol-amine synthesis in a concentration-dependent manner (thehalf-maximal effective concentration, EC50=14.5±2.1 μM),i.e., a significant increase in 14C-catecholamine synthesis wasdetected at 1.0 μM and was maximal at 100 μM (Fig. 1b).
Effect of nobiletin and/or acetylcholine on 14C-catecholaminesynthesis from [14C]tyrosine or [14C]DOPA
As shown in Fig. 2a, ACh (0.3 mM) increased the synthesis of14C-catecholamines from [14C]tyrosine by 262 % over thecontrol. The concurrent treatment of cells with nobiletininhibited the stimulatory effect of acetylcholine on 14C-cate-cholamine synthesis in a concentration (1.0–100 μM)-depen-dent manner (the half-maximal inhibitory concentration,IC50=10.0±0.1 μM). To determine which step of catechol-amine synthesis was stimulated by nobiletin, we used[14C]DOPA as a substrate instead of [14C]tyrosine. Neithernobiletin nor ACh increased the synthesis of 14C-catechol-amines from [14C]DOPA (Fig. 2b), indicating that the stimu-lation of 14C-catecholamine synthesis caused by nobiletin orACh occurs predominantly upstream of the DOPA decarbox-ylase step. Nobiletin slightly but significantly inhibited basaland ACh-induced 14C-catecholamine synthesis from[14C]DOPA probably due to a persistent secretion of 14C-catecholamines formed in the cells (Zhang et al. 2010).
Effects of nobiletin on basal and ACh-induced activityof tyrosine hydroxylase
We next examined the effect of treatment with nobiletinon tyrosine hydroxylase activity in cells. Incubationwith nobiletin (10 and 100 μM) for 10 min caused asignificant increase in tyrosine hydroxylase activity of27 and 40 %, respectively, over the activity in thecontrol (Fig. 3a). In contrast, nobiletin (100 μM)inhibited the tyrosine hydroxylase activity induced byACh (0.3 mM) (Fig. 3b).
Effects of extracellular Ca2+ deprivation and various inhibitorsof protein kinases on 14C-catecholamine synthesis inducedby nobiletin
To study the role(s) of extracellular Ca2+ in the stimulation ofcatecholamine synthesis, we examined the effect of nobiletin
Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:15–22 17
on catecholamine synthesis in the absence of extracellularCa2+. When cells were incubated with a Ca2+-free KRP me-dium, nobiletin had little effect on catecholamine synthesis(Fig. 4a), suggesting that Ca2+ is essential for the nobiletin-induced increase in 14C-catecholamine synthesis. To investi-gate the role of intracellular signals such as Ca2+ andcyclic AMP, we next used KN-93, an inhibitor ofCaMKII, U0126, an inhibitor of ERK kinase, and H-
89, an inhibitor of protein kinase A, on catecholaminesynthesis induced by nobiletin. KN-93 (10 μM) and H-89 (10 μM) significantly inhibited nobiletin-induced14C-catecholamine synthesis, although U0126 and aninactive analogue of KN-93, KN-92 (10 μM), failed toinhibit it (Fig. 4b). H-89 plus KN-93 completely inhibitedthe stimulatory effect of nobiletin on 14C-catecholaminesynthesis.
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Fig. 1 Time course (a) and concentration-response curve (b) of nobiletinfor 14C-catecholamine synthesis in cultured bovine adrenal medullary cells.a Cells (4×106/dish) were incubated with (filled circle) or without (opencircle) nobiletin (100 μM) for 10–30 min at 37 °C in 1.0 ml KRP buffercontaining L-[U-14C]tyrosine (20 μM, 1 μCi). The 14C-labeled catechol-amines formed are shown as the total 14C-catecholamines (adrenaline,noradrenaline, and dopamine). Data are expressed as the mean±SEM of
four experiments carried out in duplicate. b Cells (4×106/dish) wereincubated without (open circle ) or with various concentrations (1–300 μM) (filled circle) of nobiletin at 37 °C for 20 min in 1.0 ml KRPbuffer containing L-[U-14C]tyrosine (20 μM, 1 μCi). The 14C-catechol-amines formedweremeasured. Data are expressed asmeans±SEMof threeseparate experiments carried out in triplicate. *p<0.05, compared to control
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Fig. 2 Effects of nobiletin and/or ACh on 14C-catecholamine synthesisfrom [14C]tyrosine (a) or [14C]DOPA (b) in the cells. a Cells (4×106/dish) were stimulated with (filled circle) or without (open circle)ACh (0.3 mM) at 37 °C for 20 min in the presence of variousconcentrations of nobiletin and L-[U-14C] tyrosine (20 μM, 1 μCi).The 14C-catecholamines formed were measured. b Cells were incubated
with or without nobiletin (100 μM) and/or ACh (0.3 mM) at 37 °C for15 min in the presence of [14C]DOPA (20 μM, 0.25 μCi) for 15 min.The 14C-catecholamines formed were measured. Data are expressed asthe mean±SEM of four experiments carried out in triplicate. *p <0.05and **p <0.01, compared with control; ***p <0.05, compared withACh alone
18 Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:15–22
Effects of protein kinase inhibitors, H-89 and KN-93,on tyrosine hydroxylase activity induced by nobiletin
Nobiletin (100 μM) enhanced tyrosine hydroxylase activityby 35 % over the control (Fig. 5). H-89 (10 μM) and KN-93 (10 μM) significantly suppressed the stimulatory effectof nobiletin on tyrosine hydroxylase activity, and H-89 plusKN-93 further inhibited it.
Phosphorylation sites of tyrosine hydroxylase inducedby nobiletin
Tyrosine hydroxylase is phosphorylated at Ser19, Ser31, andSer40 by CaM kinase II, ERK, and protein kinase A, respec-tively (Dunkley et al. 2004). To determine which site(s) is
phosphorylated by nobiletin, we studied the effect of nobiletinon the Ser19, Ser31, and Ser40 phosphorylation of tyro-sine hydroxylase in the cells. As shown in Fig. 6,nobiletin (100 μM) caused a significant increase inphosphorylation of Ser19 and Ser40, but not Ser31 oftyrosine hydroxylase, whereas dibutyryl cAMP (DB-cAMP) (2 mM) increased the phosphorylation of onlySer40 of this enzyme.
Discussion
In the present study, we demonstrated that nobiletin, byitself, stimulates basal 14C-catecholamine synthesis, but itinhibits the synthesis of 14C-catecholamines induced by
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Fig. 3 Effects of various concentrations of nobiletin on basal activity (a)and ACh-induced activity (b) of tyrosine hydroxylase in the cell. Cells(106/well) were stimulated with or without nobiletin (1–100 μM) for10 min at 37 °C and then incubated for another 10 min in the presence
of L-[1-14C]tyrosine (18 μM, 0.2 μCi) with ACh (0.3 mM) (b) or withoutACh (0.3 mM) (a), and tyrosine hydroxylase activity was measured. Dataare the mean±SEM of four separate experiments carried out in triplicate.*p <0.05, compared with control; **p <0.05, compared with ACh only
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Nobiletin
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Fig. 4 Effects of deprivation of extracellular Ca2+ (a) and variousinhibitors of protein kinases (b ) on 14C-catecholamine synthesisinduced by nobiletin. a Cells (4×106/dish) were incubated withL -[U-14C]tyrosine (20 μM, 1 μCi) in the presence or absence ofnobiletin (100 μM) and extracellular Ca2+ for 20 min at 37 °C. The14C-catecholamines formed were measured. b Cells were incubated
with or without nobiletin (100 μM) and/or H-89 (10 μM), U0126(10 μM), KN-93 (10 μM), and KN-92 (10 μM) for 20 min at 37 °C.The 14C-catecholamines formed were measured. Data are expressedas means±SEM of three separate experiments carried out in tripli-cate. *p <0.05, compared with control; **p <0.05, compared withnobiletin alone
Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:15–22 19
ACh in cultured bovine adrenal medullary cells. When[14C]DOPA was used as a substrate instead of [14C]ty-rosine, nobiletin failed to stimulate the synthesis of 14C-catecholamines, suggesting that the stimulation of 14C-catecholamine synthesis induced by nobiletin occurs
predominantly before the DOPA decarboxylase step,and probably at the tyrosine hydroxylase step. Indeed,nobiletin increased tyrosine hydroxylase activity in thecells. Based on these findings, it is likely that nobiletinactivates tyrosine hydroxylase and subsequently stimulatescatecholamine synthesis.
Stimulatory effect of nobiletin on basal 14C-catecholaminesynthesis
Incubation of the cells with nobiletin caused 14C-catechol-amine synthesis in a time (20–30 min)- and concentration(1.0–100 μM)-dependent manner. In our previous study(Zhang et al. 2010), nobiletin caused catecholamine secretionthrough an increase in intracellular Ca2+ via activation by theL- and N-types of voltage-dependent Ca2+ channels and par-tially the Na+/Ca2+ exchange system in bovine adrenal med-ullary cells. In the regulation of catecholamine synthesis, Ca2+
plays an indispensable role as the coupler in the stimulus-synthesis coupling (Yanagihara et al. 1987) as well as thestimulus-secretion coupling (Douglas and Rubin 1961,1963). In the present study, deprivation of extracellular Ca2+
abolished the catecholamine synthesis induced by nobiletin,suggesting a Ca2+-dependent process. Since nobiletin (1.0–100 μM) caused catecholamine secretion in a concentration-
0
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Fig. 5 Effects of inhibitors of protein kinases on nobiletin-inducedtyrosine hydroxylase activity. Cells (106/well) were stimulated with orwithout nobiletin (100 μM) and/or H-89 (10 μM), and KN-93(10 μM) for 10 min at 37 °C and then incubated for another 10 minwith or without nobiletin (100 μM) and/or H-89 (10 μM), and KN-93(10 μM) in the presence of L-[1-14C]tyrosine (18 μM, 0.2 μCi).Tyrosine hydroxylase activity was measured. *p <0.05, comparedwith control; **p <0.05, compared to nobiletin alone
total TH
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Fig. 6 Effect of nobiletin on tyrosine hydroxylase phosphorylation atSer19, Ser31, and Ser40. Cells (2×106/dish) were incubated with orwithout nobiletin (100 μM) or dibutyryl cAMP (DB-cAMP) (2 mM)for 10 min at 37 °C. Phosphorylation at the sites Ser 19, Ser31, andSer40 of tyrosine hydroxylase (TH) was determined and expressed as
% of control tyrosine hydroxylase band [the ratio of the density ofphospho-Ser19 (a), phospho-Ser31 (b ), and phospho-Ser40 (c ) of ty-rosine hydroxylase to that of the total tyrosine hydroxylase]. Data aremeans±SEM from three separate experiments carried out intriplicate.*p <0.05 and **p <0.01, compared with control
20 Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:15–22
dependent manner similar to that of 45Ca2+ influx (Zhang et al.2010), it is likely that nobiletin stimulates catecholaminesynthesis through Ca2+ influx in the cells.
Intracellular signals of phosphorylation and activationof tyrosine hydroxylase induced by nobiletin
It has been established that the activity of tyrosine hydrox-ylase is regulated by phosphorylation (Zigmond et al. 1989)at residues of Ser19, Ser31, and Ser40 by CaM kinase II,ERK, and protein kinase A, respectively (Bobrovskayaet al. 1998; Dunkley et al. 2004). In the present study,KN-93, an inhibitor of CaM kinase II, and H-89, an inhib-itor of protein kinase A, but not U0126, an inhibitor ofERK kinase, inhibited the stimulatory effects of nobiletinon catecholamine synthesis and tyrosine hydroxylase activ-ity. Indeed, Ca2+ was reported to activate a Ca2+/calmodu-lin-dependent isozyme of adenylate cyclase (Keogh andMarley 1991) and CaM kinase II in bovine adrenal medul-lary cells (Tsutsui et al. 1994; Yanagihara et al. 1994). Inaddition, nobiletin induced the phosphorylation of tyrosinehydroxylase at Ser19 and Ser40 in the cells. Previous studiesreported that high K+-induced depolarization causes theactivation of ERK which is associated with catecholaminesecretion in bovine adrenal medullary cells (Cox andParsons 1997; Mendoza et al. 2003). However, ERK maynot be involved in the stimulatory effect of nobiletin oncatecholamine synthesis, because (1) U0126, an inhibitor ofERK kinase, did not affect the stimulatory effect ofnobiletin on catecholamine synthesis and tyrosine hydrox-ylase activity and (2) nobiletin did not stimulate the phos-phorylation of tyrosine hydroxylase at Ser31. From thesefindings, it seems that nobiletin enhances the activity oftyrosine hydroxylase via the activation of CaM kinase IIand protein kinase A, which in turn, stimulates catechol-amine synthesis in the cells. To the best of our knowledge,this is the first direct evidence showing the stimulatoryeffects of nobiletin, a citrus polymethoxy flavone, on thephosphorylation of tyrosine hydroxylase at Ser19 and Ser40
and catecholamine synthesis.
Inhibitory effect of nobiletin on the catecholamine synthesisinduced by ACh
In the present study, nobiletin inhibited tyrosine hydroxy-lase activity as well as catecholamine synthesis induced byACh. We previously reported that nobiletin suppresses22Na+ influx and 45Ca2+ influx by inhibiting nAChR-ionchannels and voltage-dependent Ca2+ channels, respec-tively (Zhang et al. 2010). It is likely that nobiletinattenuates ACh-induced catecholamine synthesis throughthe inhibition of the Na+ influx and the subsequent Ca2+
influx.
Pharmacological significance of the effects of nobiletinon catecholamine synthesis
Nobiletin is one of the flavonoids isolated from the peel ofCitrus depressa , which is often used in a Chinese traditionalherbal medicine. Nogata et al. (2006) reported the contents ofnobiletin in various citrus fruits (total tissue, 0.4∼8.1mg/100 g;peel tissue, 1.5∼18.5 mg/100 g; juice vesicle tissue, 0∼0.9 mg/100 g). Flavonoids have been reported to have diverse phar-macological actions such as neurotrophic and antidementiaactivities (Nagase et al. 2005a, b; Nakajima et al. 2007).Nobiletin also restores β-amyloid-impaired cAMP responseelement binding protein (CREB) phosphorylation and rescuesmemory deterioration in Alzheimer’s disease model rats(Matsuzaki et al. 2006). In the present study, nobiletin stimu-lated the phosphorylation and activation of tyrosine hydroxy-lase probably through CaM kinase II and protein kinase A.These findings suggest that nobiletin enhances the synthesis ofcatecholamines such as noradrenaline and dopamine in brainnoradrenergic and dopaminergic neurons as well as peripheralsympathetic neurons.
The present results further demonstrated that nobiletin in-hibits the catecholamine synthesis induced by ACh, a physio-logical secretagogue. Several lines of evidence have shown thatchronic stress responses can be associated with disease symp-toms such as peptic ulcer or cardiovascular disorders(Goldstein et al. 2003) and lead to DNA damage (Freedmanand Lefkowitz 2004; Antoni et al. 2006). Hara et al. (2011)reported that the stress hormone adrenaline stimulates β2-adrenoceptors which induces the Gs-protein-dependent activa-tion of protein kinase A and the β-arrestin-mediated signalingpathway, and then triggers DNA damage and suppresses p53levels. Taken together, it gives rise to the possibility thatnobiletin, by itself, enhances catecholamine synthesis in restingor normal states, whereas it suppresses the hyperactive cate-cholamine system induced by prolonged stress or emotionalexcitation which evokes the secretion of ACh from the splanch-nic nerves and stimulates a massive secretion of catechol-amines from the adrenal medulla. Although the clinical signif-icance of present findings showing the dual effects of nobiletinon catecholamine synthesis and secretion remains to be deter-mined, the findings may add to our understanding of nobiletinfunctions by describing new pharmacological actions of a citrusflavone, nobiletin. To confirm the effects of nobiletin on cate-cholamine synthesis and secretion, further in vivo studies of theadministration of nobiletin to animals or humans are required.
In summary, we have demonstrated that nobiletin stimulatescatecholamine synthesis through the phosphorylation of tyro-sine hydroxylase at Ser19 and Ser40, whereas it inhibits thecatecholamine synthesis induced by ACh in adrenal medullarycells and probably in sympathetic neurons. The present find-ings also add to our understanding of nobiletin’s pharmaco-logical mechanism(s) in traditional Chinese herbal medicine.
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Acknowledgments This research was supported, in part, by Grant-in-Aids (23617035, 23590159, 23617036, and 24890286) for ScientificResearch (C) from the Japan Society for the Promotion of Science.
Conflict of interest The authors have no conflict of interest to report.
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