Echinacoside Inhibits Glutamate Release by Suppressing Voltage-Dependent Ca2+ Entry and Protein Kinase C in Rat Cerebrocortical Nerve Terminals

International Journal of Molecular Sciences

Article Echinacoside Inhibits Glutamate Release by Suppressing Voltage-Dependent Ca2+ Entry and Protein Kinase C in Rat Cerebrocortical Nerve Terminals

Cheng Wei Lu 1,2, Tzu Yu Lin 1,2, Shu Kuei Huang 1 and Su Jane Wang 3,*

1 Department of Anesthesiology, Far-Eastern Memorial Hospital, Pan-Chiao District, New Taipei City 22060, Taiwan; [email protected] (C.W.L.); [email protected] (T.Y.L.); [email protected] (S.K.H.) 2 Department of Mechanical Engineering, Yuan Ze University, Taoyuan 32003, Taiwan 3 School of Medicine, Fu Jen Catholic University, No. 510, Zhongzheng Rd., Xinzhuang Dist., New Taipei 24205, Taiwan * Correspondence: [email protected]; Tel.: +886-2-29-053-465

Academic Editor: Katalin Prokai-Tatrai Received: 10 May 2016; Accepted: 20 June 2016; Published: 24 June 2016

Abstract: The glutamatergic system may be involved in the effects of neuroprotectant therapies. Echinacoside, a phenylethanoid glycoside extracted from the medicinal Chinese herb Herba Cistanche, has neuroprotective effects. This study investigated the effects of echinacoside on 4-aminopyridine-evoked glutamate release in rat cerebrocortical nerve terminals (synaptosomes). Echinacoside inhibited Ca2+-dependent, but not Ca2+-independent, 4-aminopyridine-evoked glutamate release in a concentration-dependent manner. Echinacoside also reduced the 4-aminopyridine-evoked increase in cytoplasmic free Ca2+ concentration but did not alter the synaptosomal membrane potential. The inhibitory effect of echinacoside on 4-aminopyridine-evoked glutamate release was prevented by ω-conotoxin MVIIC, a wide-spectrum blocker of Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels, but was insensitive to the intracellular Ca2+ release-inhibitors dantrolene and 7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157). Furthermore, echinacoside decreased the 4-aminopyridine-induced phosphorylation of protein kinase C, and protein kinase C inhibitors abolished the effect of echinacoside on glutamate release. According to these results, we suggest that the inhibitory effect of echinacoside on evoked glutamate release is associated with reduced voltage-dependent Ca2+ entry and subsequent suppression of protein kinase C activity.

Keywords: echinacoside; glutamate release; cerebrocortical nerve terminals; voltage-dependent Ca2+ channels; protein kinase C

1. Introduction Echinacoside is a major phenylenthanoid glycoside present in Herba Cistanche, a well-known traditional Chinese medicine used to treat forgetfulness, impotency, and chronic constipation [1]. Echinacoside possesses various bioactivities such as antioxidation, anti-inﬂammation, anticancer, hepatoprotection, and immune modulation [2–4]. Notably, echinacoside has neuroprotective effects; for example, it can protect against oxidative stress- or neurotoxin-induced neurotoxicity in primary rat cortical neurons, human neuroblastoma SH-SY5Y cells, and pheochromocytoma (PC12) cells [5–8]. Furthermore, echinacoside attenuates brain damage and improves cognitive function in animal models

Int. J. Mol. Sci. 2016, 17, 1006; doi:10.3390/ijms17071006 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2016, 17, 1006 2 of 13 of Parkinson’s disease, Alzhermer’s disease, and middle cerebral artery occlusion [9–12]. However, the mechanism through which echinacoside induces neuroprotection is not fully understood. Neuroprotection is a complex process of preserving neuronal structure and function upon toxic insults. Glutamate excitotoxicity reduction is considered a potential mechanism involved in brain neuroprotection. Glutamate, an excitatory amino acid neurotransmitter, has a crucial role in several brain functions [13]. However, overactivation of glutamate receptors under high glutamate concentrations causes intracellular Ca2+ overload, mitochondrial dysfunction, free radical production, and neuronal death [14,15]. This pathological process is implicated in numerous brain disorders including cerebral ischemia, traumatic brain injury, epilepsy, and neurodegenerative disease [16,17]. Hence, inhibitors blocking pathophysiological glutamatergic transmission are considered a potential neuroprotective drugs. Notable examples of these are glutamate receptor antagonists [18,19]; however, clinical trials for these drugs have failed because of less effectivity and undesired, or even cytotoxic side effects [20,21]. In addition to direct glutamate receptor blockade, glutamate release inhibition may be an effective strategy for neuroprotection. Several neuroprotectants (e.g., memantine and riluzole) can reduce glutamate release in rat brain tissues [22–24]. Considering the role of glutamate in excitotoxicity and the neuroprotective profile of echinacoside, the present study used isolated nerve terminals (synaptosomes) purified from the rat cerebral cortex to investigate the effect of echinacoside on glutamate release and further explored potential mechanisms. The isolated nerve terminal preparation is a well-established model for studying the presynaptic regulation of neurotransmitter release by drugs in the absence of any postsynaptic effects [25]. By using this model, we evaluated the effect of echinacoside on glutamate release, membrane potential, presynaptic Ca2+ influx, and protein kinase C activity. According to our review of the literature, this is the first report documenting the mechanism through which echinacoside inhibits endogenous glutamate release at the presynaptic level.

2. Results

2.1. Echinacoside Inhibits 4-Aminopyridine-Evoked Glutamate Release from Rat Cerebrocortical Nerve Terminals by Reducing Vesicular Exocytosis Figure1 illustrates the concentration-dependent effect of echinacoside on 4-aminopyridine-evoked glutamate release from purified rat cerebrocortical synaptosomes. In synaptosomes incubated with 1 mM CaCl2, 1 mM 4-aminopyridine evoked a glutamate release of 7.4 ˘ 0.1 nmol/mg/5 min, which was reduced by 1, 5, 10, 30, and 50 µM echinacoside to 6.5 ˘ 0.2, 5.8 ˘ 0.3, 4.8 ˘ 0.2, 4.1 ˘ 0.1, or 2.3 ˘ 0.4 nmol/mg/5 min, respectively (F(5,24) = 67.1, p = 0.000). The IC50 value for echinacoside-mediated inhibition of 4-aminopyridine-evoked glutamate release, derived from a dose-response curve, was 24 µM. Moreover, the glutamate release evoked by 1 mM 4-aminopyridine in an extracellular Ca2+-free solution containing 300 µM ethylene glycol bis(β-aminoethyl ether)-N,N,N1,N1-tetraacetic acid (EGTA) was 2.1 ˘ 0.2 nmol/mg/5 min (F(2,12) = 310.65, p = 0.000), and this Ca2+-independent component of 4-aminopyridine-evoked glutamate release was unaffected by 20 µM echinacoside (1.8 ˘ 0.2 nmol/mg/5 min; p = 0.58; Figure1). In synaptosomes treated with 0.1 µM bafilomycin A1, a vesicular transporter inhibitor [26], 4-aminopyridine-evoked glutamate release was reduced significantly (2.2 ˘ 0.2 nmol/mg/5 min; F (2,12) = 249.518, p = 0.000). In the presence of bafilomycin A1, 20 µM echinacoside failed to significantly inhibit the release of glutamate (2.1 ˘ 0.2 nmol/mg/5 min; p = 0.94; Figure1). By contrast, 10 µM DL-threo-beta-benzyl-oxyaspartate (DL-TBOA, a glutamate reuptake inhibitor) [27], increased 4-aminopyridine-evoked glutamate release to 11.8 ˘ 0.4 nmol/mg/5 min (t(8) = ´11.31, p = 0.000). Even in the presence of DL-TBOA, 20 µM echinacoside inhibited 4-aminopyridine-evoked glutamate release significantly (7.7 ˘ 0.2 nmol/mg/5 min; F(2,12) = 87.23, p = 0.000; Figure1). Int.Int. J. J. Mol. Mol. Sci. Sci.2016 2016, ,17 17,, 1006 1006 33 of of 13 13

15 6

4 ∗∗ 12 IC50 = 24 μM 2

Glutamate release (nmol/mg/5 min) min) (nmol/mg/5 release Glutamate -6 -5 -4 9 Con log[Echinacoside] (M) # ∗ ∗∗ 6 ∗∗ ∗∗ ∗∗ 3 ∗∗ ∗∗ ∗∗ ∗∗

0 Glutamate release (nmol/mg/5 min) 1 4-aminopyridine (mM) + + + + + + - - + + + + CaCl2 (1.2 mM) - 1 5 10 30 50 - 20 - 20 - 20 Echinacoside (μM) ------+ + - - - - EGTA (300 μM) ------+ + - - DL-TBOA (10 μM) ------+ + Bafilomycin A1 (0.1 μM)

Figure 1. Echinacoside inhibits 4-aminopyridine-evoked glutamate release from rat cerebrocortical nerveFigure terminals 1. Echinacoside via the Cainhibits2+-dependent 4-aminopyridine-evoked exocytotic component. glutamate Glutamate release releasefrom rat was cerebrocortical evoked by 2+ 1nerve mM 4-aminopyridine terminals via the in Ca the absence-dependent (control) exocytotic or presence component. of echinacoside Glutamate (1, release 5, 10, 30,was and evoked 50 µM), by 1 mM 4-aminopyridine in the absence (control) or presence1 1 of echinacoside (1, 5, 10, 30, and 50 µM), 300 µM ethylene glycol bis(β-aminoethyl ether)-N,N,N ,N -tetraacetic acid (EGTA) (omitting CaCl2), 300 µM ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) (omitting CaCl2), 300 µM EGTA (omitting CaCl2) and 20 µM echinacoside, 10 µM DL-threo-beta-benzyl-oxyaspartate (DL300-TBOA), µM EGTA 10 µ M(omittingDL-TBOA CaCl and2) and 20 µ M20 echinacoside,µM echinacoside, 0.1 µ M10 bafilomycinµM DL-threo-beta-benzyl-oxyaspartate A1, or 0.1 µM bafilomycin A1(DL and-TBOA), 20 µ M10 echinacoside. µM DL-TBOA Echinacoside and 20 µM was echinacoside, added 10 min 0.1 beforeµM bafilomycin depolarization A1, and,or 0.1 EGTA, µM DLbafilomycin-TBOA or bafilomycinA1 and 20 µM A1, echinacoside. 20 min prior Echinacoside to this. Results was areadded mean 10 ˘minS.E.M. before of depolarization 4–6 independent and, synaptosomalEGTA, DL-TBOA preparations. or bafilomycin *, p < 0.01,A1, 20 **, minp < 0.001priorversus to this. control Results group. are mean #, p < ± 0.05 S.E.M. versus of the4–6 DLindependent-TBOA-treated synaptosomal group. preparations. *, p < 0.01, **, p < 0.001 versus control group. #, p < 0.05 versus the DL-TBOA-treated group. 2.2. Echinacoside Reduces Cytosolic Ca2+ Concentration but Does Not Alter the Synaptosomal Membrane2.2. Echinacoside Potential Reduces Cytosolic Ca2+ Concentration but Does Not Alter the Synaptosomal Membrane Potential Synaptosome depolarization caused by 1 mM 4-aminopyridine increased Ca2+ concentration (p = 0.000;Synaptosome Table1). depolarization The application caused of 20 µbyM 1 echinacoside mM 4-aminopyridine did not significantly increased Ca affect2+ concentration basal Ca2+ concentration(p = 0.000; Table (t(8) 1). = 0.06,The papplication= 0.95) but of significantly 20 µM echinacoside reduced the did 4-aminopyridine-induced not significantly affect basal increase Ca2+ inconcentration Ca2+ concentration (t(8) = 0.06, (t(10) p = 0.95) 6.16, butp = significantly 0.000). In addition, reduced 1the mM 4-aminopyridine-induced 4-aminopyridine increased increase in in Ca2+ concentration (t(10) = 6.16, p = 0.000). In addition, 1 mM 4-aminopyridine increased in 3’,3’,3’-dipropylthiadicarbocyanine iodide [DiSC3(5)] fluorescence (p = 0.000). The addition of 20 µM echinacoside3’,3’,3’-dipropylthiadicarbocyanine did not alter the resting membraneiodide [DiSC potential3(5)] fluorescence (t(8) = 0.976, (pp == 0.000). 0.36)or The significantly addition of change 20 µM echinacoside did not alter the resting membrane potential (t(8) = 0.976, p = 0.36) or significantly the 4-aminopyridine-mediated increase in DiSC3(5) fluorescence (t(8) = ´0.014, p = 0.99; Table1). change the 4-aminopyridine-mediated increase in DiSC3(5) fluorescence (t(8) = −0.014, p = 0.99; TableTable 1). 1. Effect of echinacoside on membrane potential and cytosolic Ca2+ levels from rat cortical synaptosomes. Table 1. Effect of echinacoside on membrane potential and cytosolic Ca2+ levels from rat cortical synaptosomes.Membrane Potential (Fluorescence Units) Cytosolic [Ca2+] (nM)

BasalMembrane 4-Aminopyridine Potential (Fluore (1scence mM) Units) Basal Cytosolic 4-Aminopyridine [Ca2+] (nM) (1 mM) Control 5.6 ˘ 0.3 14.8 ˘4-Aminopyridine0.4 144.6 ˘ 3.14-Aminopyridine 219.3 ˘ 6.9 Basal Basal Echinacoside 5.2 ˘ 0.3 14.8 ˘ 0.4(1 mM) 143.7 ˘ 3.5 176.5 ˘(16.7 mM) ** EachControl value is mean ˘ S.E.M. 5.6 ± of0.3 independent experiments 14.8 ± using 0.4 synaptosomal 144.6 preparations ± 3.1 from 219.35–6 animals. ± 6.9 **, p < 0.001 versus the control group. Echinacoside 5.2 ± 0.3 14.8 ± 0.4 143.7 ± 3.5 176.5 ± 6.7 ** Each value is mean ± S.E.M. of independent experiments using synaptosomal preparations from 5–6 animals. **, p < 0.001 versus the control group.

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2.3. ReducedInt. J. Mol. Ca Sci.2+ 2016Influx, 17, 1006 through the Cav2.2 (N-Type) and Cav2.1 (P/Q-Type) Channels May Be Associated4 of 13 with the Inhibition of 4-Aminopyridine-Evoked Glutamate Release by Echinacoside 2.3. Reduced Ca2+ Influx through the Cav2.2 (N-Type) and Cav2.1 (P/Q-Type) Channels May Be Associated 2+ Figurewith the2 Inhibitionshows thatof 4-Am 2 inopyridine-EvokedµM ω-conotoxin Glutamat MVIIC,e Release an N- by and Echinacoside P/Q-type Ca channel blocker, reduced 4-aminopyridine-evoked glutamate release from 7.4 ˘ 0.2 to 2.0 ˘ 0.1 nmol/mg/5 min Figure 2 shows that 2 µM ω-conotoxin MVIIC, an N- and P/Q-type Ca2+ channel blocker, (t(9) = 25.35, p = 0.000). In the presence of ω-conotoxin MVIIC, the effect of 20 µM echinacoside on reduced 4-aminopyridine-evoked glutamate release from 7.4 ± 0.2 to 2.0 ± 0.1 nmol/mg/5 min 4-aminopyridine-evoked(t(9) = 25.35, p = 0.000). glutamate In the presence release ofwas ω-conotoxin nonsignificant MVIIC, (1.8the ˘effect0.2 of nmol/mg/5 20 µM echinacoside min; t(8) on = 1.06, 2+ p = 0.32).4-aminopyridine-evoked Dantrolene (10 µglutamateM), an inhibitor release was of nonsignificant intracellular (1.8 Ca ± 0.2release nmol/mg/5 from min; the t(8) endoplasmic = 1.06, reticulump = 0.32). [28], Dantrolene reduced 4-aminopyridine-evoked (10 µM), an inhibitor of intracellular glutamate Ca release2+ release (5.6 from˘ 0.3 the nmol/mg/5 endoplasmic min; F(2,14)reticulum = 104.95, [28],p =reduced 0.000). 4-aminopyridine-evoked However, in the presence glutamate of dantrolene, release (5.6 20 ± µ0.3M echinacosidenmol/mg/5 min; could still inhibitF(2,14) glutamate= 104.95, p = release 0.000). However, significantly in the (3.3 presence˘ 0.2 of nmol/mg/5 dantrolene, 20 min; µMp echinacoside= 0.000). Similar could still results were observedinhibit glutamate using 100releaseµM significantly 7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (3.3 ± 0.2 nmol/mg/5 min; p = 0.000). Similar results were (CGP37157),observed a membrane-permeableusing 100 µM 7-chloro-5-(2-chloroph blocker of mitochondrialeny)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one Na+/Ca2+ exchange. In the five (CGP37157), a membrane-permeable blocker of mitochondrial Na+/Ca2+ exchange. In the five examined synaptosomal preparations, 20 µM echinacoside combined with 100 µM CGP37157 reduced examined synaptosomal preparations, 20 µM echinacoside combined with 100 µM CGP37157 4-aminopyridine-evoked glutamate release by 48.3% ˘ 5.2% (F(2,13) = 136.79, p = 0.000), similar to the reduced 4-aminopyridine-evoked glutamate release by 48.3% ± 5.2% (F(2,13) = 136.79, p = 0.000), inhibitionsimilar by to echinacoside the inhibition aloneby echinacoside (46.2% ˘ alone2.3%; (46.2%p = 0.89; ± 2.3%; Figure p =2 0.89;). Figure 2).

∗∗ 6 ∗∗

∗∗

4 # # ∗∗ ∗∗

∗∗ ∗∗ 2

0 Glutamate release (nmol/mg/5min) 1 4-aminopyridine (mM) + + + + + + + + CaCl2 (1.2 mM) - + - + - + - + Echinacoside (20 μM) - - + + - - - - ω -conotoxin MVIIC (2 μM) - - - - + + - - Dantrolene (50 μM) ------+ + CGP37157 (100 μM)

FigureFigure 2. 2.Effects Effects of ofω -conotoxinω-conotoxin MVIIC,MVIIC, dantrolene,dantrolene, an andd 7-chloro-5-(2-chloropheny)-1,5- 7-chloro-5-(2-chloropheny)-1,5- dihydro-4,1-benzothiazepin-2(3H)-onedihydro-4,1-benzothiazepin-2(3H)-one (CGP37157)(CGP37157) on on the the echinacoside-mediated echinacoside-mediated inhibition inhibition of of 4-aminopyridine-evoked4-aminopyridine-evoked glutamate glutamate release. release. Glutamate Glutamate release release waswas evoked by by 1 1 mM mM 4-aminopyridine 4-aminopyridine in the absencein the (control)absence (control) or presence or presence of 20 µM of echinacoside, 20 µM echinacoside, 2 µM ω-conotoxin 2 µM ω-conotoxin MVIIC, 2MVIIC,µM ω -conotoxin2 µM MVIICω-conotoxin and 20 µM MVIIC echinacoside, and 20 10µMµ Mechinacoside, dantrolene, 10 10 µMµM dantrolene, dantrolene 10 and µM 20 dantroleneµM echinacoside, and 20 µM 100 µM CGP37157,echinacoside, or 100 100µM µM CGP37157 CGP37157, and or 20100µ µMM echinacoside.CGP37157 and Echinacoside20 µM echinacoside. was addedEchinacoside 10 min was before added 10 min before depolarization and, ω-conotoxin MVIIC, dantrolene or CGP37157, 30 min prior depolarization and, ω-conotoxin MVIIC, dantrolene or CGP37157, 30 min prior to this. Results are to this. Results are mean ± S.E.M. of 5–6 independent synaptosomal preparations. **, p < 0.001 versus mean ˘ S.E.M. of 5–6 independent synaptosomal preparations. **, p < 0.001 versus control group. control group. #, p < 0.05 versus the dantrolene- or CGP37157-treated group. #, p < 0.05 versus the dantrolene- or CGP37157-treated group. 2.4. Echinacoside Inhibits 4-Aminopyridine-Evoked Glutamate Release by Signaling through Protein Kinase C 2.4. Echinacoside Inhibits 4-Aminopyridine-Evoked Glutamate Release by Signaling through Protein Kinase C As illustrated in Figure 3, 10 µM 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) Asmaleimide illustrated (GF109203X), in Figure a general3, 10 proteinµM kinase 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) C inhibitor [29], reduced 4-aminopyridine-evoked maleimideglutamate (GF109203X), release (F(2,13) a general = 19.46, protein p = kinase0.000). CIn inhibitorthe GF109203X-treated [29], reduced synaptosomes, 4-aminopyridine-evoked 20 µM ± glutamateechinacoside release reduced (F(2,13) 4-amin = 19.46,opyridine-evokedp = 0.000). glutamate In the GF109203X-treated release by only 5.5% synaptosomes, 1.8% (p = 0.89), less 20 µM than that by echinacoside alone (42.4% ± 2.3%; p = 0.000). Similar results were obtained with echinacoside reduced 4-aminopyridine-evoked glutamate release by only 5.5% ˘ 1.8% (p = 0.89), 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile less than that by echinacoside alone (42.4% ˘ 2.3%; p = 0.000). Similar results were obtained (Go6976), a selective for Ca2+-dependent protein kinase C isoforms (α, βI, βII, γ) [29]. In the presence with 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile

(Go6976), a selective for Ca2+-dependent protein kinase C isoforms (α, βI, βII, γ)[29]. In the Int. J. Mol. Sci. 2016, 17, 1006 5 of 13

Int. J. Mol. Sci. 2016, 17, 1006 5 of 13 presence of 3 µM Go6976, 20 µM echinacoside reduced glutamate release by 11.9% ˘ 2.9% (p = 0.59),of 3signifying µM Go6976, a signiﬁcant 20 µM echinacoside reduction comparedreduced glutamate with that release by echinacoside by 11.9% alone± 2.9%(42.4% (p = 0.59),˘ 2.3%; 2+ p = 0.000;signifying Figure a significant3). By reduction contrast, compared 3 µM with rottlerin, that by echinacoside a Ca -independent alone (42.4% protein ± 2.3%; p kinase = 0.000; Cδ inhibitorFigure [30 ],3). didBy contrast, not signiﬁcantly 3 µM rottlerin, alter a Ca 4-aminopyridine2+-independent protein (1 mM)-evokedkinase Cδ inhibitor glutamate [30], did release not (p = 0.45).significantlyNevertheless, alter 4-aminopyridine in the presence (1 mM)-evoked of rottlerin, glutamate 20 µM release echinacoside (p = 0.45). effectively Nevertheless, caused in the an averagepresence inhibition of rottlerin, of 37.1% 20 µM˘ 5.6%echinacoside of the effectivel release (F(2,13)y caused an = 19.72,averagep inhibition= 0.000), of similar 37.1% ± to5.6% that of by echinacosidethe release alone (F(2,13) (p = 0.41;= 19.72, Figure p = 30.000),). In addition,similar to thethat mitogen-activatedby echinacoside alone protein (p = 0.41; kinase Figure inhibitor 3). 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one)In addition, the mitogen-activated protein kinase (PD98059) inhibitor (50 2-(2-amiµM) andno-3-methoxyphenyl)- the protein kinase A 4H-1-benzopyran-4-one) (PD98059) (50 µM) and the protein kinase A inhibitor inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89) (100 µM) reduced N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89) (100 µM) reduced µ 4-aminopyridine-evoked4-aminopyridine-evoked glutamate glutamate release release (p = 0.000).(p = 0.000). Even Even in the in presencethe presence of PD98059 of PD98059 or H89, or H89, 20 M echinacoside20 µM echinacoside effectively reduced effectively the re releaseduced the (F(2,13) release = (F(2,13) 52.3, p == 52.3, 0.000; p = Figure0.000; Figure3). 3).

∗ 6 ∗∗ ∗∗ ∗∗ # ∗∗ ∗∗ # 4 ∗∗ # ∗∗ ∗∗ ∗∗

0 Glutamaterelease (nmol/mg/5 min) 1 4-aminopyridine (mM) + + + + + + + + + + + + CaCl2 (1.2 mM) - + - + - + - + - + - + Echinacoside (20 μM) - - + + ------GF109203X (10 μM) - - - - + + ------Go6976 (3 μM) ------+ + - - - - Rottlerin (3 μM) ------+ + - - H89 (100 μM) ------+ μ + PD98059 (50 M)

FigureFigure 3. Echinacoside-mediated 3. Echinacoside-mediated inhibition inhibition of 4-aminopyridine-evokedof 4-aminopyridine-evoked glutamate release release is isblocked blocked by proteinby protein kinase Ckinase inhibitors. C inhibitors. Glutamate Glutamate release releas wase evoked was evoked by 1 mMby 1 4-aminopyridine mM 4-aminopyridine in the in absence the (control)absence or presence(control) or of presence 20 µM of 20 echinacoside, µM echinacoside, 10 10µM µM2-[1-(3-dimethylaminopropyl)indol-3- 2-[1-(3-dimethylaminopropyl)indol-3- yl]-3-(indol-3-yl)yl]-3-(indol-3-yl) maleimide maleimide (GF109203X), (GF109203X), 10 10 µMµM GF109203X GF109203X and 20 and µM 20 echinacoside,µM echinacoside, 3 µM 3 µM 5,6,7,13-tetrahydro-13-methyl-5-oxo-125,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-H-indolo[2,3-a]pyrrolo[3,4-a]pyrrolo[3,4-c]carbazole-12-propanenitrilec]carbazole-12-propanenitrile (Go6976), (Go6976),3 µM 3 µ MGo6976 Go6976 and and 20 20µMµ Mechinacoside, echinacoside, 3 µM 3 µ Mrottlerin, rottlerin, 3 µM 3 µ Mrottlerin rottlerin and and 20 20µMµ Mechinacoside, echinacoside, 100 µM N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), 100 µM H89 and 100 µM N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), 100 µM H89 and 20 µM echinacoside, 50 µM PD98059, or 50 µM PD98059 and 20 µM echinacoside. Echinacoside 20 µM echinacoside, 50 µM PD98059, or 50 µM PD98059 and 20 µM echinacoside. Echinacoside was was added 10 min before depolarization and, GF109203X, Go697, rottlerin, H89 or PD98059, 30 min added 10prior min to beforethis. Results depolarization are mean and,± S.E.M. GF109203X, of 5–6 independent Go697, rottlerin, synaptosomal H89 or preparations. PD98059, 30 *, min p < prior0.01, to this. Results**, p < are0.001 mean versus˘ controlS.E.M. group. of 5–6 #, independent p < 0.05 versus synaptosomal the rottlerin-, preparations.H89-, or PD98059-treated *, p < 0.01, group. **, p < 0.001 versus control group. #, p < 0.05 versus the rottlerin-, H89-, or PD98059-treated group. Figure 4 shows that 1 mM 4-aminopyridine increased the phosphorylation of protein kinase C Figurein synaptosomes4 shows that ( 1t(4) mM = 4-aminopyridine−6.871, p = 0.002). increased When synaptosomes the phosphorylation were pretreated of protein with kinase 20 µM C in echinacoside for 10 min before the addition of 4-aminopyridine, 4-aminopyridine induced synaptosomes (t(4) = ´6.871, p = 0.002). When synaptosomes were pretreated with 20 µM echinacoside phosphorylation of protein kinase C considerably decreased (F(2,6) = 29.202, p = 0.001). for 10 min before the addition of 4-aminopyridine, 4-aminopyridine induced phosphorylation of protein2.5. kinase Echinacoside-Mediated C considerably Inhibition decreased of Glutam (F(2,6)ate = 29.202,Release Doesp = 0.001).Not Involve a Gamma-Aminobutyric Acid Type A (GABAA) Receptor 2.5. Echinacoside-Mediated Inhibition of Glutamate Release Does Not Involve a Gamma-Aminobutyric Acid Type A (GABAIn FigureA) Receptor 5, the effect of echinacoside on 4-aminopyridine-evoked glutamate release in the absence or presence of SR95531 (an antagonist of the GABAA receptor) was compared. In addition, In100 Figure µM 5SR95531, the effect did not of significantly echinacoside alter on 4-aminopyridine 4-aminopyridine-evoked (1 mM)-evoked glutamate glutamate release release. in In the absence or presence of SR95531 (an antagonist of the GABA receptor) was compared. In addition, A 100 µM SR95531 did not signiﬁcantly alter 4-aminopyridine (1 mM)-evoked glutamate release. In the Int. J. Mol. Sci. 2016, 17, 1006 6 of 13

Int. J. Mol. Sci. 2016, 17, 1006 6 of 13 SR95531-treated synaptosomes, application of 20 µM echinacoside resulted in a 43% inhibition on 4-aminopyridine-evokedInt.the J. SR95531-treated Mol. Sci. 2016, 17, 1006 synapt glutamateosomes, releaseapplication (F(2,12) of 20 = µM 42.63, echinap =coside 0.000), resulted which in was a 43% not inhibition signiﬁcantly6 of 13 on 4-aminopyridine-evoked glutamate release (F(2,12) = 42.63, p = 0.000), which was not significantly differentthe SR95531-treated from the inhibition synapt producedosomes, application by echinacoside of 20 µM alone echina (40%;cosidep resulted= 0.000). in Aa 43% similar inhibition result was different from the inhibition produced by echinacoside alone (40%; p = 0.000). A similar result was obtainedon 4-aminopyridine-evoked with another GABAA glutamatereceptor release antagonist, (F(2,12) bicuculline = 42.63, p = (50 0.000),µM). which The was release not significantly measured in the obtained with another GABAA receptor antagonist, bicuculline (50 µM). The release measured in the presencedifferent of bicuculline from the inhibition and echinacoside produced wasby echinacoside signiﬁcantly alone different (40%; fromp = 0.000). that obtainedA similar inresult the was presence presence of bicuculline and echinacoside was significantly different from that obtained in the of bicucullineobtained with alone another (p = 0.000). GABAA receptor antagonist, bicuculline (50 µM). The release measured in the presence of bicuculline alone (p = 0.000). presence of bicuculline and echinacoside was significantly different from that obtained in the presence of bicuculline alone (p = 0.000). p-PKC

p-PKCPKC

PKC

Figure 4. Echinacoside decreases 4-aminopyridine-induced phosphorylation of protein kinase C Figure 4. Echinacoside decreases 4-aminopyridine-induced phosphorylation of protein kinase C (PKC). Phosphorylation of PKC was detected in synaptosomal lysates by Western blotting using (PKC).Figure Phosphorylation 4. Echinacoside of decreases PKC was 4-aminopyridine-in detected in synaptosomalduced phosphorylation lysates by of Western protein blottingkinase C using anti-phospho-PKC (pan) antibody. Synaptosomes were incubated for 2 min in 4-(2-hydroxyethyl)-1- anti-phospho-PKC(PKC). Phosphorylation (pan) antibody. of PKC was Synaptosomes detected in synaptosomal were incubated lysates for 2by min Western in 4-(2-hydroxyethyl)-1- blotting using piperazineethanesulfonic acid (HEPES) buffer medium that contained 1.2 mM CaCl2 at 37 °C in the anti-phospho-PKC (pan) antibody. Synaptosomes were incubated for 2 min in 4-(2-hydroxyethyl)-1-˝ piperazineethanesulfonicabsence (control) or presence acid (HEPES) of 1 mM buffer 4-amin mediumopyridine, that or 1 contained mM 4-aminopyridine 1.2 mM CaCl and2 at20 37µM C in piperazineethanesulfonic acid (HEPES) buffer medium that contained 1.2 mM CaCl2 at 37 °C in the the absenceechinacoside (control) added or 10 presence min before of the 1 mM addition 4-aminopyridine, of 4-aminopyridine. or 1 Results mM 4-aminopyridine are the mean ± S.E.M. and of 20 µM absence (control) or presence of 1 mM 4-aminopyridine, or 1 mM 4-aminopyridine and 20 µM echinacosidefive independent added10 synaptos min beforeomal preparations. the addition *, of p4-aminopyridine. < 0.01 versus control Results group. are#, p the < 0.05 mean versus˘ S.E.M. the of echinacoside added 10 min before the addition of 4-aminopyridine. Results are the mean ± S.E.M. of ﬁve independent4-aminopyridine-treated synaptosomal group. preparations. *, p < 0.01 versus control group. #, p < 0.05 versus the five independent synaptosomal preparations. *, p < 0.01 versus control group. #, p < 0.05 versus the 4-aminopyridine-treated group. 4-aminopyridine-treated group. 8

8 6

# # 6 ∗∗ ∗∗ ∗∗ 4 # # ∗∗ ∗∗ ∗∗ 4 2

2 0 Glutamate release (nmol/mg/5min) 1 4-aminopyridine (mM) 0 + + + + + + CaCl2 (1.2 mM) Glutamate release (nmol/mg/5min) - + - 1 + - + 4-aminopyridineEchinacoside (20 (mM) μM) - - + + - - μ + + + + + + CaClSR955312 (1.2 (100 mM) M) - - - - + + μ - + - + - + EchinacosideBicuculline (50 (20 M) μM) - - + + - - SR95531 (100 μM) - - - - + + μ Figure 5. Effect of the gamma-Aminobutyric acid A (GABAA) receptorBicuculline antagonists (50 M) SR95531 and bicuculline on the echinacoside-mediated inhibition of glutamate release. Glutamate release was Figure 5. Effect of the gamma-Aminobutyric acid A (GABAA) receptor antagonists SR95531 and Figureevoked 5. Effect by 1 mM of the 4-aminopyridine gamma-Aminobutyric in the absence acid (con Atrol) (GABA or in Athe) receptorpresence of antagonists 20 µM echinacoside, SR95531 and bicuculline on the echinacoside-mediated inhibition of glutamate release. Glutamate release was bicuculline100 µM onSR95531, the echinacoside-mediated 100 µM SR95531 and 20 µM inhibition echinacosi ofde, glutamate 50 µM bicuculline, release. or Glutamate 50 µM bicuculline release was evoked by 1 mM 4-aminopyridine in the absence (control) or in the presence of 20 µM echinacoside, evokedand by 20 1µM mM echinacoside. 4-aminopyridine Results inare the mean absence ± S.E.M. (control) of 5 independent or in the presence synaptosomal of 20 µpreparations.M echinacoside, 100 µM SR95531, 100 µM SR95531 and 20 µM echinacoside, 50 µM bicuculline, or 50 µM bicuculline µ**, p < 0.001 versusµ control group. #, p < 0.05µ versus the SR95531-, µor bicuculline-treated group.µ 100 andM SR95531, 20 µM echinacoside. 100 M SR95531 Results and are 20meanM ± echinacoside,S.E.M. of 5 independent 50 M bicuculline, synaptosomal or50 preparations.M bicuculline and 20**, pµ M< 0.001 echinacoside. versus control Results group. are #, pmean < 0.05 versus˘ S.E.M. the SR95531-, of 5 independent or bicuculline-treated synaptosomal group. preparations. **, p < 0.001 versus control group. #, p < 0.05 versus the SR95531-, or bicuculline-treated group.

Int. J. Mol. Sci. 2016, 17, 1006 7 of 13

3. Discussion In this study, echinacoside, an active compound in Herba Cistanche, inhibited 4-aminopyridine-evoked glutamate release in the rat cerebral cortex nerve terminals. The possible underlying mechanisms for the echinacoside-mediated inhibition of glutamate release are further investigated and discussed here.

3.1. Mechanisms Underlying Echinacoside-Mediated Inhibition of Glutamate Release Glutamate release evoked by 4-aminopyridine comprises two components: a physiologically relevant Ca2+-dependent component, which is produced through exocytosis of synaptic vesicles containing glutamate; and a Ca2+-independent component, which originates from prolonged depolarization causing a membrane potential-mediated shift of the glutamate transporter steady-state toward the outward direction, thus affecting cytosolic glutamate efflux [31]. Here, we observed that echinacoside did not significantly inhibit 4-aminopyridine-evoked glutamate release in the presence of a Ca2+-free medium (Ca2+-independent release). Furthermore, the observed echinacoside-mediated inhibition of 4-aminopyridine-evoked glutamate release was effectively prevented by bafilomycin A1 (which depletes the glutamate content of synaptic vesicles), but not by DL-TBOA (which nonselectively inhibits all excitatory amino acid transporter subtypes). These results suggest that echinacoside affects the Ca2+-dependent exocytosis of glutamate release without affecting the Ca2+-independent cytosolic efflux of glutamate through the reversal of the nerve terminal plasma membrane glutamate transporter. In synaptic terminals, Na+ channel inhibition or K+ channel activation stabilizes membrane excitability and consequently reduced the evoked Ca2+ entry and neurotransmitter release [32,33]. Therefore, the potential mechanism underlying echinacoside-mediated glutamate release inhibition involves a reduction in synaptosomal excitability. However, this possibility is untenable on the basis of two observations: (1) 4-aminopyridine-evoked membrane potential depolarization, measured with the membrane potential sensitive dye DiSC3(5) was unaffected by the addition of echinacoside; and (2) echinacoside did not affect the 4-aminopyridine-evoked Ca2+-independent glutamate release, a component of release that depends on only the membrane potential [31]. If the effect is not caused by synaptosomal excitability suppression, it may be manifested through a reduction in the 2+ activity of Cav2.2 (N-type) and Cav2.1 (P/Q-type) Ca channels coupled with glutamate exocytosis in the nerve terminals [34–36]. By using fura-2, we demonstrate that echinacoside significantly reduces the 4-aminopyridine-evoked increase in Ca2+ concentration. In addition, our data show that the inhibitory effect of echinacoside on 4-aminopyridine-evoked glutamate release decreased from 42.4% ˘ 2.3% to 12.1% ˘ 3.9% after exposure to a blocker of Cav2.2 (N-type) and Cav2.1 (P/Q-type) Ca2+ channels. Furthermore, we observed that echinacoside continuted to significantly inhibit 4-aminopyridine-evoked glutamate release in the presence of intracellular Ca2+ release inhibitors. These results indicate that the simultaneous suppression of Cav2.2 (N-type) and Cav2.1 (P/Q-type) Ca2+ channel activity is the potentially mechanism underlying echinacoside-mediated glutamate 2+ release inhibition. However, the combined activation of Cav2.2 (N-type) and Cav2.1 (P/Q-type) Ca channel activity could not block the action of echinacoside completely. Hence, other unidentified types of Ca2+ channels or other presynaptic pathways may be involved in the inhibition. For example, GABAA receptors are present at the presynaptic level, and their activation has been shown to inhibit 2+ Ca influx and glutamate release [37]. In the present study, the GABAA receptor antagonists SR95531 and bicuculline did not block echinacoside-mediated inhibition of glutamate release, suggesting that 2+ GABAA receptors are not involved in the reduction of voltage-dependent Ca channel activity and in the subsequent inhibition of glutamate release. Ca2+ entry through voltage-dependent Ca2+ channels activates several protein kinases associated with glutamate release in nerve terminals including mitogen-activated protein kinase, protein kinase C, and protein kinase A. Here, we demonstrate that protein kinase C inhibitors efficiently antagonized the echinacoside-mediated inhibition of glutamate release; nevertheless, the mitogen-activated protein kinase inhibitor PD98059 or the protein kinase A inhibitor H89 was ineffective. Furthermore, the Int. J. Mol. Sci. 2016, 17, 1006 8 of 13

4-aminopyridine-induced phosphorylation of protein kinase C decreased in synaptosomes after pretreatment with echinacoside at a concentration effective for inhibiting glutamate release. Therefore, the signaling pathway of the echinacoside-mediated glutamate release inhibition may involve protein kinase C. Protein kinase C is an important intracellular signaling system that is present at the presynaptic level and has a crucial role in neurotransmitter exocytosis. For example, several synaptic proteins involved in the synaptic vesicle trafﬁcking or recruitment and exocytosis, such as the myristoylated alanine-rich C kinase substrate, are phosphorylated by protein kinase C [38,39]. This phosporylation process can be increased by depolarization-stimulated Ca2+ entry, which facilitates glutamate release [40]. Hence, we can reasonably speculate that the inhibitory effect of echinacoside on Ca2+ entry observed here may reduce protein kinase C activity and consequently glutamate release.

3.2. Therapeutic Implications Excitotoxicity, a pathological process caused by excessive glutamate release and glutamate receptor activation, is the major cause of neuronal death in acute and chronic brain disorders such as stroke, traumatic brain injury, Parkinson’s, and Alzheimer’s diseases [13,41], and therapeutic strategies involving glutamate release inhibition may be promising neuroprotective strategies for treating such diseases. Echinacoside has been conﬁrmed to penetrate the blood-brain barrier (BBB) and exhibits neuroprotective effects in various in vivo models of neurotoxicity [8,10–12,42]. Although the mechanism of these neuroprotective effects is not completely understood, several possible mechanisms have been reported including inﬂammatory response inhibition, mitochondrial function stabilization, antioxidation, free radical scavenging, and neurotrophic function mimicking [5,9,12,42]. In the current study, the ability of echinacoside to reduce glutamate release from nerve terminals may also partly explain its neuroprotective mechanism. However, whether this effect contributes to the apparent therapeutic potential of echinacoside in brain disorders associated with glutamate excitotoxicity warrants further research.

4. Materials and Methods

4.1. Chemicals Fura-2-acetoxymethyl ester (Fura-2-AM) and 3’,3’,3’-dipropylthiadicarbocyanine iodide [DiSC3(5)] were purchased from Invitrogen (Carlsbad, CA, USA). ω-conotoxin MVIIC, rottlerin, 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide (GF109203X), 5,6,7,13-tetrahydro- 13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile (Go6976) and N-[2-(p- bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89) were purchased from Tocris Bioscience (Bristol, UK). Echinacoside, dantrolene, DL-threo-beta-benzyl-oxyaspartate (DL-TBOA), 7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157), 2-(2-amino-3- methoxyphenyl)-4H-1-benzopyran-4-one) (PD98059), ethylene glycol bis(β-aminoethyl ether)- N,N,N1,N1-tetraacetic acid (EGTA) and all other reagents were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

4.2. Animals Two-month old male Sprague–Dawley rats were used. Animals were housed under standardized environmental conditions (22 ˘ 1 ˝C; 50% relative humidity; 12 h light/dark cycle) and allowed unlimited access to food and water. The animals were killed by decapitation and the cerebral cortex rapidly removed at 4 ˝C. The experimental procedures were approved by the Fu Jen Institutional Animal Care and Utilization Committee (A10259), in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize animal suffering and to use a minimum number of animals necessary to produce reliable results. Int. J. Mol. Sci. 2016, 17, 1006 9 of 13

4.3. Synaptosomal Preparations Synaptosomes were purified from the cerebral cortex of rats on discontinuous Percoll gradients as described previously [43,44]. Briefly, the tissue was homogenized in medium containing 0.32 M sucrose (pH 7.4), the homogenate was centrifuged for 10 min at 3000ˆ g (5000 rpm in a JA 25.5 rotor; Beckman Coulter, Inc., Miami, FL, USA) and 4 ˝C, and the supernatant was centrifuged again for 12 min at 14,500ˆ g (11,000 rpm in a JA 25.5 rotor). The pellet was gently resuspended in 0.32 M sucrose (pH 7.4), and an aliquot of this synaptosomal suspension (2 mL) was placed onto a 3 mL Percoll discontinuous gradient containing 0.32 M sucrose, 1 mM EDTA, 0.25 mM DL-dithiothreitol, and 3%, 10%, and 23% Percoll (pH 7.4). After centrifugation at 32,500ˆ g (16,500 rpm in a JA 20.5 rotor) for 7 min at 4 ˝C, the synaptosomes were recovered from between the 10% and the 23% Percoll bands, and they were diluted in a final volume of 30 mL of HEPES buffer medium (140 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1 mM MgCl2¨ 6H2O, 1.2 mM Na2HPO4, 10 mM glucose, and 10 mM HEPES (pH 7.4)). Following further centrifugation at 27,000ˆ g (15,000 rpm in a JA 25.5) for 10 min, the synaptosome pellet was resuspended in 3 mL of HEPES buffer medium, and the protein content was determined using a Bradford assay. Finally, 0.5 mg of the synaptosomes suspension was diluted in 10 ml of HEPES buffer medium and centrifuged at 3000ˆ g (5000 rpm in a JA 20.1 rotor) for 10 min. The supernatant was discarded, and the pellets containing the synaptosomes were stored on ice and used within 4–6 h.

4.4. Glutamate Release Glutamate release was assayed by on-line fluorimetry as described previously [45,46]. Synaptosomal pellets were resuspended in HEPES buffer medium (0.5 mg/mL) and preincubated at 37 ˝C for 10 min in the presence of 16 µM bovine serum albumin to bind any free fatty acids released from synaptosomes during preincubation. A 2-mL aliquot of the synaptosomes was transferred to a stirred cuvette containing 2 mM NADP+, 50 units of glutamate dehydrogenase, and 1.2 mM CaCl2, and the fluorescence of NADPH was measured in a Perkin-Elmer LS-55 spectrofluorimeter (PerkinElmer Life and Analytical Sciences, Waltham, MA, USA) at excitation and emission wavelengths of 340 and 460 nm, respectively. As synaptosomes are not amenable to electrical stimulation, the potassium channel blocker 4-aminopyridine was used to stimulate glutamate release. 4-aminopyridine destabilizes the membrane potential and is thought to cause repetitive spontaneous Na+ channel-dependent depolarization that closely approximates in vivo depolarization of the synaptic terminal that leads to the activation of voltage-dependent Ca2+ channels and neurotransmitter release [47]. Data were obtained at 2 s intervals. A standard of exogenous glutamate (5 nmol) was added at the end of each experiment. The value of the fluorescence change produced by the standard addition was used to calculate the released glutamate as nanomoles of glutamate per milligram of synaptosomal protein (nmol/mg). Release values quoted in the text are levels attained at steady-state after 5 min of depolarization (nmol/mg/5 min). Cumulative data were analyzed using Lotus 1-2-3 spreadsheets (IBM, White Plains, NY, USA) and MicroCal Origin (OriginLab Corporation, Northampton, MA, USA).

4.5. Plasma Membrane Potential The plasma membrane potential was determined with a membrane-potential-sensitive dye, DiSC3(5) [48]. Synaptosomes were resuspended in HEPES buffer medium, and 2 mL aliquots ˝ were transferred to a stirred cuvette containing 5 µM DiSC3(5) at 37 C in a Perkin–Elmer LS-55 spectrofluorometer (PerkinElmer Life and Analytical Sciences, Waltham, MA, USA). After allowing the mixture to equilibrate for 3 min, the fluorescence was determined at excitation and emission wavelengths of 646 and 674 nm, respectively. Data were collected at 2 s intervals. Cumulative data were analyzed using MicroCal Origin (OriginLab Corporation, Northampton, MA, USA) and expressed in fluorescence units. Int. J. Mol. Sci. 2016, 17, 1006 10 of 13

2+ 2+ 4.6. Cytosolic Ca Concentration ([Ca ]C) 2+ 2+ The [Ca ]C was measured with the Ca indicator fura-2. Synaptosomes (0.5 mg/mL) were ˝ preincubated in HEPES buffer medium containing 5 µM fura-2 and 0.1 mM CaCl2, for 30 min at 37 C in a stirred test tube. After fura-2 loading, synaptosomes were centrifuged in a microcentrifuge for 30 s at 3000ˆ g (5000 rpm). The synaptosomal pellets were resuspended in HEPES buffer medium, and the synaptosomal suspension was stirred in a thermostatted cuvette in a Perkin-Elmer LS-55 spectroﬂuorometer (PerkinElmer Life and Analytical Sciences, Waltham, MA, USA). CaCl2 (1 mM) was added after 3 min and further additions were made after an additional 10 min. Fluorescence data were accumulated at excitation wavelengths of 340 and 380 nm (emission wavelength 505 nm) at 2+ 2 s intervals. [Ca ]C (nM) was calculated using calibration procedures [49] and equations described previously [50]. Cumulative data were analyzed using MicroCal Origin (OriginLab Corporation, Northampton, MA, USA).

4.7. Western Blotting Synaptosomes were homogenized in a lysis buffer (10 mM HEPES buffer, pH 7.4), 1% Triton X-100, and protease inhibitor mixture. Lysates were clarified by centrifugation, and protein concentration was determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane. The membranes were blocked with Tris-buffered saline that contained 5% low-fat milk and incubated with appropriate primary antibody (phospho-protein kinase C (pan), 1:3000, NOVUS Biologicals Inc., Beverly, MA, USA) overnight at 4 ˝C. After three washes in Tris-buffered saline, the membrane was then treated with the secondary horseradish peroxidase-conjugated antibody (1:3000) for 1 h at room temperature. The membranes were then washed at least three times with Tris-buffered saline and visualized using the enhanced chemiluminescence system (Amersham, Buckinghamshire, UK). An aliquot of samples was loaded and probed with anti-PKC antibody for detection of PKC as a loading control. The level of expression or phosphorylation was assessed by band density, which was quantified by densitometry. Densitometric quantification of bands was analyzed using Syngene software (Synoptics, Cambridge, UK).

4.8. Statistical Analysis Data were obtained from a single synaptosomal preparation and were not independent of one another. To test the significance of the effect of a drug versus control, a two-tailed Student’s t-test was used. When an additional comparison was required (such as whether a second treatment influenced the action of echinacoside), a one-way ANOVA followed by Tukey’s test was used. Analysis was completed via software SPSS (17.0; SPSS Inc., Chicago, IL, USA). Data are expressed as mean ˘ S.E.M.; significance was evaluated at p < 0.05 for all statistical measures.

5. Conclusions This is the first study demonstrating that echinacoside inhibits glutamate release from rat cerebrocortical synaptosomes by reducing Ca2+ influx through Cav2.2 and Cav2.1 channels, and this release inhibition is likely dependent on the suppression of the protein kinase C pathway, at least in part. The present finding is valuable because it provides a novel insight into the mechanisms of action of echinacoside in the brain.

Acknowledgments: This work was supported by a grant from the Ministry of Science and Technology (MOST 103-2320-B-030-001 MY3). Author Contributions: Tzu Yu Lin and Su Jane Wang conceived and designed the experiments; Cheng Wei Lu performed the experiments; Cheng Wei Lu and Shu Kuei Huang analyzed the data; Su Jane Wang wrote the paper. Conﬂicts of Interest: The authors declare no conﬂict of interest. Int. J. Mol. Sci. 2016, 17, 1006 11 of 13

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