Part 1：Echinacoside Inhibits Glutamate Release By Suppressing Voltage-Dependent Ca2+ Entry And Protein Kinase C in Rat Cerebrocortical Nerve Terminals

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

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

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 o-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-chlorophenyl)-1,5-dihydro-4,1-benzodiazepine-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 phenylethanoid 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 of Parkinson’s disease, Alzheimer'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, the 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 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.

Echinacoside- neuroprotection

2. Results

2.1. Echinacoside Inhibits 4-Aminopyridine-Evoked Glutamate Release from Rat Cerebrocortical Nerve Terminals by Reducing Vesicular Exocytosis

Figure 1 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 uM 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 uM. Moreover, the glutamate release evoked by 1 mM 4-aminopyridine in an extracellular Ca2+-free solution containing 300 uM ethylene glycol bis(β-aminoethyl ether)-N,N,N/,N/-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 uM echinacoside (1.8 ± 0.2 nmol/mg/5 min; p = 0.58; Figure 1). In synaptosomes treated with 0.1 uM 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 uM echinacoside failed to signiﬁcantly inhibit the release of glutamate (2.1 ± 0.2 nmol/mg/5 min; p = 0.94; Figure 1). By contrast, 10 uM 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 uM echinacoside inhibited 4-aminopyridine-evoked glutamate release signiﬁcantly (7.7 ± 0.2 nmol/mg/5 min; F(2,12) = 87.23, p = 0.000; Figure 1).

2.2 Echinacoside 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; Table 1). The application of 20 uM echinacoside did not signiﬁcantly affect basal Ca2+ concentration (t(8) = 0.06, p = 0.95) but signiﬁcantly reduced the 4-aminopyridine-induced increase 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 uM echinacoside did not alter the resting membrane potential (t(8) = 0.976, p = 0.36) or signiﬁcantly change the 4-aminopyridine-mediated increase in DiSC3(5) ﬂuorescence (t(8) = -0.014, p = 0.99; Table 1).

Echinacoside from cisatnche extract

2.3 Reduced Ca2+ Inﬂux through the Cav2.2 (N-Type) and Cav2.1 (P/Q-Type) Channels May Be Associated with the Inhibition of 4-Aminopyridine-Evoked Glutamate Release by Echinacoside

Figure 2 shows that 2 uM o-conotoxin MVIIC, an N- and P/Q-type Ca2+ channel blocker, reduced 4-aminopyridine-evoked glutamate release from 7.4 ± 0.2 to 2.0 ± 0.1 nmol/mg/5 min (t(9) = 25.35, p = 0.000). In the presence of o-conotoxin MVIIC, the effect of 20 uM echinacoside on 4-aminopyridine-evoked glutamate release was nonsigniﬁcant (1.8 2±+ 0.2 nmol/mg/5 min; t(8) = 1.06,p = 0.32). Dantrolene (10 uM), an inhibitor of intracellular Ca release from the endoplasmic

reticulum [28], reduced 4-aminopyridine-evoked glutamate release (5.6 ± 0.3 nmol/mg/5 min; F(2,14) = 104.95, p = 0.000). However, in the presence of dantrolene, 20 uM echinacoside could still inhibit glutamate release signiﬁcantly (3.3 ± 0.2 nmol/mg/5 min; p = 0.000). Similar results were observed using 100 uM 7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157), a membrane-permeable blocker of mitochondrial Na+/Ca2+ exchange. In the ﬁve examined synaptosomal preparations, 20 uM echinacoside combined with 100 uM CGP37157 reduced 4-aminopyridine-evoked glutamate release by 48.3% ± 5.2% (F(2,13) = 136.79, p = 0.000), similar to the inhibition by echinacoside alone (46.2% ± 2.3%; p = 0.89; Figure 2).

2.4 Echinacoside Inhibits 4-Aminopyridine-Evoked Glutamate Release by Signaling through Protein Kinase C

As illustrated in Figure 3, 10 uM 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide (GF109203X), a general protein kinase C inhibitor [29], reduced 4-aminopyridine-evoked glutamate release (F(2,13) = 19.46, p = 0.000). In the GF109203X-treated synaptosomes, 20 uM echinacoside reduced 4-aminopyridine-evoked glutamate release by only 5.5% ± 1.8% (p = 0.89), less than that by echinacoside alone (42.4% ± 2.3%; p = 0.000). Similar results were obtained 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, y) [29]. In the presence of 3 uM Go6976, 20 uM echinacoside reduced glutamate release by 11.9% ± 2.9% (p = 0.59), signifying a signiﬁcant reduction compared with that by echinacoside alone (42.4% 2.3%; p = 0.000; Figure 3). By contrast, 3 uM rottlerin, a Ca2+-independent protein kinase C6 inhibitor [30], did not signiﬁcantly alter 4-aminopyridine (1 mM)-evoked glutamate release (p = 0.45). Nevertheless, in the presence of rottlerin, 20 uM echinacoside effectively caused an average inhibition of 37.1% ± 5.6% of the release (F(2,13) = 19.72, p = 0.000), similar to that by echinacoside alone (p = 0.41; Figure 3). In addition, the mitogen-activated protein kinase inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one) (PD98059) (50 uM) and the protein kinase A inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89) (100 uM) reduced 4-aminopyridine-evoked glutamate release (p = 0.000). Even in the presence of PD98059 or H89, 20 uM echinacoside effectively reduced the release (F(2,13) = 52.3, p = 0.000; Figure 3).

Figure 4 shows that 1 mM 4-aminopyridine increased the phosphorylation of protein kinase C in synaptosomes (t(4) = -6.871, p = 0.002). When synaptosomes were pretreated with 20 uM echinacoside for 10 min before the addition of 4-aminopyridine, 4-aminopyridine induced phosphorylation of protein kinase C considerably decreased (F(2,6) = 29.202, p = 0.001).

2.5 Echinacoside-Mediated Inhibition of Glutamate Release Does Not Involve a Gamma-Aminobutyric Acid Type A (GABAA) Receptor

In Figure 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, 100 uM SR95531 did not signiﬁcantly alter 4-aminopyridine (1 mM)-evoked glutamate release. In the SR95531-treated synaptosomes, application of 20 uM echinacoside resulted in a 43% inhibition on 4-aminopyridine-evoked glutamate release (F(2,12) = 42.63, p = 0.000), which was not signiﬁcantly different from the inhibition produced by echinacoside alone (40%; p = 0.000). A similar result was obtained with another GABAA receptor antagonist, bicuculline (50 uM). The release measured in the presence of bicuculline and echinacoside was signiﬁcantly different from that obtained in the presence of bicuculline alone (p = 0.000).

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 activity of Cav2.2 (N-type) and Cav2.1 (P/Q-type) Ca2+ 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 release inhibition. However, the combined activation of Cav2.2 (N-type) and Cav2.1 (P/Q-type) Ca2+ 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 Ca2+ 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 GABAA receptors are not involved in the reduction of voltage-dependent Ca2+ 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 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 phosphorylation 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.

cistanche benefit