PART 2 Echinacoside Inhibits Glutamate Release By Suppressing Voltage-Dependent Ca2+ Entry And Protein Kinase C in Rat Cerebrocortical Nerve Terminals

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3. Discussion

In this study, echinacoside, an active compound in Herba Cistanche, inhibited4-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.

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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 continued 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 potential 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, that. 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 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.

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-(pbromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89) were purchased from TocrisBioscience (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 was rapidly removed at 4 ˝C. The experimental procedures were approved by the Fu Jen InstitutionalAnimal 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.

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 a 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 online 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 FL 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 FL 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 FL 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 FL fluorescence units.

4.6. Cytosolic Ca2+ Concentration ([Ca2+]C)

The [Ca2+]C was measured with the Ca2+ 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 a 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 s intervals. [Ca2+]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 sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)and transferred to the 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 an anti-PKC antibody for the detection of PKC as a loading control. The level of expression or phosphorylation was assessed by band density, which was quantified by densitometry. Densitometricquantifification 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 conflicts of interest.

Conflicts of Interest: The authors declare no conflict of interest.

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