The present study used an in vitro rat hippocampal culture model to determine the inhibitory mechanisms of GSPE in low [Mg2+]o or oxygen glucose deprivation-induced neuronal cell death. GSPE reduced the glutamate-induced [Ca2+]i increase by inhibiting the AMPA, NMDA, and DHPG-induced [Ca2+]i increase in hippocampal neurons. Synaptically mediated low [Mg2+]o-induced [Ca2+]i spikes were also inhibited by GSPE. GSPE inhibited low [Mg2+]o or oxygen glucose deprivation-induced neuronal cell death by inhibition of both [Ca2+]i increase and Ca2+-dependent NO formation.
Glutamate depolarizes membranes by an influx of Na+ (partly Ca2+) through non-NMDA receptors, which secondarily activate voltage-gated Ca2+ channels and induce Ca2+ influx . Glutamate also induces Ca2+ influx directly through NMDA receptor channels and Ca2+-permeable non-NMDA AMPA receptor channels. In the present study, GSPE inhibited glutamate, AMPA, and NMDA-induced [Ca2+]i increase, but it did not affect the depolarization-induced [Ca2+]i increase from 50 mM K+ HEPES-HBSS, suggesting that GSPE inhibits AMPA-induced [Ca2+]i increase by inhibiting Ca2+ influx directly through Ca2+-permeable AMPA receptors. In fact, Ca2+-permeable AMPA receptors are strongly expressed in hippocampal neurons, especially early in development . All these data suggested that GSPE inhibited Ca2+ influx through Ca2+-permeable AMPA channels and NMDA channels. This data are indirectly supported by other reports that flavonoids such as baicalin, baicalein, and EGCG, decreased glutamate or NMDA-induced [Ca2+]i increase [15, 31].
The group I metabotropic glutamate receptor agonist, DHPG, induces a release of Ca2+ from IP3-sensitive stores by activating PLC [25, 32]. In the present study, GSPE inhibited DHPG-induced [Ca2+]i increase. Although the working mechanism of GSPE is not obvious, GSPE may inhibit DHPG-induced Ca2+ release from IP3-sensitive stores or DHPG-induced activation of PLC. Therefore, further research is needed to determine whether proanthocyanidin inhibits release of Ca2+ from IP3-sensitive stores or metabotropic glutamate receptor-induced activation of PLC.
In the present study, GSPE inhibited glutamate-induced [Ca2+]i increase by inhibiting AMPA, NMDA, and metabotropic glutamate receptor-induced [Ca2+]i increase. Reduction of [Mg2+]o in cultured central nervous system neurons to 0.1 mM elicited [Ca2+]i spikes that depend on glutaminergic synaptic transmission [27, 29, 33]. In the present study, GSPE inhibited low [Mg2+]o-induced [Ca2+]i spikes. All these data suggest a possibility that proanthocyanidin can inhibit glutaminergic synaptic transmission in hippocampal neurons. In the present study, GSPE did not affect the depolarization-induced [Ca2+]i increase induced by high K+, which is involved in neurotransmitter release in the synaptic terminal. Thus, it is not clear whether proanthocyanidin inhibited synaptic transmission by decreasing glutamate release in presynaptic sites.
In the present study, GSPE completely inhibited low [Mg2+]o-induced NO formation, and it slightly inhibited glutamate-induced formation. GSPE reportedly has potent inhibitory action on NO production presumably through of the inhibition of Ca2+-dependent nitric oxide synthase . In neuronal cells, NO was synthesized from Ca2+-dependent enzymes, neuronal nitric oxide synthase [35, 36]. Therefore, the inhibition of excessive Ca2+ influx or Ca2+ release from intracellular stores and formation of NO by glutamate in the present study suggest that proanthocyanidin inhibits NO formation by inhibiting glutamate or low [Mg2+]o-induced [Ca2+]i increase.
Previous investigations have reported that proanthocyanidin protects multiple target organs from drug- and chemical-induced toxicity. GSPE protects cells against acetaminophen-induced hepato- and nephrotoxicity, amiodarone-induced lung toxicity, doxorubicin-induced cardiotoxicity, and dimethylnitrosamine-induced spleenotoxicity . GSPE inhibited 12-O-tetradecanoylphorbol-13-acetate and O-ethyl-S,S-dipropyl phosphorodithioate-induced brain neurotoxicity [2, 37]. Grape seed extract has also been reported to reduce brain ischemic injury in gerbils [4, 38] and rats , suggesting that the neuroprotective effects of proanthocyanidin are mediated by its antioxidant effects and antiapoptotic effects, respectively. However, there have been no reports on the underlying roles of calcium signalling or NO formation in proanthocyanidin-induced neuroprotection. GSPE inhibited low [Mg2+]o- and oxygen glucose deprivation-induced neuronal cell death as well as both [Ca2+]i increase and Ca2+-dependent NO formation. Ischemic insults have reportedly induced [Ca2+]i increase and formation of NO in neurons [10, 12, 40, 41]. In addition, proanthocyanidin blueberry extract is reported to have reversed dopamine, Aβ42, and lipopolysaccharide-induced dysregulation of Ca2+ buffering capacity, thereby inducing neuroprotection in hippocampal neurons . These results suggest that proanthocyanidin might inhibit ischemia-induced neuronal cell death by inhibiting glutamate-induced [Ca2+]i signalling and NO formation as well as antioxidant effects and antiapoptotic effects.
The daily intake of proanthocyanidins may vary from tens to several hundred mg/day depending on diet . Proanthocyanidins, especially oligomeric proanthocyanidins, are more easily absorbed and are present in blood after oral intake [21, 43]. Catechin and epicatechin are reportedly bioavailable to the brain after ingestion of oligomeric proanthocyanidin , which suggests that oligomeric proanthocyanidins can cross the blood-brain barrier and affect neuronal cells. In fact, the IH636 grape seed proanthocyanidin extract (GSPE) used in the present study was composed of more than 73% oligomeric polyphenolic compounds including monomeric, dimeric, trimeric, and tetrameric proanthocyanidin . Although the biological efficacy of GSPE has been studied previously in humans [37, 44], the bioavailablity of GSPE used in the present study remains unknown. However, it should be noted that this particular concentration of grape seed proanthocyanidin extract (GSPE) was less than or equal to the serum concentration in humans following intake of 200 mg/kg proanthocyanidins or oligomeric proanthocyanidins . These data suggest a possibility that IH636 grape seed proanthocyanidin extract (GSPE) can induce neuroprotection after intake of oligomeric proanthocyanidin in humans as well as animals.