Ca2+-regulated expression of high affinity methylaminoisobutryic acid transport in hippocampal neurons inhibited by riluzole and novel neuroprotective aminothiazoles

Abstract

High affinity methylaminoisobutyric acid(MeAIB)/glutamine(Gln) transport activity regulated by neuronal firing occurs at the plasma membrane in mature rat hippocampal neuron-enriched cultures. Spontaneous Ca²⁺-regulated transport activity was similarly inhibited by riluzole, a benzothiazole anticonvulsant agent, and by novel naphthalenyl substituted aminothiazole derivatives such as SKA-378. Here, we report that spontaneous transport activity is stimulated by 4-aminopyridine (4-AP) and that phorbol-myristate acetate (PMA) increases high K⁺ stimulated transport activity that is inhibited by staurosporine. 4-AP-stimulated spontaneous and PMA-stimulated high K⁺-induced transport is not present at 7 days in vitro (DIV) and is maximal by DIV∼21. The relative affinity for MeAIB is similar for spontaneous and high K⁺-stimulated transport (K_m ∼ 50 μM) suggesting that a single transporter is involved. While riluzole and SKA-378 inhibit spontaneous transport with equal potency (IC₅₀ ∼ 1 μM), they exhibit decreased (∼3-5 X) potency for 4-AP-stimulated spontaneous transport. Interestingly, high K⁺-stimulated MeAIB transport displays lower and differential sensitivity to the two compounds. SKA-378-related halogenated derivatives of SKA-75 (SKA-219, SKA-377 and SKA-375) preferentially inhibit high K⁺-induced expression of MeAIB transport activity at the plasma membrane (IC₅₀ < 25 μM), compared to SKA-75 and riluzole (IC₅₀ > 100 μM). Ca²⁺-dependent spontaneous and high K⁺-stimulated MeAIB transport activity is blocked by ω-conotoxin MVIIC, ω-agatoxin IVA, ω-agatoxin TK (IC₅₀ ∼ 500 nM) or cadmium ion (IC₅₀ ∼ 20 μM) demonstrating that P/Q-type Ca_V channels that are required for activity-regulated presynaptic vesicular glutamate (Glu) release are also required for high-affinity MeAIB transport expression at the plasma membrane. We suggest that neural activity driven and Ca²⁺ dependent trafficking of the high affinity MeAIB transporter to the plasma membrane is a unique target to understand mechanisms of Glu/Gln recycling in synapses and acute neuroprotection against excitotoxic presynaptic Glu induced neural injury.

Keywords: Glutamate/glutamine cycle; Hippocampal neurons; Methylaminoisobutyric acid/glutamine transporter; Riluzole; SKA-378; Vesicle recycling.

Fig. 1

Spontaneous and high K ⁺ -stimulated expression of high affinity MeAIB transport activity at the plasma membrane in hippocampal neuronal cultures. A, 4-AP stimulates spontaneous (Spont) transport activity (200 μM, 15 min) and is dependent on neural network activity: TTX (1 μM), Ver (25 μM), N/A – NBQX (10 μM)/AP5 (50 μM), GABA (2 mM). Spontaneous expression of transport activity requires external Ca²⁺ ions (-Ca²⁺). Statistical values are compared to spontaneous transport activity alone. B, Developmental increase in 4-AP stimulated spontaneous transport is the same as spontaneous transport alone and occurs during the 2nd and 3rd post dissection (E19) weeks in vitro. Background values (-Ca²⁺) were subtracted. C, Pretreatment with PMA (50 nM, 10 min) stimulates expression of transport activity after high K⁺ exposure (60 mM, 5 min) that is selectively blocked by staurosporine (stauro, 200 nM). The inactive 4-alphaPMA does not stimulate transport. High K⁺-expression of transport activity also requires external Ca²⁺ ion (-Ca²⁺). Statistical values are compared to high K⁺-stimulated transport alone. D, Developmental increase for PMA stimulation of transport activity after high K⁺ exposure occurs is the same as high K⁺ stimulation alone and occurs during the 2nd and 3rd post dissection weeks in vitro. Background values (-Ca²⁺) were subtracted. A value of p < 0.05 is regarded as statistically significant (**p < 0.01, ****p < 0.0001).

Fig. 2

Chemical structures of riluzole, SKA-75, SKA-378, SKA-219, SKA-377 and SKA-375.

Fig. 3

Potency of riluzole, SKA-75 and halogenated derivatives of SKA-75 on spontaneous and high K⁺expression of MeAIB transport activity. A, Concentration response curves reveal that SKA-378 (green), SKA-219 (purple), SKA-377 (orange), and SKA-375 (black) similarly inhibit spontaneous high affinity MeAIB transport activity similar to riluzole (blue). The curve is shifted to the right for SKA-75 (red), yet the IC₅₀ value is still in the low μM range. B, Concentration response curves for all compounds are shifted to the right in the presence of 4-AP (200 μM) with IC₅₀ values 3-5X greater than for spontaneous transport alone. C, Concentration response curves for all compounds are further shifted to the right (5-50X) with PMA but now riluzole joins SKA-75 as least potent. D, Following high K⁺-exposure and stimulation with PMA, concentration response curves show that all halogenated SKA-75 derivatives are more potent than SKA-75 or riluzole. All IC₅₀ values in A-D are compared to SKA-378. A value of p < 0.05 is regarded as statistically significant (****p < 0.0001). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Non-competitive inhibition by SKA-378 of Ca²⁺-dependent high K⁺ stimulation of high affinity MeAIB transport activity. A, Michaelis-Menten kinetics of MeAIB uptake in the presence of SKA-378 (25 μM). Background was measured in the absence of Ca²⁺ ions at each MeAIB concentration and was subtracted. Representative kinetic experiment is shown and was repeated twice. B, Eadie-Hofstee scatchard analysis shows that the -slope (K_m) is not affected by SKA-378 while the initial velocity (V, V_max) of transport is reduced by ∼50% by SKA-378. C, Concentration response curves for inhibition of spontaneous and high K⁺ stimulated transport by P/Q calcium channel (Ca_V) antagonists ω-conotoxin MVIIC (MVIIC) and cadmium ions (Cd²⁺). MVIIC or Cd²⁺ were present only during the high K⁺-depolarization period to block expression of MeAIB transport activity to the plasma membrane. Background uptake was determined by omitting Ca²⁺ ion during spontaneous uptake or during the high K⁺-stimulation period and were subtracted. The potency of MVIIC to inhibit spontaneous vs. high K⁺-stimulated transport was not statistically different (p > 0.99). The potency of Cd²⁺ to inhibit spontaneous vs. high K⁺-stimulated transport was not statistically different (p = 0.27). Values are results from 3 independent neuronal cultures.

Fig. 5

Model of the Glu/Gln cycle between neurons and astrocytes that could recycle Glu for neurotransmission indicates where the neural activity regulated MeAIB/Gln transport system we describe further here operates (blue) that supports the concept of a Ca²⁺-dependent pathway for cycling of this high affinity transporter under spontaneous and by high K⁺ stimulation in synaptic terminals for exocytotic release. Our data indicate that SKA-378 targets an unidentified site (X) to inhibit Ca²⁺-dependent exocytosis and recycling of MeAIB transport activity to the plasma membrane in hippocampal neurons >5X more potently than riluzole. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)