Functional identification of activity-regulated, high-affinity glutamine transport in hippocampal neurons inhibited by riluzole

Abstract

Glutamine (Gln) is considered the preferred precursor for the neurotransmitter pool of glutamate (Glu), the major excitatory transmitter in the mammalian CNS. Here, an activity-regulated, high-affinity Gln transport system is described in developing and mature neuron-enriched hippocampal cultures that is potently inhibited by riluzole (IC₅₀ 1.3 ± 0.5 μM), an anti-glutamatergic drug, and is blocked by low concentrations of 2-(methylamino)isobutyrate (MeAIB), a system A transport inhibitor. K⁺ -stimulated MeAIB transport displays an affinity (K_m ) for MeAIB of 37 ± 1.2 μM, saturates at ~ 200 μM, is dependent on extracellular Ca²⁺ , and is blocked by inhibition of voltage-gated Ca²⁺ channels. Spontaneous MeAIB transport is also dependent on extracellullar Ca²⁺ and voltage-gated calcium channels, but is also blocked by the Na⁺ channel blocker tetrodotoxin, by Glu receptor antagonists, and by GABA indicating its dependence on intact neural circuits driven by endogenous glutamatergic activity. The transport of MeAIB itself does not rely on Ca²⁺ , but on Na⁺ ions, and is pH sensitive. Activity-regulated, riluzole-sensitive spontaneous and K⁺ -stimulated transport is minimal at 7-8 days in vitro, coordinately induced during the next 2 weeks and is maximally expressed by days in vitro > 20; the known period for maturation of the Glu/Gln cycle and regulated pre-synaptic Glu release. Competition analyses with various amino acids indicate that Gln is the most likely physiological substrate. Activity-regulated Gln/MeAIB transport is not observed in astrocytes. The functional identification of activity-regulated, high-affinity, riluzole-sensitive Gln/MeAIB transport in hippocampal neurons may have important ramifications in the neurobiology of activity-stimulated pre-synaptic Glu release, the Glu/Gln cycle between astrocytes and neurons, and neuronal Glu-induced excitotoxicity. Cover Image for this issue: doi: 10.1111/jnc.13805.

Keywords: activity-dependent regulation; excitotoxicity; glutamate/glutamine cycle; neuronal glutamine transporter; neuroprotection; neurotransmitter cycling.

Fig. 1. Ca²⁺-dependent ³H-Gln transport in hippocampal neurons is stimulated by brief high K⁺-induced depolarization

A relatively low concentration of ³H-Gln (20 µM) in the uptake medium is used to isolate a high affinity K⁺-stimulated Gln transport system in cultured hippocampal neurons. Abundant uptake of ³H-Gln by neurons is observed following 5 min incubation in normal Krebs buffer without Ca²⁺ (N buffer) but ³H-Gln transport is not significantly affected by inclusion of MeAIB (5 mM). The uptake of ³H-Gln increases ~25% by brief exposure (5 min) to high K⁺-depolarization (plus Ca²⁺) of axon terminals (K), which is blocked by inclusion of MeAIB (5 mM) in the uptake buffer (K-M). The MeAIB-sensitive portion of ³H-Gln transport increases ~8X fold following K⁺-depolarization (M sen, +Ca²⁺). The transport of ³H-Gln is conducted in N buffer at DIV 14. Uptake values obtained at 4°C were less than 5% and were subtracted. N, normal Krebs buffer without Ca²⁺ and with EGTA (1 mM). N-M, Same buffer with MeAIB (5 mM). K, Krebs buffer containing Ca²⁺ (1.2 mM) and with Na⁺ replaced with high K⁺ (60 mM, 5 min). K-M, Same K⁺-buffer with MeAIB (5 mM). M-sen, MeAIB-sensitive transport with same buffers. Data are the mean +/− S.E. values for n=3 independent experiments. F_5,18 = 59.4, p < 0.01 (main effect). *p = 0.04 (K vs K-M), ** p = 0.003 (M_sen+Ca vs M_sen-Ca), N.S. not significant.

Fig. 2. Ca²⁺-dependent ¹⁴C-MeAIB transport is stimulated by brief high K⁺-induced depolarization and exists spontaneously in neurons, but not in astrocytes

Hippocampal neurons (DIV 14) were first rinsed in N buffer that does not contain Ca²⁺, but EGTA (1 mM). For K⁺-stimulated uptake, cells are exposed to high K⁺ (60 mM) (with or without 1.2 mM CaCl₂) for 5 min at 37°C. Cells were then rinsed in N buffer and ¹⁴C-MeAIB uptake is performed for 5 min in N buffer (37°C). For spontaneous uptake, cells are first rinsed in N buffer and then incubated (5 min, 37°C) in N buffer or in normal Krebs medium with Ca²⁺ (1.2 mM) containing MeAIB (20 µM). Uptake values obtained at 4°C were less than 5% and were subtracted. K⁺, high K⁺ depolarization. N, Krebs buffer without Ca²⁺ and with EGTA. –Ca, 5 min pre-treatment in same buffers without Ca²⁺ (with 1 mM EGTA). Ver, Verapamil (20 µM) is included during the 5 min K⁺-depolarization period in Ca²⁺ containing high K⁺ buffer. Astrocytes pretreated with high K⁺ depolarization (plus Ca²⁺) do not transport significant amounts of ¹⁴C-MeAIB (20 µM, 5 min, 37°C). Data are the mean +/− S.E. for n=3 independent experiments. F_7,24 = 25.5, p < 0.01 (main effect) ** p < 0.001.

Fig. 3. Ca²⁺-dependent, high affinity ¹⁴C-MeAIB transport in hippocampal neurons is dependent on Na⁺ ions and is pH-sensitive

Cultures are exposed to high K⁺ (60 mM) (plus 1.2 mM CaCl₂) for 5 min at 37°C, then rinsed in N buffer, and ¹⁴C-MeAIB (20 µM) uptake is performed (5 min, 37°C) in: A, N buffer or N buffer that contains 145 mM LiCl, instead of Na⁺, or B, N buffer at pH 8.2, 7.4, or 6.0. Uptake values obtained at 4°C were less than 5% and were subtracted. Data are the mean +/− S.E. values for n=3 independent experiments. F_4,18 = 73.1, p < 0.01 (main effect). ** p < 0.01.

Fig. 4. Kinetic analysis of Ca²⁺-dependent K⁺-stimulated ¹⁴C-MeAIB transport in hippocampal neurons

A, Saturation isotherm of initial uptake velocity (3 min) of ¹⁴C-MeAIB (3.9 – 500 µM) in hippocampal neuron-enriched primary cultures (DIV 14). B, Lineweaver-Burk analysis of initial uptake velocity. Cells are exposed to high K⁺ (60 mM) (with 1.2 mM CaCl₂) for 5 min at 37°C, then rinsed in N buffer, and ¹⁴C-MeAIB uptake is performed in N buffer (37°C). Uptake values obtained at 4°C were less than 5% and were subtracted. The K_m and V_max value for MeAIB was calculated from the nonlinear regression of specific MeAIB uptake velocity versus the extracellular concentration of MeAIB. Data are from a representative experiment performed in duplicate, and K_m values are derived from n=4 independent experiments.

Fig. 5. Substrate competition profiles for Ca²⁺-dependent, high affinity ¹⁴C-MeAIB transport

Cultures are exposed to high K⁺ (60 mM plus 1.2 mM CaCl₂) for 5 min at 37°C, then are rinsed in N buffer, and ¹⁴C-MeAIB (20 µM) uptake is performed in N buffer (5 min, 37°C). Transport is conducted in the absence or presence of various amino acid competitive substrates (2 mM). Uptake values obtained at 4°C were less than 5% and were subtracted. Subtracted data were normalized to ¹⁴C-MeAIB uptake in the absence of unlabeled amino acids. N’N-DMG, N’N dimethylglycine. AIB, aminoisobutyric acid. BCH, 2-amino-2-norbornane-carboxylic acid. Cyclo-D-ser, cyclo-D-serine. Data are the mean +/− S.E. values from n=3–5 independent experiments. **p < 0.01, indicates significant differences from uptake in the absence of unlabeled substrate. ***p < 0.01) indicate amino acids (ala, pro, sarcosine, his, MeAIB, Gln) that reduce ¹⁴C-MeAIB transport by greater than 90%. N.S., not significant.

Fig. 6. Spontaneous ¹⁴C-MeAIB transport requires extracellular Ca²⁺ and is blocked by inhibitors of VGCCs, Na⁺ channel activity, glutamatergic transmission via AMPA and NMDA receptors, and by GABA

Cultures (DIV 14) were rinsed in N buffer and then preincubated in Krebs buffer with Ca²⁺ with or without inhibitors (2 min) and then in the same buffers containing ¹⁴C-MeAIB (20 µM) at 37°C (5 min). Uptake values obtained at 4°C were less than 5% and were subtracted. Con, control. Ver, verapamil 20 µM, A/N, AP5, 50 µM / NBQX 10 µM, TTX, tetrodotoxin 1 µM; MCPG, α-methyl-4-carboxyphenylglycine, 1 mM. Selected amino acids (2 mM) included with ¹⁴C-MeAIB during transport reveal that spontaneous ¹⁴C-MeAIB transport exhibits that same substrate preference to K⁺-stimulated transport, except for GABA, which is inhibitory due to the blockade of excitatory glutamatergic transmission. Data are the mean +/− S.E. values from n=3 independent experiments. F_12,39 = 571 (main effect), p < 0.0001. **p < 0.0001.

Fig. 7. Spontaneous Ca²⁺-regulated ¹⁴C-MeAIB transport is potently and preferentially blocked by riluzole, an anti-glutamatergic drug

Spontaneous Ca²⁺-dependent ¹⁴C-MeAIB transport (20 µM) is measured (15 min, 37°C) with and without various anti-glutamatergic drugs, following their brief (2 min) pre-incubation at 37°C. Spontaneous transport in Ca²⁺-free Krebs buffer was measured in all independent experiments and is generally less than 15% of Ca²⁺-containing control samples, was not affected by any of the drugs tested, and was subtracted as the negative background control. The IC₅₀ value for riluzole (black circles) and phenytoin (open squares) inhibition of ¹⁴C-MeAIB transport was calculated from dose response curves. Data shown are from a representative experiment, and the experiment was repeated n=4 times.

Fig. 8. Developmental up-regulation of the expression of activity-dependent spontaneous and K⁺-stimulated Ca²⁺ sensitive ¹⁴C-MeAIB transport

Spontaneous and K⁺-stimulated ¹⁴C-MeAIB transport (20 µM) is measured (5 min, 37°C) in hippocampal neuron-enriched cultures at different days in vitro (DIV 8, 12, 14, 16, and 20). N, normal Krebs buffer without Ca²⁺ and with EGTA. N+C, same buffer with Ca²⁺ (1.2 mM) and without EGTA. K, Krebs buffer Na⁺ replaced with high K⁺ (60 mM) but without Ca²⁺ and with EGTA. K+C, Same K⁺ buffer with Ca²⁺ and without EGTA. Ca²⁺-dependent spontaneous and K⁺-stimulated transport are controlled by inclusion of MeAIB (5 mM). Background levels at 4°C are consistently at <100 pmol/well. MeAIB (5 mM) subtracted data are the mean +/− S.E. values from n=3 independent experiments. F_9,72 = 481, p < 0.0001 (main effect). * p < 0.05, ** p < 0.001.