Control of glutamate release by calcium channels and kappa-opioid receptors in rodent and primate striatum

Abstract

The modulation of depolarization (4-aminopyridine, 2 mM)-evoked endogenous glutamate release by kappa-opioid receptor activation and blockade of voltage-dependent Ca2+ -channels has been investigated in synaptosomes prepared from rat and marmoset striatum. 4-Aminopyridine (4-AP)-stimulated, Ca2+ -dependent glutamate release was inhibited by enadoline, a selective kappa-opioid receptor agonist, in a concentration-dependent and norbinaltorphimine (nor-BNI, selective kappa-opioid receptor antagonist)-sensitive manner in rat (IC50 = 4.4+/-0.4 microM) and marmoset (IC50 = 2.9+/-0.7 microM) striatal synaptosomes. However, in the marmoset, there was a significant (approximately 23%) nor-BNI-insensitive component. In rat striatal synaptosomes, the Ca2+ -channel antagonists omega-agatoxin-IVA (P/Q-type blocker), omega-conotoxin-MVIIC (N/P/Q-type blocker) and omega-conotoxin-GVIA (N-type blocker) reduced 4-AP-stimulated, Ca2+ -dependent glutamate release in a concentration-dependent manner with IC50 values of 6.5+/-0.9 nM, 75.5+5.9 nM and 106.5+/-8.7 nM, respectively. In marmoset striatal synaptosomes, 4-AP-stimulated, Ca2+ -dependent glutamate release was significantly inhibited by omega-agatoxin-IVA (30 nM, 57.6+/-2.3%, inhibition), omega-conotoxin-MVIIC (300 nM, 57.8+/-3.1%) and omega-conotoxin-GVIA (1 microM, 56.7+/-2%). Studies utilizing combinations of Ca2+ -channel antagonists suggests that in the rat striatum, two relatively distinct pools of glutamate, released by activation of either P or Q-type Ca2+ -channels, exist. In contrast, in the primate there is much overlap between the glutamate released by P and Q-type Ca2+ -channel activation. Studies using combinations of enadoline and the Ca2+ -channel antagonists suggest that enadoline-induced inhibition of glutamate release occurs primarily via reduction of Ca2+ -influx through P-type Ca2+ -channels in the rat but via N-type Ca2+ -channels in the marmoset. In conclusion, the results presented suggest that there are species differences in the control of glutamate release by kappa-opioid receptors and Ca2+ -channels.

Figure 1

The κ-opioid receptor agonist, enadoline, inhibits 4-AP-stimulated, Ca²⁺-dependent glutamate release in rat and marmoset striatal synaptosomes. To determine the effect of κ-opioid receptor activation on 4-AP-stimulated, Ca²⁺-dependent glutamate release in rat and marmoset striatal synaptosomes, enadoline (0.1–300 μM) or vehicle was added to the synaptosome preparation 5 min prior to addition of 4-AP. The Ca²⁺-dependent release of glutamate was then followed for a further 220 s. Vehicle effects have been subtracted from results. Values are expressed as mean±s.e.mean; ^*P<0.05, ^**P<0.01, ^***P<0.001, n=3–12 experiments (one-way ANOVA, followed by Tukey-Kramer multiple comparisons test).

Figure 2

Enadoline-induced inhibition of 4-AP-stimulated, Ca²⁺-dependent glutamate release in rat and marmoset striatal synaptosomes is mediated by κ-opioid receptor activation. To determine the receptor involved in enadoline-induced inhibition of 4-AP-stimulated, Ca²⁺-dependent glutamate release in rat and marmoset striatal synaptosomes, nor-BNI (5 μM), cyprodime (10 μM), naltrindole (20 μM) or vehicle were added together with enadoline to the synaptosome preparation 5 min prior to addition of 4-AP. The Ca²⁺-dependent release of glutamate was then followed for a further 220 s. Values are expressed as mean±s.e.mean; ^*P<0.05, ^**P<0.01, ^***P<0.001, compared to vehicle, $$P<0.01, $$$P<0.001 compared to enadoline+vehicle, n=3–12 experiments (one-way ANOVA, followed by Tukey-Kramer multiple comparisons test).

Figure 3

Ca²⁺-channel antagonists inhibit 4-AP-stimulated, Ca²⁺-dependent glutamate release in rat and marmoset striatal synaptosomes. To determine The effect of Ca²⁺-channel antagonists on 4-AP-stimulated, Ca²⁺-dependent glutamate release in rat (a) and (b) marmoset striatal synaptosomes, ω-aga-IVA (rat, 0.1–300 nM; marmoset, 30 nM), ω-con GVIA (rat, 1–3000 nM; marmoset 1 μM), ω-con-MVIIC (rat, 1–3000 nM; marmoset, 300 nM), nifedipine (rat, 1–30 μM; marmoset, 10 μM) or vehicle were added to the synaptosome preparation 5 min prior to addition of 4-AP. The Ca²⁺-dependent release of glutamate was then followed for a further 220 s. Vehicle effects have been subtracted from results. Values are expressed as mean±s.e.mean. (a) In rat striatal synaptosomes, each antagonist (apart from nifedipine) inhibited 4-AP-stimulated, Ca²⁺-dependent glutamate release in a concentration-dependent manner (one-way ANOVA, followed by Tukey-Kramer multiple comparisons test; n=3–8, significance levels have been omitted for clarity). (b) In marmoset striatal synaptosomes, each antagonist (apart from nifedipine) inhibited 4-AP-stimulated, Ca²⁺-dependent glutamate release, ^***P<0.001, compared to control, n=3–6 experiments (one-way ANOVA, followed by Dunnett's test).