ABSTRACT
Title
Modulation of kainate receptors by extracellular cGMP
Authors
D. Frattaroli1, M.C. Mazzotta 1, C. Cervetto 1, M. Passalacqua 1 G. Maura1,2,3, and M. Marcoli1,2
1 Department of Experimental Medicine, Section of Pharmacology and Toxicology and Section of Biochemistry, University of Genoa, Genoa, Italy; 2 Center of Excellence for Biomedical Research, University of Genoa, Genoa, Italy; 3 National Institute of Neuroscience, INN, Turin, Italy
1 Department of Experimental Medicine, Section of Pharmacology and Toxicology and Section of Biochemistry, University of Genoa, Genoa, Italy; 2 Center of Excellence for Biomedical Research, University of Genoa, Genoa, Italy; 3 National Institute of Neuroscience, INN, Turin, Italy
Abstract
In the cerebellum cGMP produced in response to the activation of glutamate transmission functions as a key intracellular messenger at parallel fiber-Purkinje cell synapses. Although cGMP can be transported from intracellular to the extracellular compartment through membrane transporters, and extracellular cGMP is considered a reliable measure of cerebellar glutamate transmission activation in in vivo models, roles for extracellular cGMP have been scarcely searched for. We investigated on the ability of extracellular cGMP to affect AMPA/kainate receptor responses in the adult rat cerebellum by using superfused purified synaptosomes, allowing direct pharmacological characterization of presynaptic release-regulating receptors. Additional experiments on the role of endogenously synthesized cGMP were performed in different experimental conditions, i.e in synaptosomes under static incubation (allowing accumulation of compounds in the extracellular compartment) and in superfused slices (retaining complexity of neuron-glial networks and allowing accumulation of compounds in the presynaptic receptor biophase).
Briefly, purified synaptosomes prepared from adult rat cerebellum by homogenization and stratification on a discontinuous Percoll gradient (analysed for purity by confocal microscopy), were loaded with [3H]D-aspartate, transferred to parallel superfusion chambers at 37°C, and exposed to ionotropic glutamate receptor agonists in the presence or not of glutamate receptor antagonists, inhibitors of AMPA or kainate receptor desensitization, or inhibitors of cGMP production/transport. In a subset of experiments, a period of static incubation was used to allow accumulation of neuroactive compounds in the incubation medium, or [3H]D-aspartate release was measured from 400-mm cerebellar slices.Endogenous glutamate was measured by HPLC and fluorimetric detection.
Confocal analysis with the neuronal marker MAP-2 and non-neuronal markers GFAP, integrin a-M or RIP confirmed that synaptosomes represented a purified preparation of nerve terminals, not contamined by astrocytes, oligodendrocytes or microglia. Activity of the glutamate receptor agonists AMPA and kainate, of inhibitors of the AMPA or kainate receptor desensitization cyclothiazide or concanavalin A, of the AMPA or kainate receptor antagonists CNQX, GYKI 52466 or NS-102 indicated that both AMPA and kainate receptors including the GluR6 subunit (EC50 value for kainate, in the presence of 30 mM GYKI 52466 and 3 mM concanavalin A, 62.7 mM ) functioned as autoreceptors activating glutamate release from terminals of cerebellar parallel/climbing fibers. Cyclic GMP acting at an extracellular site selectively inhibited the glutamate release evoked by kainate receptor activation (IC50 value against [3H]D-aspartate and endogenous glutamate efflux: 36.3 and 15.1 mM, respectively) from parallel/climbing fibers. When substances produced intracellularly and transported across the membrane were allowed to accumulate in the extracellular compartment (synaptosomes under static incubation), the cerebellar nerve terminals appeared a source for cGMP affecting the kainate autoreceptor response, as indicated by the capability of ODQ (inhibitor of cGMP production) or probenecid (inhibitor of cGMP transporters) to increase the response to kainate receptor activation. In superfused slices, where neuron-glial networks and control of synapse functioning was retained allowing biophase formation at presynaptic autoreceptors, endogenously synthesized cGMP appeared to be transported extracellularly and reach active concentrations in the biophase of presynaptic kainate autoreceptors.
It is concluded that endogenously synthesized cGMP in cerebellar nerve terminals might be the physiological source for the extracellular cGMP, andinhibition of kainate-evoked glutamate release from terminals of parallel/climbing fibers by extracellular cGMP might be a physiologically relevant process in the cerebellum.
Briefly, purified synaptosomes prepared from adult rat cerebellum by homogenization and stratification on a discontinuous Percoll gradient (analysed for purity by confocal microscopy), were loaded with [3H]D-aspartate, transferred to parallel superfusion chambers at 37°C, and exposed to ionotropic glutamate receptor agonists in the presence or not of glutamate receptor antagonists, inhibitors of AMPA or kainate receptor desensitization, or inhibitors of cGMP production/transport. In a subset of experiments, a period of static incubation was used to allow accumulation of neuroactive compounds in the incubation medium, or [3H]D-aspartate release was measured from 400-mm cerebellar slices.Endogenous glutamate was measured by HPLC and fluorimetric detection.
Confocal analysis with the neuronal marker MAP-2 and non-neuronal markers GFAP, integrin a-M or RIP confirmed that synaptosomes represented a purified preparation of nerve terminals, not contamined by astrocytes, oligodendrocytes or microglia. Activity of the glutamate receptor agonists AMPA and kainate, of inhibitors of the AMPA or kainate receptor desensitization cyclothiazide or concanavalin A, of the AMPA or kainate receptor antagonists CNQX, GYKI 52466 or NS-102 indicated that both AMPA and kainate receptors including the GluR6 subunit (EC50 value for kainate, in the presence of 30 mM GYKI 52466 and 3 mM concanavalin A, 62.7 mM ) functioned as autoreceptors activating glutamate release from terminals of cerebellar parallel/climbing fibers. Cyclic GMP acting at an extracellular site selectively inhibited the glutamate release evoked by kainate receptor activation (IC50 value against [3H]D-aspartate and endogenous glutamate efflux: 36.3 and 15.1 mM, respectively) from parallel/climbing fibers. When substances produced intracellularly and transported across the membrane were allowed to accumulate in the extracellular compartment (synaptosomes under static incubation), the cerebellar nerve terminals appeared a source for cGMP affecting the kainate autoreceptor response, as indicated by the capability of ODQ (inhibitor of cGMP production) or probenecid (inhibitor of cGMP transporters) to increase the response to kainate receptor activation. In superfused slices, where neuron-glial networks and control of synapse functioning was retained allowing biophase formation at presynaptic autoreceptors, endogenously synthesized cGMP appeared to be transported extracellularly and reach active concentrations in the biophase of presynaptic kainate autoreceptors.
It is concluded that endogenously synthesized cGMP in cerebellar nerve terminals might be the physiological source for the extracellular cGMP, andinhibition of kainate-evoked glutamate release from terminals of parallel/climbing fibers by extracellular cGMP might be a physiologically relevant process in the cerebellum.