THE BLOCKADE OF LOW AFFINITY NEUROTENSIN RECEPTOR ALTERS MITOCHONDRIAL BIOENERGETICS AND THE TRIAD NMDA RECEPTOR, PSD-95 AND nNOS PROTEINS

A.A. GUTNISKY; A.G. KARADAYIAN; G. RODRÍGUEZ DE LORES ARNAIZ; S. LORES ARNAIZ; J. MIRANDA

Villa Carlos Paz, Córdoba.

Congreso; XXXIV Reunión Anual de la Sociedad Argentina de Investigación en Neurociencias (SAN); 2019

Sociedad Argentina de Investigación en Neurociencias (SAN)

Neurotensin is known to inhibit neuronal Na+, K+ -ATPase, an effect that is rescued by nitric oxide (NO) synthase inhibition (López Ordieres et al., 2011). In previous work we have shown the impairment of nitric oxide (NO) production and mitochondrial alterations after the blockade of low affinity neurotensin receptor (NTS2) by levocabastine. The activities of mitochondrial respiratory chain complexes I-IV were severely diminished by levocabastine administration, being this effect independent from the regulation of NO synthesis (Lores Arnaiz et al., 2017). A functional interaction between neuronal nitric oxide synthase (nNOS) and NR2B subunit of N-methyl-D-aspartic acid (NMDA) receptor through assembly post-synaptic density protein 95 (PSD-95) has been described at the synapses. In this study, mitochondrial function was characterized after both in vivo and in vitro levobacastine treatment. In addition, protein expression of nNOS, PSD-95, NMDA NR2B subunit and β-actin were determined after levocabastine administration.Materials and methodsExperimental design: Wistar rats were injected (i.p.) with levocabastine (50 μg/kg) or saline solution (controls) and were decapitated after 18 hours. Crude mitochondrial fractions from cerebral cortex were obtained by differential centrifugation (A), while synaptosomal membrane fractions were isolated by differential and sucrose gradient centrifugation (B). For in vitro assays, control mitochondrial fractions were incubated with 1 μM levocabastine.Mitochondrial function parameters:Oxygen consumption was assayed by high-resolution respirometry. Mitochondrial membrane potential was measured by flow cytomety using TMRE probe. ATP production rate was determined by a chemiluminescent assay using luciferine-luciferase.Protein expression: nNOS, PSD-95, NMDA-R NR2B subunit and -actin were analyzed in synaptosomal membranes by Western blot assays.Summary and conclusions?Mitochondrial respiratory rates were significantly impaired by in vivo and in vitro levocabastine treatment.?Levocabastine administration induced mitochondrial depolarization and decreased ATP production rates both in vivo and in vitro.?Levocabastine treatment significantly decreased nNOS, PSD-95, NMDA-receptor NR2B subunit and -actin protein expression at synaptosomal membranes, thus suggesting alterations in protein-protein interactions between PSD-95, NMDA-receptor and nNOS, as well as in neuron cytoskeleton.Results suggest that the blockade of NTS2 receptor by levocabastine leads to an important mitochondrial dysfunction. Bioenergetics impairment and ATP depletion induced by levocabastine at the synaptic level might play a role in the disassembly between PSD-95, NMDA-receptor and nNOS with the consequent decrease in NO levels. Alterations in the actin cytoskeleton may also compromise the assembly of proteins, as well as mitochondrial morphology network.