Pharmacological Characterization of the Newly Synthesized 5-Amino-N-butyl-2-(4-ethoxyphenoxy)-benzamide Hydrochloride (BED) as a Potent NCX3 Inhibitor That Worsens Anoxic Injury in Cortical Neurons, Organotypic Hippocampal Cultures, and Ischemic Brain

Agnese Secondo, Giuseppe Pignataro, Paolo Ambrosino, Anna Pannaccione, Pasquale Molinaro, Francesca Boscia, Maria Cantile, Ornella Cuomo, Alba Esposito, Maria Josè Sisalli, Antonella Scorziello, Natascia Guida, Serenella Anzilotti, Ferdinando Fiorino, Beatrice Severino, Vincenzo Santagada, Giuseppe Caliendo, Gianfranco Di Renzo, Lucio Annunziato

Research output: Contribution to journalArticle

Abstract

The Na+/Ca2+ exchanger (NCX), a 10-transmembrane domain protein mainly involved in the regulation of intracellular Ca2+ homeostasis, plays a crucial role in cerebral ischemia. In the present paper, we characterized the effect of the newly synthesized compound 5-amino-N-butyl-2-(4-ethoxyphenoxy)-benzamide hydrochloride (BED) on the activity of the three NCX isoforms and on the evolution of cerebral ischemia. BED inhibited NCX isoform 3 (NCX3) activity (IC50 = 1.9 nM) recorded with the help of single-cell microflorimetry, 45Ca2+ radiotracer fluxes, and patch-clamp in whole-cell configuration. Furthermore, this drug displayed negligible effect on NCX2, the other isoform expressed within the CNS, and it failed to modulate the ubiquitously expressed NCX1 isoform. Concerning the molecular site of action, the use of chimera strategy and deletion mutagenesis showed that α1 and α2 repeats of NCX3 represented relevant molecular determinants for BED inhibitory action, whereas the intracellular regulatory f-loop was not involved. At 10 nM, BED worsened the damage induced by oxygen/glucose deprivation (OGD) followed by reoxygenation in cortical neurons through a dysregulation of [Ca2+]i. Furthermore, at the same concentration, BED significantly enhanced cell death in CA3 subregion of hippocampal organotypic slices exposed to OGD and aggravated infarct injury after transient middle cerebral artery occlusion in mice. These results showed that the newly synthesized 5-amino-N-butyl-2-(4-ethoxyphenoxy)-benzamide hydrochloride is one of the most potent inhibitor of NCX3 so far identified, representing an useful tool to dissect the role played by NCX3 in the control of Ca2+ homeostasis under physiological and pathological conditions.

Original languageEnglish
Pages (from-to)1361-1370
Number of pages10
JournalACS Chemical Neuroscience
Volume6
Issue number8
DOIs
Publication statusPublished - Aug 19 2015

Keywords

  • cerebral ischemia
  • NCX isoforms
  • NCX3 inhibitor
  • OGD
  • Sodium calcium exchanger

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Physiology
  • Cognitive Neuroscience

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    Secondo, A., Pignataro, G., Ambrosino, P., Pannaccione, A., Molinaro, P., Boscia, F., Cantile, M., Cuomo, O., Esposito, A., Sisalli, M. J., Scorziello, A., Guida, N., Anzilotti, S., Fiorino, F., Severino, B., Santagada, V., Caliendo, G., Di Renzo, G., & Annunziato, L. (2015). Pharmacological Characterization of the Newly Synthesized 5-Amino-N-butyl-2-(4-ethoxyphenoxy)-benzamide Hydrochloride (BED) as a Potent NCX3 Inhibitor That Worsens Anoxic Injury in Cortical Neurons, Organotypic Hippocampal Cultures, and Ischemic Brain. ACS Chemical Neuroscience, 6(8), 1361-1370. https://doi.org/10.1021/acschemneuro.5b00043