Neurounina-1, a novel compound that increases Na+/Ca2+ exchanger activity, effectively protects against stroke damage.
Previous studies have demonstrated that the knockdown or knockout of the three Na(+)/Ca(2+) exchanger (NCX) isoforms, NCX1, NCX2, and NCX3, worsens ischemic brain damage. This suggests that the activation of these antiporters exerts a neuroprotective action against stroke damage. However, drugs able to increase the activity of NCXs are not yet available. We have here succeeded in synthesizing a new compound, named neurounina-1 (7-nitro-5-phenyl-1-(pyrrolidin-1-ylmethyl)-1H-benzo[e][1,4]diazepin-2(3H)-one), provided with an high lipophilicity index and able to increase NCX activity. Ca(2+) radiotracer, Fura-2 microfluorimetry, and patch-clamp techniques revealed that neurounina-1 stimulated NCX1 and NCX2 activities with an EC(50) in the picomolar to low nanomolar range, whereas it did not affect NCX3 activity. Furthermore, by using chimera strategy and site-directed mutagenesis, three specific molecular determinants of NCX1 responsible for neurounina-1 activity were identified in the α-repeats. Interestingly, NCX3 became responsive to neurounina-1 when both α-repeats were replaced with the corresponding regions of NCX1. In vitro studies showed that 10 nM neurounina-1 reduced cell death of primary cortical neurons exposed to oxygen-glucose deprivation followed by reoxygenation. Moreover, in vitro, neurounina-1 also reduced γ-aminobutyric acid (GABA) release, enhanced GABA(A) currents, and inhibited both glutamate release and N-methyl-d-aspartate receptors. More important, neurounina-1 proved to have a wide therapeutic window in vivo. Indeed, when administered at doses of 0.003 to 30 μg/kg i.p., it was able to reduce the infarct volume of mice subjected to transient middle cerebral artery occlusion even up to 3 to 5 hours after stroke onset. Collectively, the present study shows that neurounina-1 exerts a remarkable neuroprotective effect during stroke and increases NCX1 and NCX2 activities.
Division of Pharmacology, Department of Neuroscience, School of Medicine, "Federico II" University of Naples, Via Pansini 5, 80131 Naples, Italy.
Pasquale
P
Molinaro
Maria
M
Cantile
Ornella
O
Cuomo
Agnese
A
Secondo
Anna
A
Pannaccione
Paolo
P
Ambrosino
Giuseppe
G
Pignataro
Ferdinando
F
Fiorino
Beatrice
B
Severino
Elena
E
Gatta
Maria José
MJ
Sisalli
Marco
M
Milanese
Antonella
A
Scorziello
Giambattista
G
Bonanno
Mauro
M
Robello
Vincenzo
V
Santagada
Giuseppe
G
Caliendo
Gianfranco
G
Di Renzo
Lucio
L
Annunziato
D000818:Animals; D001570:Benzodiazepinones / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D006224:Cricetinae; D004285:Dogs; D005442:Flumazenil / Q000494:pharmacology; D058787:GABA-A Receptor Antagonists / Q000494:pharmacology; D018698:Glutamic Acid / Q000378:metabolism; D020244:Infarction, Middle Cerebral Artery / Q000150:complications; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009154:Mutation; D009474:Neurons / Q000187:drug effects / Q000473:pathology; D018696:Neuroprotective Agents / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D011759:Pyrrolidines / Q000494:pharmacology*; D051381:Rats; D011963:Receptors, GABA-A / Q000502:physiology; D016194:Receptors, N-Methyl-D-Aspartate / Q000819:agonists; D019831:Sodium-Calcium Exchanger / Q000235:genetics / Q000378:metabolism*; D020521:Stroke / Q000209:etiology / Q000473:pathology / Q000517:prevention & control*; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
C579559:7-nitro-5-phenyl-1-(pyrrolidin-1-ylmethyl)-1H-benzo(e)(1,4)diazepin-2(3H)-one; D001570:Benzodiazepinones; D058787:GABA-A Receptor Antagonists; D018696:Neuroprotective Agents; D011759:Pyrrolidines; D011963:Receptors, GABA-A; D016194:Receptors, N-Methyl-D-Aspartate; D019831:Sodium-Calcium Exchanger; D018698:Glutamic Acid; D005442:Flumazenil; D005680:gamma-Aminobutyric Acid; D002118:Calcium