Previous studies have demonstrated that the knockdown or knockout of the three Na+/Ca2+ 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. Ca2+ 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 a-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 GABAA 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. Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics.
Neurounina-1, a novel compound that increases Na+/Ca 2+ exchanger activity, effectively protects against stroke damage
Ambrosino, Paolo;
2013-01-01
Abstract
Previous studies have demonstrated that the knockdown or knockout of the three Na+/Ca2+ 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. Ca2+ 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 a-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 GABAA 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. Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
