A Comparative Study of the Neuroprotective Properties of Cardiotonic Steroids in a Model of NMDA Receptor Hyperactivation in Cortical Neurons

Cardiotonic steroids (CTS), classical inhibitors of the Na/K-ATPase (NKA), exert diverse effects in excitable cells depending on their concentration and NKA isoform specificity. While high CTS concentrations inhibit ion transport, low nanomolar levels can activate NKA and trigger signaling pathways associated with neuroprotection. However, it remains unclear whether this effect is unique to ouabain or shared by other CTS. Here, we compared the effects of ouabain, digoxin, digitoxin, marinobufagenin, and rostafuroxin on intracellular Ca2+ dynamics in rat cortical neurons subjected to NMDA receptor activation, modeling glutamate-induced excitotoxicity. Using Fluo-8 calcium imaging, we found that 1 nM ouabain and digitoxin significantly reduced NMDA-evoked Ca2+ responses by 24 and 61%, respectively (p < 0.01), whereas digoxin, MBG, and rostafuroxin had no significant effects. The neuroprotective-like actions of ouabain and digitoxin may reflect their higher affinity for the neuronal NKA α3 isoform, which is functionally coupled to the sodium-calcium exchanger and critical for Ca2+ homeostasis during excitatory stress. In contrast, rostafuroxin, a non-inhibitory CTS antagonist, did not modify NMDA-induced Ca2+ signals, consistent with its inability to modulate NKA transport or signaling.