Abstract <p>Cardiotonic steroids (CTS), classical inhibitors of the Na/K-ATPase(NKA), exert diverse effects in excitable cells depending on theirconcentration and NKA isoform specificity. While high CTS concentrationsinhibit ion transport, low nanomolar levels can activate NKA andtrigger signaling pathways associated with neuroprotection. However,it remains unclear whether this effect is unique to ouabain or sharedby other CTS. Here, we compared the effects of ouabain, digoxin,digitoxin, marinobufagenin, and rostafuroxin on intracellular Ca²⁺ dynamicsin rat cortical neurons subjected to NMDA receptor activation, modelingglutamate-induced excitotoxicity. Using Fluo-8 calcium imaging,we found that 1 nM ouabain and digitoxin significantly reduced NMDA-evokedCa²⁺ responses by 24&#xa0;and 61%, respectively(<i>p</i> &lt; 0.01), whereas digoxin,MBG, and rostafuroxin had no significant effects. The neuroprotective-likeactions of ouabain and digitoxin may reflect their higher affinityfor the neuronal NKA α3 isoform, which is functionally coupled tothe sodium-calcium exchanger and critical for Ca²⁺ homeostasisduring excitatory stress. In contrast, rostafuroxin, a non-inhibitoryCTS antagonist, did not modify NMDA-induced Ca²⁺ signals, consistentwith its inability to modulate NKA transport or signaling.</p>