(Na++K+)-ATPase (NKA) mediates positive inotropy in the heart. Extensive studies have demonstrated that the reverse-mode NA(+)/Ca2+-exchanger (NCX) plays a critical role in increasing intracellular Ca2+ concentration through the inhibition of NKA-induced positive inotropy by cardiac glycosides. Little is known about the nature of the NCX functional mode in the activation of NKA-induced positive inotropy. Here, we examined the effect of an NKA activator SSA412 antibody on Ca-45 influx in isolated rat myocytes and found that KB-R7943, a NCX reverse-mode inhibitor, fails to inhibit the activation of NKA-induced Ca-45 influx, suggesting that the Ca2+ influx via the reverse-mode NCX does not mediate this process. Nifedipine, an L-type Ca2+ channel (LTCC) inhibitor, completely blocks the activation of NKA-induced Ca-45 influx, suggesting that the LTCC is responsible for the moderate increase in intracellular Ca2+. In contrast, the inhibition of NKA by ouabain induces 4.7-fold Ca-45 influx compared with the condition of activation of NKA. Moreover, approximately 70% of ouabain-induced Ca-45 influx was obstructed by KB-R7943 and only 30% was impeded by nifedipine, indicating that both the LTCC and the NCX contribute to the rise in intracellular Ca2+ and that the NCX reverse-mode is the major source for the Ca-45 influx induced by the inhibition of NKA. This study provides direct evidence to demonstrate that the activation of NKA-induced Ca2+ increase is independent of the reverse-mode NCX and pinpoints a mechanistic distinction between the activation and inhibition of the NKA-mediated Ca2+ influx path ways in cardiomyocytes. (C) 2011 Elsevier Inc. All rights reserved.