Comparison of Digitalis Sensitivities of Na⁺/K⁺-ATPases from Human and Pig Kidneys

Abstract

Digitalis drugs are selective inhibitors of the plasma membrane Na+/K+-ATPase. There are many studies on molecular mechanisms of digitalis interaction with purified pig kidney enzyme, with the tacit assumption that it is a good model of human kidney enzyme. However, previous studies on crude or recombinant human kidney enzymes are limited, and have not resulted in consistent findings on their digitalis sensitivities. Hence, we prepared comparably purified enzymes from human and pig kidneys and determined inhibitory constants of digoxin, ouabain, ouabagenin, bufalin, and marinobufagenin (MBG) on enzyme activity under optimal turnover conditions. We found that each compound had the same potency against the two enzymes, indicating that (i) the pig enzyme is an appropriate model of the human enzyme, and (ii) prior discrepant findings on human kidney enzymes were either due to structural differences between the natural and recombinant enzymes or because potencies were determined using binding constants of digitalis for enzymes under nonphysiological conditions. In conjunction with previous findings, our newly determined inhibitory constants of digitalis compounds for human kidney enzymes indicate that (i) of the compounds that have long been advocated to be endogenous hormones, only bufalin and MBG may act as such at kidney tubules, and (ii) beneficial effects of digoxin, the only digitalis with extensive clinical use, does not involve its inhibitory effect on renal tubular Na+/K+-ATPase.

Disrupted Plasma Membrane Protein Homeostasis in a Xenopus Laevis Model of Retinitis Pigmentosa

“…The degree of NKA␣ downregulation was 83.4 Ϯ 11.2% in rods expressing a high-level of Rho Q344ter -Dend2. This degree of downregulation was similar to the extent of NKA inhibition (ϳ 80%; Gable et al, 2017) by the inhibitor concentrations (3.5 M for digoxin and 1 M for ouabain) introduced in this study. Thus, those studies strongly suggest that NKA downregulation induced by rhodopsin mislocalization is sufficient to cause rod OS shortening and loss.…”

“…As with the studies of digoxin, photoreceptor outer segments were visualized by P/rds and XAP-2 immunofluorescence. Ouabain is a steroidal glycoside more potent and selective than digoxin (Gable et al, 2017), whereas istaroxime is a steroidal but non-glycoside molecule (De Munari et al, 2003). After 7 d of treatment with ouabain, rod OSs were 17-23.5% ( p Ͻ 0.001) shorter and 12-22.5% fewer ( p Ͻ 0.05) on average compared with age-matched untreated retinas (Table 1).…”

Telocinobufagin and Marinobufagin Produce Different Effects in LLC-PK1 Cells: A Case of Functional Selectivity of Bufadienolides

“…In contrast to most mammalian cells, renal epithelial cells basically express one isozyme in abundance, α1β1, and serve as a good model to evaluate the effect of CTS without the interference of other isozymes. Moreover, pig kidney α1β1 is sensitive to CTS and has been widely used to dissect binding and Na + /K + -ATPase-mediated signaling properties of these compounds and is an appropriate model of human (kidney) Na + /K + -ATPase [ 41 ]. We first evaluated the inhibitory potency of telocinobufagin and marinobufagin on the enzymatic activity of pig kidney Na + /K + -ATPase.…”

Skeletal Muscle Na,K-ATPase as a Target for Circulating Ouabain

“…Ouabain is known to be able activate the α2 Na,K-ATPase [29] as well as the α1 Na,K-ATPase [32][33][34][35][36]. These data are still controversial and in other experiments on non-cellular Na,K-ATPase preparations, such effects were not observed [37][38][39]. In our study, no ouabain-induced stimulation of the Na,K-ATPase activity in human RBC, purified lamb kidney and Torpedo membrane preparations (expressing the α1 Na,K-ATPase isozyme only) was observed (Figure 4).…”