Depth of the Steroid Core Location Determines the Mode of Na,K-ATPase Inhibition by Cardiotonic Steroids

Cardiotonic steroids (CTSs) are specific inhibitors of Na,K-ATPase (NKA). They induce diverse physiological effects and were investigated as potential drugs in heart diseases, hypertension, neuroinflammation, antiviral and cancer therapy. Here, we compared the inhibition mode and binding of CTSs, such as ouabain, digoxin and marinobufagenin to NKA from pig and rat kidneys, containing CTSs-sensitive (α1S) and -resistant (α1R) α1-subunit, respectively. Marinobufagenin in contrast to ouabain and digoxin interacted with α1S-NKA reversibly, and its binding constant was reduced due to the decrease in the deepening in the CTSs-binding site and a lower number of contacts between the site and the inhibitor. The formation of a hydrogen bond between Arg111 and Asp122 in α1R-NKA induced the reduction in CTSs’ steroid core deepening that led to the reversible inhibition of α1R-NKA by ouabain and digoxin and the absence of marinobufagenin’s effect on α1R-NKA activity. Our results elucidate that the difference in signaling, and cytotoxic effects of CTSs may be due to the distinction in the deepening of CTSs into the binding side that, in turn, is a result of a bent-in inhibitor steroid core (marinobufagenin in α1S-NKA) or the change of the width of CTSs-binding cavity (all CTSs in α1R-NKA).

Cellular Mechanisms Underlying the Low Cardiotoxicity of Istaroxime

Background Istaroxime is an inhibitor of Na + /K + ATPase with proven efficacy to increase cardiac contractility and to accelerate relaxation attributable to a relief in phospholamban‐dependent inhibition of the sarcoplasmic reticulum Ca 2+ ATPase. We have previously shown that pharmacologic Na + /K + ATPase inhibition promotes calcium/calmodulin‐dependent kinase II activation, which mediates both cardiomyocyte death and arrhythmias. Here, we aim to compare the cardiotoxic effects promoted by classic pharmacologic Na + /K + ATPase inhibition versus istaroxime. Methods and Results Ventricular cardiomyocytes were treated with ouabain or istaroxime at previously tested equi‐inotropic concentrations to compare their impact on cell viability, apoptosis, and calcium/calmodulin‐dependent kinase II activation. In contrast to ouabain, istaroxime neither promoted calcium/calmodulin‐dependent kinase II activation nor cardiomyocyte death. In addition, we explored the differential behavior promoted by ouabain and istaroxime on spontaneous diastolic Ca 2+ release. In rat cardiomyocytes, istaroxime did not significantly increase Ca 2+ spark and wave frequency but increased the proportion of aborted Ca 2+ waves. Further insight was provided by studying cardiomyocytes from mice that do not express phospholamban. In this model, the lower Ca 2+ wave incidence observed with istaroxime remains present, suggesting that istaroxime‐dependent relief on phospholamban‐dependent sarcoplasmic reticulum Ca 2+ ATPase 2A inhibition is not the unique mechanism underlying the low arrhythmogenic profile of this drug. Conclusions Our results indicate that, different from ouabain, istaroxime can reach a significant inotropic effect without leading to calcium/calmodulin‐dependent kinase II–dependent cardiomyocyte death. Additionally, we provide novel insights regarding the low arrhythmogenic impact of istaroxime on cardiac Ca 2+ handling.

Vacuolar H+-ATPase dysfunction rescues intralumenal vesicle cargo sorting in yeast lacking PI(3,5)P2 or Doa4

Endosomes undergo a maturation process highlighted by a reduction in lumenal pH, a conversion of surface markers that prime endosome-lysosome fusion, and the sequestration of ubiquitinated transmembrane protein cargoes within intralumenal vesicles (ILVs). We investigated ILV cargo sorting in mutant strains of the budding yeast Saccharomyces cerevisiae that are deficient for either the lysosomal/vacuolar signaling lipid PI(3,5)P2 or the Doa4 ubiquitin hydrolase that deubiquitinates ILV cargoes. Disruption of PI(3,5)P2 synthesis or Doa4 function causes a defect in the sorting of a subset of ILV cargoes. We show that these cargo-sorting defects are suppressed by mutations that disrupt Vph1, which is a subunit of Vacuolar H+-ATPase (V-ATPase) complexes that acidify late endosomes and vacuoles. We further show that Vph1 dysfunction increases endosome abundance and disrupts vacuolar localization of Ypt7 and Vps41, two critical mediators of endosome-vacuole fusion. Because V-ATPase inhibition attenuates endosome-vacuole fusion and rescues the ILV cargo-sorting defects in yeast lacking PI(3,5)P2 and Doa4 activity, our results suggest that the V-ATPase performs a role in the coordination of ILV cargo sorting with the membrane fusion machinery.

IN VITRO ANTI-ULCER ACTIVITY OF DIFFERENT EXTRACTS OF CISSUS QUADRANGULARIS

Aim:The aim of this study was undertaken to assessin vitro H+-K+ ATPase inhibitory activity of different extracts of Cissus quadrangularis Linn. Materials and Methods: Phytochemical and Phyto-analytical studies like total phenolic compound and total flavonoid contents from extract were quantified and H+-K+ ATPase inhibition assay was performed in presence of different concentrations(10,20,50 and 100 μg/ml) of standard (omeprazole),chloroform and methanolic extract. Results:The extract has shown dose dependent significant (*P < 0.05) proton pump inhibitory activity in the goat gastric mucosal homogenate which was compared to standard Omeprazole. Conclusions: Hence, from this study we have concluded that methanolic extract of C.quadrangularis has more inhibitory effect than chloroform extract.

THE ROLE OF PROTON ATPase SPECIFIC INHIBITOR $N,N'$-DICYCLOHEXYLCARBODIIMIDE AND EXTERNAL FORMATE CONCENTRATION ON $E.~COLI$ GROWTH DURING MIXED CARBON SOURCES FERMENTATION AT DIFFERENT pHs.

This research is focused on the investigation of specific growth rate changes of $E.~coli$ wild type and mutant strains with defect of Hyd, FDH enzymes and FhlA regulatory protein in the presence of $N,N'$-dicyclohexylcarbodiimide (DCCD) and external formate various concentration during co-fermentation of glucose, glycerol and formate at pHs $5.5-7.5.$ The highest value of SGR was observed at pH 7.5. It was revealed that SGR depends on external formate concentration at all pHs. DCCD inhibitory effect was shown mainly at pH 7.5 and partially at pH 6.5 and 5.5. In the case of the F0F1-ATPase inhibition FhlA compensatory effect on SGR was revealed.

Molecular basis of V-ATPase inhibition by bafilomycin A1

Pharmacological inhibition of vacuolar-type H+-ATPase (V-ATPase) by its specific inhibitor can abrogate tumor metastasis, prevent autophagy, and reduce cellular signaling responses. Bafilomycin A1, a member of macrolide antibiotics and an autophagy inhibitor, serves as a specific and potent V-ATPases inhibitor. Although there are many V-ATPase structures reported, the molecular basis of specific inhibitors on V-ATPase remains unknown. Here, we report the cryo-EM structure of bafilomycin A1 bound intact bovine V-ATPase at an overall resolution of 3.6-Å. The structure reveals six bafilomycin A1 molecules bound to the c-ring. One bafilomycin A1 molecule engages with two c subunits and disrupts the interactions between the c-ring and subunit a, thereby preventing proton translocation. Structural and sequence analyses demonstrate that the bafilomycin A1-binding residues are conserved in yeast and mammalian species and the 7’-hydroxyl group of bafilomycin A1 acts as a unique feature recognized by subunit c.

Ethanol Extract of Spondias mombin L. Leaves Exhibits Antidiarrhoeal Activity by Stimulation of Na+–K+ ATPase, Inhibition of Prostaglandins or Suppression of Nitric Oxide

This study was to justify the acclaimed antidiarrhoeal activity of ethanol extract of Spondias mombin leaves (EESML) and to suggest probable mechanism of action.EESML was screened for its secondary metabolites. The diarrhoeal models involved randomized Wistar rats in 5 groups of 6 animals each. Animals in groups A&B (negative and positive control) were treated with normal saline and loperamide respectively while those in groups C, D and E received 100, 200 and 400 mg/kg body weight of EESML respectively.EESML contained saponins, alkaloids, flavonoids, tannins, steroids, phenolics and glycosides. EESML lengthened the onset time of diarrhoea and as well caused reductions in the number, fresh weight and total number of wet feaces; and increase in the inhibition of defecations. EESML increased the intestinal activity of Na+–K+ ATPase; the concentrations of intestinal Na+, K+, Cl-, total protein and glucose but decreased the concentration of nitric oxide of the diarrhoeal rats. The intestinal fluid concentrations of Na+, K+, Cl- were dose dependently increased by EESML. EESML also increased the length of the small intestine.EESML possess antidiarrhoeal activity owing to the secondary metabolites, ability to enhance Na+–K+ATPase activity and electrolytes as well as suppression of nitric oxide.

Sarcoplasmic reticular Ca2+-ATPase inhibition paradoxically upregulates murine skeletal muscle Nav1.4 function

Skeletal muscle Na+ channels possess Ca2+- and calmodulin-binding sites implicated in Nav1.4 current (INa) downregulation following ryanodine receptor (RyR1) activation produced by exchange protein directly activated by cyclic AMP or caffeine challenge, effects abrogated by the RyR1-antagonist dantrolene which itself increased INa. These findings were attributed to actions of consequently altered cytosolic Ca2+, [Ca2+]i, on Nav1.4. We extend the latter hypothesis employing cyclopiazonic acid (CPA) challenge, which similarly increases [Ca2+]i, but through contrastingly inhibiting sarcoplasmic reticular (SR) Ca2+-ATPase. Loose patch clamping determined Na+ current (INa) families in intact native murine gastrocnemius skeletal myocytes, minimising artefactual [Ca2+]i perturbations. A bespoke flow system permitted continuous INa comparisons through graded depolarizing steps in identical stable membrane patches before and following solution change. In contrast to the previous studies modifying RyR1 activity, and imposing control solution changes, CPA (0.1 and 1 µM) produced persistent increases in INa within 1–4 min of introduction. CPA pre-treatment additionally abrogated previously reported reductions in INa produced by 0.5 mM caffeine. Plots of peak current against voltage excursion demonstrated that 1 µM CPA increased maximum INa by ~ 30%. It only slightly decreased half-maximal activating voltages (V0.5) and steepness factors (k), by 2 mV and 0.7, in contrast to the V0.5 and k shifts reported with direct RyR1 modification. These paradoxical findings complement previously reported downregulatory effects on Nav1.4 of RyR1-agonist mediated increases in bulk cytosolic [Ca2+]. They implicate possible local tubule-sarcoplasmic triadic domains containing reduced [Ca2+]TSR in the observed upregulation of Nav1.4 function following CPA-induced SR Ca2+ depletion.

Steroid Glycosides Hyrcanoside and Deglucohyrcanoside: On Isolation, Structural Identification, and Anticancer Activity

Cardiac glycosides (CGs) represent a group of sundry compounds of natural origin. Most CGs are potent inhibitors of Na+/K+-ATPase, and some are routinely utilized in the treatment of various cardiac conditions. Biological activities of other lesser known CGs have not been fully explored yet. Interestingly, the anticancer potential of some CGs was revealed and thereby, some of these compounds are now being evaluated for drug repositioning. However, high systemic toxicity and low cancer cell selectivity of the clinically used CGs have severely limited their utilization in cancer treatment so far. Therefore, in this study, we have focused on two poorly described CGs: hyrcanoside and deglucohyrcanoside. We elaborated on their isolation, structural identification, and cytotoxicity evaluation in a panel of cancerous and noncancerous cell lines, and on their potential to induce cell cycle arrest in the G2/M phase. The activity of hyrcanoside and deglucohyrcanoside was compared to three other CGs: ouabain, digitoxin, and cymarin. Furthermore, by in silico modeling, interaction of these CGs with Na+/K+-ATPase was also studied. Hopefully, these compounds could serve not only as a research tool for Na+/K+-ATPase inhibition, but also as novel cancer therapeutics.