Ouabain (Octahydrate)

Ouabain Octahydrate (Acocantherine) is a plant-derived cardiac glycoside and also an inhibitor of Na⁺,K⁺-ATPase. Ouabain Octahydrate binds to the Na⁺,K⁺-ATPase α subunit, inhibits pump activity at high concentrations, and triggers caveolin-1-dependent signaling cascades at low concentrations. Ouabain Octahydrate stimulates the phosphorylation of Erk1/2, Akt, Bad, p53, c-Src and Src kinase, activates caspase 3, generates reactive oxygen species (ROS), elevates cytoplasmic calcium levels, regulates tight junction function, alters the expression of claudin, and assembles the Na⁺,K⁺-ATPase-Src-caveolin-1 signaling complex. Ouabain Octahydrate induces cell apoptosis, oncosis, cell death, DNA synthesis, proliferation, NF-κB activation, calcium oscillation, CD69 expression, cytokine production, nitric oxide production, Na⁺,K⁺-ATPase endocytosis and positive inotropic effects, while inhibiting lymphocyte proliferation, renin release and ventricular responses induced by atrial fibrillation. Ouabain Octahydrate can be used in studies related to neuroblastoma, hypertension, atrial fibrillation and adverse renal developmental programming^{[1][2][3][4][5][6][7][8][9][10][11]}. In Vitro:Ouabain (10 nM-10 μM; 30 min pre-incubation plus 30 min ⁸⁶RbCl incubation) Octahydrate inhibits Na⁺,K⁺-ATPase-mediated ⁸⁶Rb⁺ uptake in SH-SY5Y human neuroblastoma cells with an EC₅₀ of 5.5 μM, showing minimal inhibition at concentrations ≤100 nM^[1].
Ouabain (100 nM-10 μM; 6 h) Octahydrate alters cytosolic Na⁺ and K⁺ levels in SH-SY5Y human neuroblastoma cells after 6 h of incubation, with significant changes observed at 1 μM and 10 μM, but no effect at 100 nM^[1].
Ouabain (10 nM-10 μM; 6 h) Octahydrate induces concentration-dependent phosphorylation of Akt at Ser⁴⁷³ in SH-SY5Y human neuroblastoma cells after 6 h of incubation, with a maximum 35% increase at 10 μM^[1].
Ouabain (10 nM-10 μM; 6 h) Octahydrate induces concentration-dependent phosphorylation of Bad at Ser¹¹² in SH-SY5Y human neuroblastoma cells after 6 h of incubation, with a ~2.7-fold increase at 10 μM^[1].
Ouabain (10 nM-10 μM; 6 h) Octahydrate down-regulates total Bcl-2, phosphorylated Bcl-2 (Ser70), and Bcl-XL protein levels in a concentration-dependent manner in SH-SY5Y human neuroblastoma cells after 6 h of incubation, with significant reductions observed at 1 μM and 10 μM^[1].
Ouabain (10 nM-10 μM; 6 h) Octahydrate induces concentration-dependent release of cytochrome c into the cytosol of SH-SY5Y human neuroblastoma cells after 6 h of incubation, with a maximum ~3.5-fold increase at 1 μM^[1].
Ouabain (10 nM-10 μM; 6 h) Octahydrate activates caspase 3 in a concentration-dependent manner in SH-SY5Y human neuroblastoma cells after 6 h of incubation, with detectable activation at concentrations ≥100 nM^[1].
Ouabain (10 nM-10 μM; 6 h) Octahydrate induces concentration-dependent phosphorylation of p53 at Ser15 in SH-SY5Y human neuroblastoma cells after 6 h of incubation, with a ~75% increase at 1 μM and 10 μM^[1].
Ouabain (0.1-1 nM; 5 days) Octahydrate induces increased proliferation of canine saphenous vein VSMCs, while 10 nM Ouabain reduces proliferation compared to lower concentrations^[7].
Ouabain (1 nM; 5-60 min) Octahydrate induces rapid tyrosine phosphorylation of multiple proteins, including the EGFR, in serum-starved canine saphenous vein VSMCs, with maximal phosphorylation observed at 5 min^[7].
Ouabain (1 nM; 5-15 min) Octahydrate induces robust MAPK42/44 phosphorylation in serum-starved canine saphenous vein VSMCs, with maximal activation at 15 min, while 10 nM Ouabain produces only minimal activation^[7].
Ouabain (1 nM; 5 min) Octahydrate induces a 2-fold increase in EGFR phosphorylation in serum-starved canine saphenous vein VSMCs, which is dependent on Src kinase activity^[7].
Ouabain (1 nM; 5 min) Octahydrate induces increased Src phosphorylation in serum-starved canine saphenous vein VSMCs within 5 min of stimulation^[7].
Ouabain (10 nM-10 μM; 30 min) Octahydrate increases intracellular ROS and cytosolic Ca²⁺ levels in SH-SY5Y human neuroblastoma cells after 30 min of incubation, with significant effects observed at 1 μM and 10 μM, but no effect at 10 nM or 100 nM^[1].
Ouabain (10 nM-1 μM; 3 h) Octahydrate at 10 nM does not inhibit K⁺ pumping in MDCK cells, whereas 1 μM Ouabain does^[2].
Ouabain (10 nM-1 μM; 1-3 days) Octahydrate at 10 nM does not disrupt intracellular K⁺ balance in MDCK cells, whereas 1 μM Ouabain reduces intracellular K⁺ content after 1 day^[2].
Ouabain (10-50 nM; 1-3 days) Octahydrate does not induce toxic effects including cell detachment, apoptosis, or necrosis in MDCK cells, and causes a small increase in protein content at 10 nM after 2 days^[2].
Ouabain (10-50 nM; 3 days) Octahydrate increases TER in a concentration-dependent manner in MDCK cell monolayers, with 80% and 281% increases after 3 days, respectively^[2].
Ouabain (10-50 nM; 2-3 days) Octahydrate reduces paracellular permeability to 3 kDa dextran in MDCK cell monolayers, with 10 nM causing 38.2% and 55.8% reductions after 2 and 3 days, respectively, and no concentration-dependent effect on this parameter^[2].
Ouabain (10-50 nM; 1-3 days) Octahydrate at 10 nM increases claudin 1, claudin 2, and claudin 4 protein content in a time-dependent manner in MDCK cells, while leaving occludin expression unchanged; 50 nM Ouabain does not alter occludin expression^[2].
Ouabain (10 nM; 2 days) Octahydrate modifies the subcellular distribution of claudins 1, 2, and 4 but not occludin in MDCK cells^[2].
Ouabain (10 nM) Octahydrate activates the claudin-1 promoter in MDCK cells^[2].
Ouabain (10 nM; 28 h) Octahydrate increases claudin 1 and claudin 2 mRNA levels in MDCK cells^[2].
Ouabain (10 nM; 2 days) Octahydrate increases TER in MDCK cell monolayers only when applied to the basolateral side^[2].
Ouabain (10 nM; 3 min) Octahydrate activates c-Src (via increased Y-418 phosphorylation after 3 min) and ERK1/2 in MDCK cells, with ERK1/2 phosphorylation dependent on c-Src activity^[2].
Ouabain (10 nM; 2-3 days) Octahydrate increases claudin 1 content in MDCK cells via c-Src and ERK1/2 activation, and increases claudin 4 content via ERK1/2 activation only, after 2 and 3 days of treatment, respectively^[2].
Ouabain (10 nM) Octahydrate increases TER in MDCK cell monolayers via activation of both c-Src and partially ERK1/2, and reduces nonionic paracellular permeability (JDEX) via ERK1/2 activation only^[2].
Ouabain (25 nM-1 μM; 2 min-15 min) Octahydrate regulates the interaction between Na⁺/K⁺-ATPase and caveolin-1 in pig LLC-PK1 cells in a time- and dose-dependent manner, with maximal effects at 1 μM Ouabain for 5 min and significant stimulation observed at concentrations as low as 25 nM^[3].
Ouabain (1 μM; 2 min-15 min) Octahydrate stimulates Src-dependent tyrosine phosphorylation of caveolin-1 at Tyr-14 and promotes formation of the Na⁺/K⁺-ATPase-Src-caveolin-1 signaling complex in pig LLC-PK1 cells^[3].
Ouabain (10 nM-10 μM; 5 min) Octahydrate induces Src-dependent tyrosine phosphorylation of proteins and formation of the Na⁺/K⁺-ATPase-Src-caveolin-1 signaling complex specifically in isolated caveolae fractions from pig LLC-PK1 cells, but not in non-caveolar membrane fractions^[3].
Ouabain (1 μM; 5 min-30 min) Octahydrate -induced ERK1/2 activation in pig LLC-PK1 cells depends on caveolar Cholesterol (Cholesterol (from animal)) (HY-N0322), with activity restored upon Cholesterol repletion, while Ouabain-induced ERK activation in human 293 cells is partially dependent on caveolin-1 expression^[3].
Ouabain (100 nM-1 mM; 6 h) Octahydrate induces plasma membrane depolarization in murine thymocytes, lymph node lymphocytes, and spleen lymphocytes at 1 mM but not at 100 nM after 6 h of incubation^[4].
Ouabain (1 mM; 6 h) Octahydrate synergizes with 100 nM Dexamethasone (HY-14648) to enhance plasma membrane depolarization in murine thymocytes, lymph node lymphocytes, and spleen lymphocytes after 6 h of co-incubation^[4].
Ouabain (30 nM) Octahydrate induces intracellular calcium mobilization in murine thymocytes via a mechanism independent of Na,K-ATPase pump inhibition^[4].
Ouabain Octahydrate modulates murine bone marrow hematopoietic progenitor cell activity in a concentration-dependent manner, inhibiting CFU-S and CFU-C formation while enhancing BFU-E and CFU-E proliferation^[4].
Ouabain (50 nM; 12 h) Octahydrate reduces Ouabain-sensitive ⁸⁶Rb uptake by 58% in LLC-PK1 cells, with full recovery of uptake observed within 24 hours of Ouabain washout^[5].
Ouabain (50 nM; 12 h) Octahydrate reduces plasmalemmal Na/K-ATPase α1 and β1 subunit density by ~60% in LLC-PK1 cells, with full recovery of surface subunit density observed within 24 hours of Ouabain washout^[5].
Ouabain (50 nM; 12 h) Octahydrate induces internalization of Na/K-ATPase α1 subunits and translocation of some subunits to the nucleus in LLC-PK1 cells^[5].
Ouabain (50 nM; 12 h) Octahydrate stimulates translocation of biotinylated cell surface Na/K-ATPase α1 subunits to the nucleus in LLC-PK1 cells^[5].
Ouabain (50 nM; 1 h) Octahydrate increases Na/K-ATPase α1 subunit content in clathrin-coated vesicles by 174% in LLC-PK1 cells^[5].
Ouabain (50 nM; 1 h) Octahydrate enhances protein-protein interactions between Na/K-ATPase α1 subunits, AP-2 α subunits, and clathrin heavy chains in LLC-PK1 cells^[5].
Ouabain (50 nM; 2 h) Octahydrate increases Na/K-ATPase α1 subunit content in early and late endosomes in LLC-PK1 cells^[5].
Ouabain (50 nM; 2 h) Octahydrate induces accumulation of Na/K-ATPase α1 subunits in early endosomes in LLC-PK1 cells, which is attenuated by inhibition of Src kinase via PP2^[5].
Ouabain (50 nM) Octahydrate activates PI(3)K in LLC-PK1 cells, with significant increases in activity observed at 15 and 30 minutes post-exposure^[5].
Ouabain (50 nM) Octahydrate induces endocytosis and nuclear translocation of Na/K-ATPase α1 subunits in LLC-PK1 cells, which is attenuated by inhibition of clathrin-mediated endocytosis or PI(3)K^[5].
Ouabain (10 μM) Octahydrate induces accumulation of Na/K-ATPase α1 subunits in early and late endosomes in Src-reconstituted SYF⁺ cells, but not in Src-deficient SYF^- cells^[5].
Ouabain Octahydrate induces CD69 expression in murine thymocytes without causing plasma membrane depolarization^[4].
Ouabain (100 nM; 6 h-18 h) Octahydrate reduces NFATc1 expression in resting murine thymocytes and inhibits Con A-induced NFATc1 expression in murine thymocytes after 6 h and 18 h of incubation^[4].
Ouabain (10 nM-100 nM; 50 min-18 h) Octahydrate inhibits Con A (Concanavalin A) (HY-P2149)-induced p38 phosphorylation in murine thymocytes at concentrations as low as 10 nM, while 100 nM Ouabain alone does not induce p38 phosphorylation at any tested incubation time (50 min, 2 h, 6 h, 18 h)^[4].
Ouabain Octahydrate induces apoptosis in activated lymphocytes and the human Jurkat T lymphocyte cell line, likely through exacerbation of activation-induced cell death^[4].
Ouabain (1 mM) Octahydrate protects rat vascular smooth muscle cells from apoptosis induced by growth factor withdrawal via a Na⁺-dependent, Ca²⁺-independent mechanism that upregulates antiapoptotic genes including mortalin, with maximal antiapoptotic activity observed at 1 mM Ouabain^[8].
Ouabain Octahydrate dose-dependently inhibits Na,K-ATPase activity in primary rat proximal tubule cells, with near-maximal inhibition achieved at 2000 μM^[6].
Ouabain (50-500 μM; 20 min, 10-100 nM; 3 h, 2000 μM; 15 min) Octahydrate induces regular, low-frequency intracellular calcium oscillations in primary rat proximal tubule cells at concentrations causing partial Na,K-ATPase inhibition (50-500 μM) and at nanomolar concentrations (10-100 nM) with prolonged exposure, while full Na,K-ATPase inhibition at 2000 μM causes a sustained calcium increase without oscillations^[6].
Ouabain (100-250 μM; 30 min) Octahydrate induces a sustained, dose-dependent increase in intracellular sodium levels in primary rat proximal tubule cells^[6].
Ouabain (1 nM; 10 min) Octahydrate does not significantly inhibit sodium pump activity in canine saphenous vein VSMCs, while 0.1 mM Ouabain completely inhibits pump activity^[7].
Ouabain (250 μM) Octahydrate activates NF-κB in primary rat proximal tubule cells via induced intracellular calcium oscillations, as blocking these oscillations prevents NF-κB nuclear translocation and IκBα reduction^[6].
Ouabain (0.1-1 nM; 24 h) Octahydrate induces increased DNA synthesis in canine saphenous vein VSMCs, with 1 nM Ouabain producing a 32% increase in BrdUrd uptake, while 10 nM Ouabain has no effect^[7].
Ouabain (3 μM; 24 h) Octahydrate induces oncotic cell death in non-rodent cells expressing α1S-Na,K-ATPase via a Na⁺/K⁺-independent mechanism involving α1S-specific conformational changes and p38 MAPK activation, with 3 μM Ouabain causing massive death after 24 h; this effect is absent in cells expressing rodent α1R-Na,K-ATPase and can be inhibited by modest intracellular acidification^[8]. In Vivo:Ouabain (13.9-27.8 μg/kg per day; i.p.; loading dose on day 1 followed by daily maintenance dose; 6-8 weeks) Octahydrate produces sustained, dose-dependent hypertension in normotensive rats, with greater potency in rats with higher degrees of reduced renal mass, mediated by increased peripheral vascular resistance, and significantly elevates kidney tissue Ouabain Octahydrate levels and reduces plasma renin activity in reduced renal mass rats^[9].
Ouabain (0.025 mg/kg; i.v.; single infusion over 30 seconds) Octahydrate produces a late-phase plasma concentration half-life of 18 hours in healthy mongrel dogs^[10].
Ouabain (15 μg/kg per day; s.c.; continuous; from day 2 of pregnancy through gestation) Octahydrate completely rescues the low nephron endowment caused by maternal low-protein diet in 4-week-old Sprague-Dawley rat offspring^[11].