Ouabain-induced Increase Research Articles

Augmented Ouabain-Induced Vascular Response Reduces Cardiac Efficiency in Mice with Migraine-Associated Mutation in the Na+, K+-ATPase α2-Isoform

Heterozygous mice (α2+/G301R mice) for the migraine-associated mutation (G301R) in the Na+,K+-ATPase α2-isoform have decreased expression of cardiovascular α2-isoform. The α2+/G301R mice exhibit a pro-contractile vascular phenotype associated with decreased left ventricular ejection fraction. However, the integrated functional cardiovascular consequences of this phenotype remain to be addressed in vivo. We hypothesized that the vascular response to α2-isoform-specific inhibition of the Na+,K+-ATPase by ouabain is augmented in α2+/G301R mice leading to reduced cardiac efficiency. Thus, we aimed to assess the functional contribution of the α2-isoform to in vivo cardiovascular function of wild-type (WT) and α2+/G301R mice. Blood pressure, stroke volume, heart rate, total peripheral resistance, arterial dP/dt, and systolic time intervals were assessed in anesthetized WT and α2+/G301R mice. To address rate-dependent cardiac changes, cardiovascular variables were compared before and after intraperitoneal injection of ouabain (1.5 mg/kg) or vehicle during atrial pacing. The α2+/G301R mice showed an enhanced ouabain-induced increase in total peripheral resistance associated with reduced efficiency of systolic development compared to WT. When the hearts were paced, ouabain reduced stroke volume in α2+/G301R mice. In conclusion, the ouabain-induced vascular response was augmented in α2+/G301R mice with consequent suppression of cardiac function.

Augmentation of Ouabain-Induced Increase in Heart Muscle Contractility by Akt Inhibitor MK-2206.

Cardiac steroids (CSs), such as ouabain and digoxin, increase the force of contraction of heart muscle and are used for the treatment of congestive heart failure (CHF). However, their small therapeutic window limits their use. It is well established that Na+, K+-ATPase inhibition mediates CS-induced increase in heart contractility. Recently, the involvement of intracellular signal transduction was implicated in this effect. The aim of the present study was to test the hypothesis that combined treatment with ouabain and Akt inhibitor (MK-2206) augments ouabain-induced inotropy in mammalian models. We demonstrate that the combined treatment led to an ouabain-induced increase in contractility at concentrations at which ouabain alone was ineffective. This was shown in 3 experimental systems: neonatal primary rat cardiomyocytes, a Langendorff preparation, and an in vivo myocardial infarction induced by left anterior descending coronary artery (LAD) ligation. Furthermore, cell viability experiments revealed that this treatment protected primary cardiomyocytes from MK-2206 toxicity and in vivo reduced the size of scar tissue 10 days post-LAD ligation. We propose that Akt activity imposes a constant inhibitory force on muscle contraction, which is attenuated by low concentrations of MK-2206, resulting in potentiation of the ouabain effect. This demonstration of the increase in the CS effect advocates the development of the combined treatment in CHF.

Involvement of IL-1β and IL-6 in antiarrhythmic properties of atorvastatin in ouabain-induced arrhythmia in rats

Purpose: Evidence show that statins possess wide beneficial cardioprotective and anti-inflammatory effects; therefore, in the present experiment, we investigated the antiarrhythmic properties of atorvastatin in ouabain-induced arrhythmia in isolated rat atria and the role of several inflammatory cytokines in this effect.Materials and methods: Male rats were pretreated with either of atorvastatin (10 mg/kg) or vehicle, orally once daily for 6 weeks. After induction of anesthesia, we isolated the atria and after incubation with ouabain, time of onset of arrhythmia and asystole as well as atrial beating rate and contractile force were recorded. We also measured the atrial levels of IL-1β, IL-6, and TNF-α after the injection of ouabain to animals.Results: Pretreatment with atorvastatin significantly delayed the onset of arrhythmia and asystole compared with vehicle-treated group (p < .01, p < .001, respectively). Incubation of ouabain boosted both atrial beating rate and contractile force in vehicle-treated group (p < .05), while these responses in atorvastatin-treated group were not significant (p > .05). Injection of ouabain elevated the atrial levels of IL-1β, IL-6, and TNF-α, while pretreatment of animals with atorvastatin could reverse the ouabain-induced increase in atrial IL-1β and IL-6 (p < .01 and p < .05, respectively).Conclusions: It is concluded that observed antiarrhythmic effects of atorvastatin might be attributed to modulation of some inflammatory cytokines, at least IL-1β and IL-6.

Involvement of Inflammatory Cytokines in Antiarrhythmic Effects of Clofibrate in Ouabain-Induced Arrhythmia in Isolated Rat Atria.

Considering the cardioprotective and anti-inflammatory properties of clofibrate, the aim of the present experiment was to investigate the involvement of local and systemic inflammatory cytokines in possible antiarrhythmic effects of clofibrate in ouabain-induced arrhythmia in rats. Rats were orally treated with clofibrate (300 mg/kg), and ouabain (0.56 mg/kg) was administered to animals intraperitoneally. After induction of anesthesia, the atria were isolated and the onset of arrhythmia and asystole was recorded. The levels of inflammatory cytokines in atria were also measured. Clofibrate significantly postponed the onset of arrhythmia and asystole when compared to control group (P ≤ 0.05 and P ≤ 0.01, resp.). While ouabain significantly increased the atrial beating rate in control group (P ≤ 0.05), same treatment did not show similar effect in clofibrate-treated group (P > 0.05). Injection of ouabain significantly increased the atrial and systemic levels of all studied inflammatory cytokines (P ≤ 0.05). Pretreatment with clofibrate could attenuate the ouabain-induced elevation of IL-6 and TNF-α in atria (P ≤ 0.01 and P ≤ 0.05, resp.), as well as ouabain-induced increase in IL-6 in plasma (P ≤ 0.05). Based on our findings, clofibrate may possess antiarrhythmic properties through mitigating the local and systemic inflammatory factors including IL-6 and TNF-α.

CAMP induction by ouabain promotes endothelin-1 secretion via MAPK/ERK signaling in beating rabbit atria.

Adenosine 3',5'-cyclic monophosphate (cAMP) participates in the regulation of numerous cellular functions, including the Na+-K+-ATPase (sodium pump). Ouabain, used in the treatment of several heart diseases, is known to increase cAMP levels but its effects on the atrium are not understood. The aim of the present study was to examine the effect of ouabain on the regulation of atrial cAMP production and its roles in atrial endothelin-1 (ET-1) secretion in isolated perfused beating rabbit atria. Our results showed that ouabain (3.0 µmol/L) significantly increased atrial dynamics and cAMP levels during recovery period. The ouabain-increased atrial dynamics was blocked by KB-R7943 (3.0 µmol/L), an inhibitor for reverse mode of Na+-Ca2+ exchangers (NCX), but did not by L-type Ca2+ channel blocker nifedipine (1.0 µmol/L) or protein kinase A (PKA) selective inhibitor H-89 (3.0 µmol/L). Ouabain also enhanced atrial intracellular cAMP production in response to forskolin and theophyline (100.0 µmol/L), an inhibitor of phosphodiesterase, potentiated the ouabain-induced increase in cAMP. Ouabain and 8-Bromo-cAMP (0.5 µmol/L) markedly increased atrial ET-1 secretion, which was blocked by H-89 and by PD98059 (30 µmol/L), an inhibitor of extracellular-signal-regulated kinase (ERK) without changing ouabain-induced atrial dynamics. Our results demonstrated that ouabain increases atrial cAMP levels and promotes atrial ET-1 secretion via the mitogen-activated protein kinase (MAPK)/ERK signaling pathway. These findings may explain the development of cardiac hypertrophy in response to digitalis-like compounds.

Structural determinants for the ouabain-stimulated increase in Na–K ATPase activity

Recent studies suggest that at low concentrations, ouabain increases Na–K ATPase and NHE1 activity and activates the Src signaling cascade in proximal tubule cells. Our laboratory demonstrated that low concentrations of ouabain increase blood pressure in rats. We hypothesize that ouabain-induced increase in blood pressure and Na–K ATPase activity requires NHE1 activity and association. To test this hypothesis we treated rats with ouabain (1μgkg body wt−1day−1) for 9days in the presence or absence of the NHE1 inhibitor, zoniporide. Ouabain stimulated a significant increase in blood pressure which was prevented by zoniporide. Using NHE1-expressing Human Kidney cells 2 (HK2), 8 (HK8) and 11 (HK11) and Mouse Kidney cells from Wild type (WT) and NHE1 knock-out mice (SWE) cell lines, we show that ouabain stimulated Na–K ATPase activity and surface expression in a Src-dependent manner in NHE1-expressing cells but not in NHE1-deplete cells. Zoniporide prevented ouabain-induced stimulation of 86Rb uptake in the NHE1-expressing cells. FRET and TIRF microscopy showed that ouabain increased association between GFP-NHE1 and mCherry-Na–K ATPase transfected into NHE1-deficient SWE cells. Mutational analysis demonstrated that the caveolin binding motif (CBM) of Na–K ATPase α1 is required for translocation of both Na–K ATPase α1 and NHE1 to the basolateral membrane. Mutations in activity or scaffold domains of NHE1 resulted in loss of ouabain-mediated regulation of Na–K ATPase. These results support that NHE1 is required for the ouabain-induced increase in blood pressure, and that the caveolin binding motif of Na–K ATPase α1 as well as the activity and scaffolding domains of NHE1 are required for their functional association.

Modification of Ca2+-handling in cardiomyocytes by redox sensitive mechanisms in response to ouabain

We examined the role of redox-sensitive signal transduction mechanisms in modifying the changes in [Ca(2+)](i) produced by ouabain upon incubating adult rat cardiomyocytes with antioxidants or inhibitors of different protein kinases and monitoring alterations in fura-2 fluorescence. Ouabain increased basal [Ca(2+)](i), augmented the KCl-induced increase in [Ca(2+)](i), and promoted oxyradical production in cardiomyocytes. These actions of ouabain were attenuated by an oxyradical scavenging mixture (superoxide dismutase plus catalase), and the antioxidants (N-acetyl-L-cysteine and N-(2-mercaptoproprionyl)glycine). An inhibitor of MAP kinase (PD98059) depressed the ouabain-induced increase in [Ca(2+)], whereas inhibitors of tyrosine kinase (tyrphostin and genistein) and PI3 kinase (Wortmannin and LV294002) enhanced the ouabain-induced increase in [Ca(2+)](i). Inhibitors of protein kinase C (calphostin and bisindolylmalaimide) augmented the ouabain-induced increase in [Ca(2+)](i), whereas stimulation of protein kinase C by a phorbol ester (phorbol 12-myristate 13-acetate) depressed the action of ouabain. These results suggest that ouabain-induced inhibition of Na (+)-K(+) ATPase may alter the redox status of cardiomyocytes through the production of oxyradicals, and increase the activities of various protein kinases. Thus, these redox-sensitive signal transduction mechanisms involving different protein kinases may modify Ca(2+)-handling sites in cardiomyocytes and determine the magnitude of net increase in [Ca(2+)](i) in response to ouabain.

Calcium-Calmodulin Kinase II Mediates Digitalis-Induced Arrhythmias

Background— Digitalis-induced Na + accumulation results in an increase in Ca 2+ i via the Na + /Ca 2+ exchanger, leading to enhanced sarcoplasmic reticulum (SR) Ca 2+ load, responsible for the positive inotropic and toxic arrhythmogenic effects of glycosides. A digitalis-induced increase in Ca 2+ i could also activate calcium-calmodulin kinase II (CaMKII), which has been shown to have proarrhythmic effects. Here, we investigate whether CaMKII underlies digitalis-induced arrhythmias and the subcellular mechanisms involved. Methods and Results— In paced rat ventricular myocytes (0.5 Hz), 50 μmol/L ouabain increased contraction amplitude by 160±5%. In the absence of electric stimulation, ouabain promoted spontaneous contractile activity and Ca 2+ waves. Ouabain activated CaMKII (p-CaMKII), which phosphorylated its downstream targets, phospholamban (PLN) (Thr17) and ryanodine receptor (RyR) (Ser2814). Ouabain-induced spontaneous activity was prevented by inhibiting CaMKII with 2.5 μmol/L KN93 but not by 2.5 μmol/L of the inactive analog, KN92. Similar results were obtained using the CaMKII inhibitor, autocamtide-2 related inhibitory peptide (AIP) (1 to 2.5 μmol/L), and in myocytes from transgenic mice expressing SR-targeted AIP. Consistently, CaMKII overexpression exacerbated ouabain-induced spontaneous contractile activity. Ouabain was associated with an increase in SR Ca 2+ content and Ca 2+ spark frequency, indicative of enhanced SR Ca 2+ leak. KN93 suppressed the ouabain-induced increase in Ca 2+ spark frequency without affecting SR Ca 2+ content. Similar results were obtained with digoxin. In vivo, ouabain-induced arrhythmias were prevented by KN93 and absent in SR-AIP mice. Conclusions— These results show for the first time that CaMKII mediates ouabain-induced arrhythmic/toxic effects. We suggest that CaMKII-dependent phosphorylation of the RyR, resulting in Ca 2+ leak from the SR, is the underlying mechanism involved.

Intracerebroventricular administration of ouabain, a Na/K-ATPase inhibitor, activates tyrosine hydroxylase through extracellular signal-regulated kinase in rat striatum

Alteration in dopamine neurotransmission has been reported to be involved in the mania of bipolar disorder. Tyrosine hydroxylase (TH) is the rate-limiting enzyme that is crucial for dopamine biosynthesis, and its activity is tightly regulated by phosphorylation at multiple N-terminal serine residues. Previously, we have reported that intracerebroventricular (ICV) injection of ouabain, a selective Na/K-ATPase inhibitor, induces hyperactivity in rats that mimics manic symptoms related to the activation of extracellular signal-regulated protein kinase1/2 (ERK1/2), which plays crucial roles in the modulation of TH phosphorylation. In this study, we investigated the effects of ICV injection of ouabain on TH phosphorylation in rat striatum and the involvement of ERK1/2 in ouabain-induced TH activation. ICV ouabain induced an acute dose-dependent increase in locomotor activity and in TH phosphorylation in rat striatum. TH phosphorylation at Ser19 was significantly increased with 100, 500, and 1000 μM ouabain, and phosphorylation at Ser31 and Ser40 was significantly increased with 500 and 1000 μM. We also found that ICV pretreatment with U0126, a specific MEK1/2 inhibitor, attenuated the 1000 μM ouabain-induced increase in TH phosphorylation at Ser19, Ser31, and Ser40, as well as the hyperactivity of rats. Moreover, the increased phosphorylation of TH (Ser19, Ser31, and Ser40) was maintained until 8 h after single administration ouabain was accompanied by increased phosphorylation of ERK1/2 (Thr202/Tyr204) and p90RSK (Thr359/Ser363). These findings imply that TH activation of the ERK1/2 signal pathway could play an important role in ouabain-induced hyperactivity of rats, a mania model.

Reducing the Late Sodium Current Improves Cardiac Function during Sodium Pump Inhibition by Ouabain

Inhibition by cardiac glycosides of Na(+), K(+)-ATPase reduces sodium efflux from myocytes and may lead to Na(+) and Ca(2+) overload and detrimental effects on mechanical function, energy metabolism, and electrical activity. We hypothesized that inhibition of sodium persistent inward current (late I(Na)) would reduce ouabain's effect to cause cellular Na(+) loading and its detrimental metabolic (decrease of ATP) and functional (arrhythmias, contracture) effects. Therefore, we determined effects of ouabain on concentrations of intracellular sodium (Na(+)(i)) and high-energy phosphates using (23)Na and (31)P NMR, the amplitude of late I(Na) using the whole-cell patch-clamp technique, and contractility and electrical activity of guinea pig isolated hearts, papillary muscles, and ventricular myocytes in the absence and presence of inhibitors of late I(Na). Ouabain (1-1.3 μM) increased Na(+)(i) and late I(Na) of guinea pig isolated hearts and myocytes by 3.7- and 4.2-fold, respectively. The late I(Na) inhibitors ranolazine and tetrodotoxin significantly reduced ouabain-stimulated increases in Na(+)(i) and late I(Na). Reductions of ATP and phosphocreatine contents and increased diastolic tension in ouabain-treated hearts were also markedly attenuated by ranolazine. Furthermore, the ouabain-induced increase of late I(Na) was also attenuated by the Ca(2+)-calmodulin-dependent kinase I inhibitors KN-93 [N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphonamide] and autocamide-2 related inhibitory peptide, but not by KN-92 [2-[N-(4'-methoxybenzenesulfonyl)]amino-N-(4'-chlorophenyl)-2-propenyl-N-methylbenzylamine phosphate]. We conclude that ouabain-induced Na(+) and Ca(2+) overload is ameliorated by the inhibition of late I(Na).

CAMKII Mediates Cardiac Glycoside Toxicity

The positive inotropic effect produced by inhibition of Na+/K+ ATPase (NKA) with digitalis has been used for the treatment of heart failure (HF) for over 200 years. However, digitalis toxicity-related death in HF patients undermines the beneficial effect of digitalis treatment. Digitalis-induced Na+ accumulation results in an increase in Ca2+i via the reverse mode of the Na+/Ca2+ exchanger (NCX) leading to enhanced SR Ca2+ load. The resulting increase in SR Ca2+ load would then be responsible for the positive inotropic effect and the toxic arrhythmogenic effects of glycosides. Digitalis-induced increase in Ca2+i could also activate CaMKII which has been shown to have proarrhythmic effects. Here we investigate whether CaMKII underlies glycoside arrhythmic effects and if so, which are the subcellular mechanisms involved. In electrically paced rat ventricular myocytes (0.5 Hz), 50 µM ouabain increased contraction amplitude by 160 ± 5%. Continued exposure to ouabain resulted in spontaneous contractile activity and Ca2+ waves which persisted even in the absence of electrical stimulation. Ouabain treatment was associated with activation of CaMKII (P-CaMKII) and phosphorilation of CaMKII downstream targets, site Tr17 of phospholamban and site Tr 2815 of the ryanodine receptor (RyR). Ouabain-induced spontaneous activity was prevented by inhibition of CaMKII with 1 µM KN93 but not by 1 µM of the inactive analogue KN92. Similar results were obtained using the structurally different CaMKII inhibitor, AIP (1-2.5µM). Ouabain treatment was associated with an increase in SR Ca2+ content and Ca2+ spark frequency, indicative of enhanced SR Ca2+ leak. KN93 suppressed the ouabain-induced increase in Ca2+ spark frequency without affecting SR content. These results suggest that CaMKII mediates ouabain-induced arrhythmic/toxic effects probably by phophorylating its SR targets. We speculate that CaMKII mediated phosphorylation of the RyR resulting Ca2+ leak from the SR could be the underlying mechanism involved.

Involvement of sarcoplasmic reticulum in changing intracellular calcium due to Na+/K+-ATPase inhibition in cardiomyocytes

Earlier studies have demonstrated that ouabain-induced increase in [Ca2+]i, as a consequence of sarcolemma (SL) Na+/K+-ATPase inhibition, is associated with activation of both the SL Na+/Ca2+ exchanger and SL Ca2+ channels. In view of the importance of sarcoplasmic reticulum (SR) in the regulation of [Ca2+]i, this study examined the role of SR in ouabain-induced increase in [Ca2+]i in both quiescent and KCl-depolarized cardiomyocytes. For this purpose, adult rat cardiomyocytes were loaded with fura-2 and ouabain-induced changes in [Ca2+]i were monitored upon treatment with or without different agents that are known to influence Ca2+ handling by the intracellular organelles. Ouabain not only increased the basal [Ca2+]i and augmented KCl-induced increase in [Ca2+]i but also produced similar effects on the ATP-induced increase in [Ca2+]i. Treatments of cardiomyocytes with caffeine, ryanodine, or cyclopiazonic acid, which affect SR Ca2+ stores, attenuated the ouabain-induced increase in basal Ca2+ as well as augmentation of the KCl response. Both ryanodine and cyclopiazonic acid produced additional effects, when used in combination with a SL Ca2+ channel inhibitor (verapamil), but not with a Na+/Ca2+ exchange inhibitor (KB-R7943). Inhibitors of Ca2+/calmodulin kinase, protein kinase A, and inositol-3-phosphate receptors were also observed to depress the ouabain-induced increase in [Ca2+]i in cardiomyocytes. On the other hand, mitochondrial Ca2+ transport inhibitors did not exert any effect on the ouabain-induced alterations in [Ca2+]i in cardiomyocytes. Furthermore, ouabain did not show any direct effect on the Ca2+ uptake and Ca2+ release activities of SR or mitochondria. These results suggest an indirect involvement of SR Ca2+ stores in the ouabain-induced increase in [Ca2+]i in cardiomyocytes and indicate the participation of both Ca2+-induced Ca2+ release and regulatory mechanisms in this action.

Na+-dependent SR Ca2+ overload induces arrhythmogenic events in mouse cardiomyocytes with a human CPVT mutation

Mutations in the cardiac ryanodine receptor Ca(2+) release channel, RyR2, underlie catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited life-threatening arrhythmia. CPVT is triggered by spontaneous RyR2-mediated sarcoplasmic reticulum (SR) Ca(2+) release in response to SR Ca(2+) overload during beta-adrenergic stimulation. However, whether elevated SR Ca(2+) content--in the absence of protein kinase A activation--affects RyR2 function and arrhythmogenesis in CPVT remains elusive. Isolated murine ventricular myocytes harbouring a human RyR2 mutation (RyR2(R4496C+/-)) associated with CPVT were investigated in the absence and presence of 1 micromol/L JTV-519 (RyR2 stabilizer) followed by 100 micromol/L ouabain intervention to increase cytosolic [Na(+)] and SR Ca(2+) load. Changes in membrane potential and intracellular [Ca(2+)] were monitored with whole-cell patch-clamping and confocal Ca(2+) imaging, respectively. At baseline, action potentials (APs), Ca(2+) transients, fractional SR Ca(2+) release, and SR Ca(2+) load were comparable in wild-type (WT) and RyR2(R4496C+/-) myocytes. Ouabain evoked significant increases in diastolic [Ca(2+)], peak systolic [Ca(2+)], fractional SR Ca(2+) release, and SR Ca(2+) content that were quantitatively similar in WT and RyR2(R4496C+/-) myocytes. Ouabain also induced arrhythmogenic events, i.e. spontaneous Ca(2+) waves, delayed afterdepolarizations and spontaneous APs, in both groups. However, the ouabain-induced increase in the frequency of arrhythmogenic events was dramatically larger in RyR2(R4496C+/-) when compared with WT myocytes. JTV-519 greatly reduced the frequency of ouabain-induced arrhythmogenic events. The elevation of SR Ca(2+) load--in the absence of beta-adrenergic stimulation--is sufficient to increase the propensity for triggered arrhythmias in RyR2(R4496C+/-) cardiomyocytes. Stabilization of RyR2 by JTV-519 effectively reduces these triggered arrhythmias.

Ca 2+ influx mechanisms in caveolae vesicles of pulmonary smooth muscle plasma membrane under inhibition of α 2β 1 isozyme of Na +/K +-ATPase by ouabain

Aims We sought to determine the mechanisms of an increase in Ca 2+ level in caveolae vesicles in pulmonary smooth muscle plasma membrane during Na +/K +-ATPase inhibition by ouabain. Main methods The caveolae vesicles isolated by density gradient centrifugation were characterized by electron microscopic and immunologic studies and determined ouabain induced increase in Na + and Ca 2+ levels in the vesicles with fluorescent probes, SBFI-AM and Fura2-AM, respectively. Key findings We identified the α 2β 1 and α 1β 1 isozymes of Na +/K +-ATPase in caveolae vesicles, and only the α 1β 1 isozyme in noncaveolae fraction of the plasma membrane. The α 2-isoform contributes solely to the enzyme inhibition in the caveolae vesicles at 40 nM ouabain. Methylisobutylamiloride (Na +/H +-exchange inhibitor) and tetrodotoxin (voltage-gated Na +-channel inhibitor) pretreatment prevented ouabain induced increase in Na + and Ca 2+ levels. Ouabain induced increase in Ca 2+ level was markedly, but not completely, inhibited by KB-R7943 (reverse-mode Na +/Ca 2+-exchange inhibitor) and verapamil (L-type Ca 2+-channel inhibitor). However, pretreatment with tetrodotoxin in conjunction with KB-R7943 and verapamil blunted ouabain induced increase in Ca 2+ level in the caveolae vesicles, indicating that apart from Na +/Ca +-exchanger and L-type Ca 2+-channels, “slip-mode conductance” of Na + channels could also be involved in this scenario. Significance Inhibition of α 2 isoform of Na +/K +-ATPase by ouabain plays a crucial role in modulating the Ca 2+ influx regulatory components in the caveolae microdomain for marked increase in (Ca 2+) i in the smooth muscle, which could be important for the manifestation of pulmonary hypertension.

Sodium pumps: ouabain, ion transport, and signaling in hypertension

TO THE EDITOR: A recent Editorial Focus in this journal (7) addresses the newly recognized role of the sodium pump as both an ion transport protein and a hormone receptor with its own signaling cascade. The relative importance of the cardiotonic steroids (CTS) as endogenous signaling hormones versus their ability to inhibit the sodium pump is of great interest. Indeed, the article noted that “although all digitalislike compounds inhibit Na-K-ATPase-mediated transport, they differ considerably in terms of their hypertensive effects.” The author concludes that “these findings do not support the prevailing hypothesis that the pathophysiological effect of digitalis results from inhibition of . . . transport activity.” This implies that the hypertensive effect of CTS may be due to the action of these agents as regulators of signal transduction, entirely unrelated “to its (Na-K-ATPase’s) role as an ion pump.” The discoveries related to Src kinase-mediated signal transduction by Na-K-ATPase add a new dimension to the view that the endogenous cardiotonic steroids comprise a novel hormone system. Nevertheless, the view that CTS induced hypertension is unrelated to inhibition of ion transport is inconsistent with published reports. Regrettably, the Editorial Focus does not cite key articles that support the conclusion that CTS hypertension is secondary to inhibition of sodium pumps. It is true that the tendency of certain CTS to induce hypertension does not correlate with their relative potency as Na-KATPase inhibitors (8, 9). The most obvious comparison is that ouabain induces hypertension while digoxin (1, 9) and a digitoxigenin derivative, PST-2238 (4), are antihypertensive. This need not imply that ouabain’s effect on blood pressure is unrelated to inhibition of Na transport. Instead, these data are consistent with the interpretation that some CTS have mixed agonist/antagonist properties. Viability of the hypothesis that enhancement of cytoplasmic Ca 2 responses by ouabain is related to inhibition of Na-KATPase relies on the secondary effect of Na on Na/Ca exchange. There is now direct evidence that elevation of blood pressure is a consequence of reduced transport activity of the ouabain-sensitive Na-K-ATPase 2-isoform (2, 3, 10) and the resultant Ca 2 gain mediated by Na/Ca exchange (3, 5, 10). Moreover, reduced 2 expression leads to murine hypertension (10). The vascular myogenic effect of that model is prevented by the Na/Ca exchange inhibitor SEA0400 (10). A key role for Na/Ca exchange is revealed by the observation that SEA0400 lowers blood pressure in ouabain-induced hypertension and other forms of salt-dependent hypertension, but not salt-independent hypertension (5). These findings demonstrate that ouabain’s hypertensive effect depends on its inhibition of the Na-K-ATPase and Ca 2 gain via Na/Ca exchange. Thus Src signaling, alone, appears to be insufficient to raise blood pressure in ouabain-induced hypertension although an accessory role is not excluded. Comparison of the effect of reduced expression of the 2 Na-K-ATPase (6) with that of low-dose ouabain on cell signaling may be a useful way to determine the roles of altered Na and Ca 2 in mediating the effects of ouabain on Src kinase signaling.

Mitochondrial Ca2+-activated K+channels more efficiently reduce mitochondrial Ca2+overload in rat ventricular myocytes

We investigated the role of the mitochondrial ATP-sensitive K(+) (K(ATP)) channel, the mitochondrial big-conductance Ca(2+)-activated K(+) (BK(Ca)) channel, and the mitochondrial permeability transition pore (MPTP) in the ouabain-induced increase of mitochondrial Ca(2+) in native rat ventricular myocytes by loading cells with rhod 2-AM. To overload mitochondrial Ca(2+), we pretreated cells with ouabain before applying mitochondrial K(ATP) or BK(Ca) channel and/or MPTP opener. Ouabain (1 mM) increased the rhod 2-sensitive fluorescence intensity (160 +/- 5.0% of control), which was dramatically decreased to the control level on application of diazoxide and NS-1619 in a dose-dependent manner (half-inhibition concentrations of 78.3 and 7.78 muM for diazoxide and NS-1619, respectively). This effect was reversed by selective inhibition of the mitochondrial K(ATP) channel by 5-hydroxydecanoate, the mitochondrial BK(Ca) channel by paxilline, and the MPTP by cyclosporin A. Although diazoxide did not efficiently reduce mitochondrial Ca(2+) during prolonged exposure to ouabain, NS-1619 reduced mitochondrial Ca(2+). These results suggest that although mitochondrial BK(Ca) and K(ATP) channels contribute to reduction of ouabain-induced mitochondrial Ca(2+) overload, activation of the mitochondrial BK(Ca) channel more efficiently reduces ouabain-induced mitochondrial Ca(2+) overload in our experimental model.

Sarcolemmal cation channels and exchangers modify the increase in intracellular calcium in cardiomyocytes on inhibiting Na+-K+-ATPase

Although inhibition of the sarcolemmal (SL) Na(+)-K(+)-ATPase is known to cause an increase in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) by stimulating the SL Na(+)/Ca(2+) exchanger (NCX), the involvement of other SL sites in inducing this increase in [Ca(2+)](i) is not fully understood. Isolated rat cardiomyocytes were treated with or without different agents that modify Ca(2+) movements by affecting various SL sites and were then exposed to ouabain. Ouabain was observed to increase the basal levels of both [Ca(2+)](i) and intracellular Na(+) concentration ([Na(+)](i)) as well as to augment the KCl-induced increases in both [Ca(2+)](i) and [Na(+)](i) in a concentration-dependent manner. The ouabain-induced changes in [Na(+)](i) and [Ca(2+)](i) were attenuated by treatment with inhibitors of SL Na(+)/H(+) exchanger and SL Na(+) channels. Both the ouabain-induced increase in basal [Ca(2+)](i) and augmentation of the KCl response were markedly decreased when cardiomyocytes were exposed to 0-10 mM Na(+). Inhibitors of SL NCX depressed but decreasing extracellular Na(+) from 105-35 mM augmented the ouabain-induced increase in basal [Ca(2+)](i) and the KCl response. Not only was the increase in [Ca(2+)](i) by ouabain dependent on the extracellular Ca(2+) concentration, but it was also attenuated by inhibitors of SL L-type Ca(2+) channels and store-operated Ca(2+) channels (SOC). Unlike the SL L-type Ca(2+)-channel blocker, the blockers of SL Na(+) channel and SL SOC, when used in combination with SL NCX inhibitor, showed additive effects in reducing the ouabain-induced increase in basal [Ca(2+)](i). These results support the view that in addition to SL NCX, SL L-type Ca(2+) channels and SL SOC may be involved in raising [Ca(2+)](i) on inhibition of the SL Na(+)-K(+)-ATPase by ouabain. Furthermore, both SL Na(+)/H(+) exchanger and Na(+) channels play a critical role in the ouabain-induced Ca(2+) increase in cardiomyocytes.

Bepridil, an Antiarrhythmic Drug, Opens Mitochondrial KATPChannels, Blocks Sarcolemmal KATPChannels, and Confers Cardioprotection

Bepridil, which is clinically useful in the treatment of arrhythmias, has been reported to inhibit sarcolemmal ATP-sensitive K(+) (sarcK(ATP)) channels. However, the effect of bepridil on mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels remains unclear. The objective of the present study was to determine whether bepridil activates mitoK(ATP) channels and confers cardioprotection. SarcK(ATP) channels composed of Kir6.2+SUR2A in human embryonic kidney (HEK) 293 cells were examined using the patch-clamp technique. Flavoprotein fluorescence in guinea pig ventricular cells and matrix volume in isolated rat heart mitochondria were measured to assay mitoK(ATP) channel activity. Mitochondrial Ca(2+) concentration ([Ca(2+)](m)) was measured by loading cells with rhod-2 fluorescence. Coronary-perfused guinea pig ventricular muscles were subjected to 35-min no-flow ischemia followed by 60-min reperfusion. Bepridil (10 microM) completely inhibited the pinacidil-induced Kir6.2+SUR2A channel current expressed in HEK 293 cells. Bepridil reversibly oxidized the flavoprotein and increased mitochondrial matrix volume in a concentration-dependent manner. Furthermore, bepridil significantly attenuated the ouabain-induced increase of [Ca(2+)](m). Pretreatment with bepridil for 5 min before ischemia improved the recovery of developed tension measured after 60 min of reperfusion. These effects of bepridil were abolished by the mitoK(ATP) channel blocker 5-hydroxydecanoate (500 microM) and by the nonselective K(ATP) channel blocker glisoxepide (10 microM). Our results indicate that bepridil is an opener of mitoK(ATP) channels but an inhibitor of sarcK(ATP) channels and exerts a direct cardioprotective effect on native cardiac myocytes. This is the first report of a unique modulator of K(ATP) channels; bepridil would be expected to mitigate ischemic injury while blunting arrhythmias.

Positive inotropic and negative chronotropic effects of proton pump inhibitors in isolated rat atrium

The effects of three specific H +/K +-ATPase inhibitors (omeprazole, lansoprazole and SCH 28080 (2-methyl-8-(phenylmethoxy)-imidazo[1,2-a] pyridine-3-acetonitrile)) were investigated on the mechanical and electrophysiological properties of rat atrium, in vitro. Omeprazole (100–300 μM), lansoprazole (100–300 μM) and SCH 28080 (10–100 μM) increased the amplitude of contractions and decreased the beating rate. These effects are reversible, reproducible and correlated with their order of potency as gastric H +/K +-ATPase inhibitors; SCH 28080 > omeprazole = lansoprazole. Cardiac effects of proton pump inhibitors were not inhibited with phentolamine (5 μM), propranolol (15 μM), atropine (1 μM), ouabain (2 μM), theophylline (300 μM) and milrinone (100 μM). Ouabain-induced increase in beating rate and contracture development were antagonized by H +/K +-ATPase inhibitors. Ouabain increased the positive inotropic effect of H +/K +-ATPase inhibitors. Lansoprazole (300 μM) significantly prolonged the duration of action potentials in rat atrial cells. H +/K +-ATPase may play a crucial role in the mechanical and electrophysiological properties of rat atrial myocardium.

The alpha2-isoform of Na-K-ATPase mediates ouabain-induced hypertension in mice and increased vascular contractility in vitro.

Although ouabain is known to induce hypertension, the mechanism of how this cardiac glycoside affects blood pressure is uncertain. The present study demonstrates that the alpha2-isoform of the Na-K-ATPase mediates the pressor effects of ouabain in mice. To accomplish this, we analyzed the effect of ouabain on blood pressure in wild-type mice, where the alpha2-isoform is sensitive to ouabain, and genetically engineered mice expressing a ouabain-insensitive alpha2-isoform of the Na-K-ATPase. Thus differences in the response to ouabain between these two genotypes can only be attributed to the alpha2-isoform of Na-K-ATPase. As the alpha1-isoform is naturally resistant to ouabain in rodents, it will not be inhibited by ouabain in either genotype. Whereas prolonged administration of ouabain increased levels of ouabain in serum from both wild-type and targeted animals, hypertension developed only in wild-type mice. In addition, bolus intravenous infusion of ouabain increased the systolic, mean arterial, and left ventricular blood pressure in only wild-type anesthetized mice. In vitro, ouabain increased vascular tone and thereby phenylephrine-induced contraction of the aorta in intact and endothelium-denuded wild-type mice but in alpha2-resistant mice. Ouabain also increased the magnitude of the spontaneous contractions of portal vein and the basal tone of the intact aorta from only wild-type mice. The increase in aortic basal tone was dependent on the presence of endothelium. Our studies also demonstrate that the alpha2-isoform of Na-K-ATPase mediates the ouabain-induced increase in vascular contractility. This could play a role in the development and maintenance of ouabain-induced hypertension.