Ouabain-induced Increase Research Articles

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.

Ouabain treatment is associated with upregulation of phosphatase inhibitor-1 and Na +/Ca 2+-exchanger and β-adrenergic sensitization in rat hearts

Cardiac glycosides are widely used in the treatment of congestive heart failure. While the mechanism of the positive inotropic effect after acute application of cardiac glycosides is explained by blockade of the Na +/K +-pump, little is known about consequences of a prolonged therapy. Here male Wistar rats were treated for 4 days with continuous infusions of ouabain (6.5 mg/kg/day) or 0.9% NaCl (control) via osmotic minipumps. Electrically driven (1 Hz, 35 °C) papillary muscles from ouabain-treated rats exhibited shorter relaxation time (−15%) and a twofold increase in the sensitivity for the positive inotropic effect of isoprenaline. The density and affinity of β 1- and β 2-adrenoceptors as well as mRNA and protein levels of stimulatory (G sα) and inhibitory (G iα-2, G iα-3) G-proteins were unaffected by ouabain. Similarly, SR-Ca 2+-ATPase 2A, phospholamban, ryanodine-receptor expression as well as the oxalate-stimulated 45Ca-uptake of membrane vesicles remained unchanged. However, mRNA abundance of the protein phosphatase inhibitor-1 (I-1) and the Na +/Ca 2+-exchanger (NCX) were increased by 52% and 26%, respectively. I-1 plays an amplifier role in cardiac signaling. Downregulation of I-1 in human heart failure is associated with desensitization of the β-adrenergic signaling pathway. The present data suggest that the ouabain-induced increase in I-1 expression might be at least partly responsible for the increased isoprenaline sensitivity and increased expression of NCX for the accelerated relaxation after chronic ouabain in this model.

Ouabain stimulates unidirectional and net potassium efflux in resting mammalian skeletal muscle

The present study compared ouabain-sensitive unidirectional K+ flux into (JinK) and out of (JoutK) perfused rat hindlimb skeletal muscle in situ and mouse flexor digitorum brevis (FDB) in vitro. In situ, 5 mM ouabain inhibited 54 +/- 4% of the total JinK in 28 +/- 1 min, and increased the net and unidirectional efflux of K+ within 4 min. In contrast, 1.8 mM ouabain inhibited 40 +/- 8% of the total JinK in 38 +/- 2 min, but did not significantly affect JoutK. In vitro, 1.8 and 0.2 mM ouabain decreased JinK to a greater extent (83 +/- 5%) than in situ, but did not significantly affect 42K loss rate compared with controls. The increase in unidirectional K+ efflux (JoutK) with 5 mM ouabain in situ was attributed to increased K+ efflux through cation channels, since addition of barium (1 mM) to ouabain-perfused muscles returned JoutK to baseline values within 12 min. Perfusion with 5 mM ouabain plus 2 mM tetracaine for 30 min decreased JinK 46 +/- 9% (0.30 +/- 0.03 to 0.16 +/- 0.02 micromol x min(-1) x g(-1)), however tetracaine was unable to abolish the ouabain-induced increase in unidirectional K+ efflux. In both rat hindlimb and mouse FDB, tetracaine had no effect on JoutK. Perfusion of hindlimb muscle with 0.1 mM tetrodotoxin (TTX, a Na+ channel blocker) decreased JinK by 15 +/- 1%, but had no effect on JoutK; subsequent addition of ouabain (5 mM) decreased JinK a further 32 +/- 2%. The ouabain-induced increase in unidirectional K+ efflux did not occur when TTX was perfused prior to and during perfusion with 5 mM ouabain. We conclude that 5 mM ouabain increases the unidirectional efflux of K+ from skeletal muscle through a barium and TTX-sensitive pathway, suggestive of voltage sensitive Na+ channels, in addition to inhibiting Na+/K+-ATPase activity.

Ouabain stimulates unidirectional and net potassium efflux in resting mammalian skeletal muscle

The present study compared ouabain-sensitive unidirectional K+ flux into (JinK) and out of (JoutK) perfused rat hindlimb skeletal muscle in situ and mouse flexor digitorum brevis (FDB) in vitro. In situ, 5 mM ouabain inhibited 54 ± 4% of the total JinK in 28 ± 1 min, and increased the net and unidirectional efflux of K+ within 4 min. In contrast, 1.8 mM ouabain inhibited 40 ± 8% of the total JinK in 38 ± 2 min, but did not significantly affect JoutK. In vitro, 1.8 and 0.2 mM ouabain decreased JinK to a greater extent (83 ± 5%) than in situ, but did not significantly affect 42K loss rate compared with controls. The increase in unidirectional K+ efflux (JoutK) with 5 mM ouabain in situ was attributed to increased K+ efflux through cation channels, since addition of barium (1 mM) to ouabain-perfused muscles returned JoutK to baseline values within 12 min. Perfusion with 5 mM ouabain plus 2 mM tetracaine for 30 min decreased JinK 46 ± 9% (0.30 ± 0.03 to 0.16 ± 0.02 µmol·min–1·g–1), however tetracaine was unable to abolish the ouabain-induced increase in unidirectional K+ efflux. In both rat hindlimb and mouse FDB, tetracaine had no effect on JoutK. Perfusion of hindlimb muscle with 0.1 mM tetrodotoxin (TTX, a Na+ channel blocker) decreased JinK by 15 ± 1%, but had no effect on JoutK; subsequent addition of ouabain (5 mM) decreased JinK a further 32 ± 2%. The ouabain-induced increase in unidirectional K+ efflux did not occur when TTX was perfused prior to and during perfusion with 5 mM ouabain. We conclude that 5 mM ouabain increases the unidirectional efflux of K+ from skeletal muscle through a barium and TTX-sensitive pathway, suggestive of voltage sensitive Na+ channels, in addition to inhibiting Na+/K+-ATPase activity.Key words: cardiac glycoside, Na,K pump, K+ channels, Na+ channels, perfused rat hindlimb, flexor digitorum brevis, TTX, barium, tetracaine.

Ouabain Interaction with Cardiac Na+/K+-ATPase Initiates Signal Cascades Independent of Changes in Intracellular Na+ and Ca2+ Concentrations

We have shown previously that partial inhibition of the cardiac myocyte Na(+)/K(+)-ATPase activates signal pathways that regulate myocyte growth and growth-related genes and that increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) and reactive oxygen species (ROS) are two essential second messengers within these pathways. The aim of this work was to explore the relation between [Ca(2+)](i) and ROS. When myocytes were in a Ca(2+)-free medium, ouabain caused no change in [Ca(2+)](i), but it increased ROS as it did when the cells were in a Ca(2+)-containing medium. Ouabain-induced increase in ROS also occurred under conditions where there was little or no change in [Na(+)](i). Exposure of myocytes in Ca(2+)-free medium to monensin did not increase ROS. Increase in protein tyrosine phosphorylation, an early event induced by ouabain, was also independent of changes in [Ca(2+)](i) and [Na(+)](i). Ouabain-induced generation of ROS in myocytes was antagonized by genistein, a dominant negative Ras, and myxothiazol/diphenyleneiodonium, indicating a mitochondrial origin for the Ras-dependent ROS generation. These findings, along with our previous data, indicate that increases in [Ca(2+)](i) and ROS in cardiac myocytes are induced by two parallel pathways initiated at the plasma membrane: One being the ouabain-altered transient interactions of a fraction of the Na(+)/K(+)-ATPase with neighboring proteins (Src, growth factor receptors, adaptor proteins, and Ras) leading to ROS generation, and the other, inhibition of the transport function of another fraction of the Na(+)/K(+)-ATPase leading to rise in [Ca(2+)](i). Evidently, the gene regulatory effects of ouabain in cardiac myocytes require the downstream collaborations of ROS and [Ca(2+)](i).

Involvement of Src and Epidermal Growth Factor Receptor in the Signal-transducing Function of Na+/K+-ATPase

Nontoxic concentrations of ouabain, causing partial inhibition of the cardiac myocyte Na(+)/K(+)-ATPase, induce hypertrophy and several growth-related genes through signal pathways that include the activation of Ras and p42/44 mitogen-activated protein kinase (MAPK). The aim of this work was to examine the ouabain-induced events upstream of the Ras/MAPK cascade. Treatment of myocytes with genistein antagonized ouabain-induced activation of the MAPK, suggesting that protein tyrosine phosphorylation has a role. Tyrosine phosphorylation of several myocyte proteins was increased rapidly upon cell exposure to ouabain. Lowering of extracellular K(+) had a similar ouabain-like effect. Ouabain also increased protein tyrosine phosphorylation in A7r5, HeLa, and L929 cells. In cardiac myocytes and A7r5 cells, herbimycin A antagonized the ouabain-induced increase in protein tyrosine phosphorylation and MAPK activation. In both cell types, ouabain stimulated Src kinase activity, Src translocation to the Triton-insoluble fraction, Src association with the epidermal growth factor receptor, and the tyrosine phosphorylation of this receptor on site(s) other than its major autophosphorylation site, Tyr(1173). The findings suggest that (a) the ouabain-induced activation of Src and the Src-induced phosphorylation of the growth factor receptor provide the scaffolding for the recruitment of adaptor proteins and Ras and the activation of Ras/MAPK cascade; and (b) the activation of such pathways may be a common feature of the signal-transducing function of Na(+)/K(+)-ATPase in most cells.

Increases in K+ conductance and Ca2+ influx under high glucose with suppressed Na+/K+-pump activity in rat myelinated nerve fibers.

To test the combined effect of high glucose and decreased Na+/K+-pump activity, a condition which closely mimics the diabetic state, on nerve ionic currents, changes in action potential and membrane current induced by high glucose in the presence of ouabain were investigated using voltage clamp analysis in rat single myelinated nerve fibers. In the presence of 0.1 mM ouabain, 30 mM glucose caused a progressive increase in the delayed K+ current as well as persistent decreases in action potential and Na+ current, suggesting that Na+/K+ pump plays an important role in preventing the increase in the K+ current. The latter increase was suppressed by a blocker of Ca2+-activated K+ channels. Two types of voltage-dependent Ca2+ channel blockers (L and N-type) as well as a Na+/Ca2+-exchange blocker diminished the ouabain-induced increase in K+ conductance. These results suggest that high glucose with suppressed Na+/K+ pump activity might induce an increase of Ca2+ influx through either Ca2+ channels or reverse Na+/Ca2+-exchange, possibly leading to the elevation of Ca2+-activated voltage-dependent K+ channels. Both a decrease in inward Na+ current and an increase in K+ conductance may result in decreased nerve conduction. In addition, a possible increase of axoplasmic Ca2+ concentration may lead to axonal degeneration. These results provide a clue for understanding the pathophysiologic mechanism of diabetic neuropathy.

Characteristics of sodium uptake across the basolateral membrane of oxyntic cells

To characterize further serosal Na uptake into gastric oxyntic cells under resting conditions, cellular element concentrations were determined in isolated frog (Rana temporaria) gastric mucosae using electron microprobe analysis. The epithelia were kept short circuited in Ussing-type chambers, and element analysis was performed on freeze-dried cryosections. After ouabain (10(-4) M), the [Na] in oxyntic cells increased within 30 to 60 minutes from approximately 25 to 100 mmol/kg wet wt, and [K] decreased similarly (from 100 to 25 mmol/kg wet wt). These changes occurred regardless of whether the basolateral incubation medium contained HCO3 or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) as buffers. When, prior to the addition of ouabain, 10(-3) M amiloride was applied to the serosal side to inhibit the Na-H antiporter, the ouabain-induced increase in cellular [Na] was prevented completely in HEPES-, but not in HCO3-Ringer. The data are compatible with the notion that Na is taken up by a Na-H antiporter and a Na-HCO3 symporter. At least under these experimental conditions, these transporters seem to contribute substantially to basolateral Na uptake in oxyntic cells.

Ouabain reduces net acid secretion and increases pHi by inhibiting NH4+ uptake on rat tIMCD Na(+)-K(+)-ATPase.

In the rat terminal inner medullary collecting duct (tIMCD), Na+ pump inhibition reduces transepithelial net acid secretion (JtAMM) [JH = total CO2 absorption (JtCO2)+ total ammonia secretion] and increases resting intracellular pH (pHi). The increase in pHi and reduction in JH that follow ouabain addition do not occur in the absence of NH4+ nor when NH4+ is substituted with another weak base. The purpose of this study was to explore the mechanism of the NH4(+)-dependent reduction in JtCO2 and increase in pHi that follow ouabain addition. We hypothesized that NH4+ enters the tIMCD cell through the Na(+)-K(+)-ATPase with proton release in the cytosol. To test this hypothesis, tIMCDs were dissected from deoxycorticosterone-treated rats and perfused in vitro with symmetrical physiological saline solutions containing 6 mM NH4Cl. Since K+ and NH4+ compete for a common binding site on the Na+ pump, increasing extracellular K+ should limit NH4+ (and hence net H+) uptake by the Na+ pump. Upon increasing extracellular K+ concentration from 3 to 12 mM, the NH4(+)-dependent, ouabain-induced increase in pHi and reduction in JtCO2 were attenuated. In the presence but not in the absence of NH4+, reducing Na+ pump activity by limiting Na+ entry reduced JtCO2 and attenuated ouabain-induced alkalinization. Ouabain-induced alkalinization was not dependent on the presence of HCO3-/CO2 and was not reproduced with BaCl2 or bumetanide addition. Therefore, ouabain-induced alkalinization is not mediated by the Na(+)-K(+)-2Cl- cotransporter or a HCO3- transporter and is not mediated by changes in membrane potential. In conclusion, on the basolateral membrane of the tIMCD cell, NH4+ uptake is mediated by the Na(+)-K(+)-ATPase. These data provide an explanation for the reduction in net acid secretion in the tIMCD observed following administration of amiloride or with dietary K+ loading.

Isoform-specific up-regulation by ouabain of Na+,K+-ATPase in cultured rat astrocytes.

There are two alpha-subunit isoforms (alpha1 and alpha2) and two beta-subunit isoforms (beta1 and beta2) of Na+,K+-ATPase in astrocytes, but the functional heterodimer composition is not known. Ouabain (0.5-1.0 mM) increased the levels of alpha1 and beta1 mRNAs, whereas it decreased those of alpha2 and beta2 mRNAs in cultured rat astrocytes. The increases in alpha1 and beta1 mRNAs were observed at 6-48 h after addition of the inhibitor. Immunochemical analyses showed that ouabain increased alpha1 and beta1, but not alpha2 and beta2, proteins, and that the isoforms in control and ouabain-treated cultures were of glial origin. Low extracellular K+ and monensin (20 microM) mimicked the effect of ouabain on alpha1 mRNA. The ouabain-induced increase in alpha1 mRNA was blocked by the protein synthesis inhibitor cycloheximide (10 microM), the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (30 microM), and the calcineurin inhibitor FK506 (1 nM). These findings indicate that chronic inhibition of Na+,K+-ATPase up-regulates the alpha1 and beta1, but not alpha2 and beta2, isoforms in astrocytes, suggesting a functional coupling of alpha1beta1 complex. They also suggest that intracellular Na+, Ca2+, and calcineurin may be involved in ouabain-induced up-regulation of the enzyme in astrocytes.

Dual Effect of Digitalis Glycosides on Norepinephrine Release from Human Atrial Tissue and Bovine Adrenal Chromaffin Cells: Differential Dependence on [Na+]iand [Ca2+

It was the aim of the present study (1) to characterize the influence of Na+/K+-ATPase inhibition by the digitalis glycoside ouabain on both spontaneous and nicotine-evoked norepinephrine release from the human heart; and (2) to further investigate the role of glycoside-induced changes in [Na+]iand [Ca2+]i(determined by microfluorimetry) for catecholamine release. The latter experiments were performed in bovine adrenal medullary chromaffin cells (BCC), an established cell culture model for sympathetic nerves. Ouabain (1–1000μmol/l) exerted a dual effect on norepinephrine release (determined by HPLC) from incubated human atrial tissue: (I) Ouabain induced a concentration-dependent increase in norepinephrine release, that was calcium-independent and almost completely prevented by blockade of the uptake1-carrier by desipramine (1μmol/l). The characteristics of this release process are consistent with a non-exocytotic mechanism. (II) In addition, ouabain augmented the nicotine-evoked (1–100μmol/l) calcium-dependent norepinephrine release, which can be considered to be exocytotic. Na+/K+-ATPase inhibition also reduced the threshold concentration of nicotine from 10 to 1μmol/l and it delayed the rapid tachyphylaxis of its norepinephrine releasing effect in human atrial tissue. In BCC, ouabain increased [Na+]i, [Ca2+]iand [3H] -norepinephrine release in parallel. Under calcium-free conditions, not only the ouabain-induced increase in [Na+]i, but also [3H]-norepinephrine release were enhanced. The ouabain-induced [3H]-norepinephrine release was always closely related to changes in [Na+]i, indicating a key role of [Na+]ifor this calcium-independent non-exocytotic norepinephrine release. In addition, pretreatment with ouabain (1 mmol/l) augmented the nicotine-evoked (0.1–10μmol/l) increments in [Na+]i, [Ca2+]iand [3H]-norepinephrine release. As nicotine-induced norepinephrine release depends on an increase in both [Na+]iand [Ca2+]i, these findings are indicative of an ouabain-mediated facilitation of exocytosis. In conclusion, increasing [Na+]iand [Ca2+]iinhibition of Na+/K+-ATPase by ouabain triggers non-exocytotic norepinephrine release, and facilitates nicotine-evoked exocytotic norepinephrine release.

Effect of the transmembrane gradient of magnesium and sodium on the regulation of cytosolic free magnesium concentration in human platelets.

1. To clarify the mechanism by which cytosolic free Mg2+ concentrations ([Mg2+]i) are regulated in human platelets, we investigated platelet membrane permeability to Mg2+ by altering extracellular Mg2+ concentrations, and tested the existence of a Na(+)-Mg2+ exchanger by manipulating the transmembrane Na+ gradient. 2. Platelet [Mg2+]i was 421 +/- 52 mumol/l in healthy men. [Mg2+]i remained constant during a 120 min exposure to nominally zero (low) or 5 mmol/l (high) external Mg2+ concentrations. 3. Preincubation of platelets with 10(-4) mol/l ouabain effectively decreased the transmembrane Na+ gradient. The ouabain-induced increase in [Mg2+]i was statistically significant after 30 min of exposure (14.6 +/- 2.0%) and reached 24.6 +/- 4.5% after 60 min. Similarly, the replacement of extracellular Na+ with N-methyl-D-glucamine significantly increased [Mg2+]i by 48.5 +/- 3.9% and 78.8 +/- 12.5%, respectively. 4. These results suggest that [Mg2+]i is well controlled in the presence of large transmembrane Mg2+ concentration gradients, and a Na(+)-Mg2+ exchanger may be involved in the regulation of [Mg2+]i in human platelets.

Effect of Na,K-ATPase inhibition on cytosolic free calcium ions in vascular smooth muscle cells of spontaneously hypertensive and normotensive rats

To investigate the role of Na(+)-Ca2+ exchange in the regulation of cytosolic free Ca2+ and the pathogenesis of primary hypertension. Cytosolic free Ca2+ ([Ca2+]i) in cultured vascular smooth muscle cells from normotensive and spontaneously hypertensive rats of the Münster strain was measured using the fluorescent dye fura-2 after inhibition of Na+,K+ATPase by ouabain and after addition of angiotensin II. [Ca2+]i showed a rapid increase together with a depolarization of membrane potential as measured by merocyanine 540. The ouabain-induced increase in [Ca2+]i was blocked in Ca(2+)-free medium and by nifedipine, but incubation with the inhibitor of the Na(+)-Ca2+ exchange, NiCl2, did not diminish the effect of ouabain. Likewise, in Na(+)-free medium the response to ouabain was not suppressed. The angiotensin II-induced changes in [Ca2+]i were diminished in Ca(2+)-free medium and by nifedipine, but enhanced by NiCl2. The increase in [Ca2+]i after Na+,K+ ATPase inhibition is not due to a modulation of Na(+)-Ca2+ exchange, but to a Ca2+ influx through Ca2+ channels. Changes in Na(+)-Ca2+ exchange caused by Na+,K+ ATPase inhibition may not play an important role in vascular smooth muscle cells of spontaneously hypertensive rats.

Effects of ouabain on muscle tension and intracellular Ca 2+ level in guinea-pig aorta

The effects of ouabain on muscle tension and the intracellular Ca 2+ level ([Ca 2+] i) were examined in guinea-pig aorta loaded with fura-2. Ouabain caused a gradual and sustained increase in both [Ca 2+] i and muscle tension. There was a positive correlation between these two parameters. In Ca 2+-free solution, ouabain did not affect either [Ca 2+] i or muscle tension, suggesting that the ouabain-induced increase in [Ca 2+] i was not due to Ca 2+ release from storage sites. The ouabain-induced increase in [Ca 2+] i and muscle tension was inhibited by Ni 2+, which inhibits the Na +/Ca 2+ exchanger, but not by verapamil. Furthermore, anionic and cationic amphiphiles were used as modulators of the Na +/Ca 2+ exchanger. Sodium dodecyl sulfate accelerated the responses to ouabain, whereas dodecyltrimethylammonium bromide inhibited them. These results suggest that in the guinea-pig aorta, ouabain induces contraction by increasing the Ca 2+ influx through the Na +/Ca 2+ exchanger on the plasma membrane, but not through verapamil-sensitive Ca 2+ channels.

Cultured chick-embryo heart cells respond differently to ouabain as measured by the increase in their intracellular Na + concentration

Using digital imaging microscopy with the sodium-sensitive fluorescent indicator sodium-binding benzofuran isopptalate (SBFI), we examined the cytosolic free sodium ion concentration ([Na +] i) in single chick-embryo heart cells. The distribution of the [Na +] i was homogeneous within one cell, but we found a wide cell to cell variation in the range of 3 to 18 mM [Na +] i. In contrast to former experiments showing a heterogeneity of chick-embryo heart cells with respect to their [Ca 2+] i (Ahlemeyer et al. (1992) Eur. J. Biochem. 205. 269–275), we could not distinguish cell populations with different [Na +] i. We found a lognormal distribution of the resting [Na +] i with a median value of 8.8 mM with a standard deviation of 4.5 mM ( n=90). After the addition of varying concentrations of ouabain, we found a biphasic dose-response curve as measured by the increase in [Na +] i. Ouabain showed its half-maximal effect on the [Na +] i between 10 −9 M and 10 −8 M and at 4.3·10 −6 M under steady-state conditions. The finding of a heterogeneity of chick-embryo heart cells with respect to their ouabain-induced increase in [Na +] i is consistent with our previous observations of cells differing in their [Ca 2+] i and in the sensitivity of their sodium pumps to cardiac glycosides.

On the Mechanism of Ouabain‐Induced Release of Acetylcholine from Synaptosomes

Ouabain (5 x 10(-8)-5 x 10(-4) M) was confirmed to cause a dose-dependent increase in [3H]acetylcholine ([3H]ACh) release, cytosolic free Ca2+ concentration ([Ca2+]i), and 22Na+ uptake in cerebrocortical synaptosomes of rats in the presence of extracellular Ca2+. Ouabain also caused a dose-dependent decrease in membrane potential. In a low-Na+ (10 mM) medium, ouabain failed to increase [3H]ACh release and [Ca2+]i. Tetrodotoxin (10(-6) M) had no effect on the ouabain-induced increase in both [3H]ACh release and [Ca2+]i but abolished the increase in 22Na+ uptake and partially inhibited the depolarizing effect. Verapamil (10(-6)-5 x 10(-4) M) inhibited the ouabain-induced increase in both [3H]ACh release and [Ca2+]i in a dose-dependent manner. Removal of extracellular Ca2+ abolished the effect of ouabain on [Ca2+]i but not on [3H]ACh release and 22Na+ uptake, regardless of the presence or absence of EGTA. In the absence of extracellular Ca2+, 10 mM Mg2+ blocked ouabain-induced [3H]ACh release, which was resistant to verapamil. These results suggest that ouabain can increase ACh release from synaptosomes without the preceding increases in intracellular Ca2+ and/or Na+ content. It seems likely that the removal of extracellular Ca2+ unmasks mechanisms of ouabain action different from those operating in the presence of Ca2+.

Sodium entry routes in principal and intercalated cells of the isolated perfused cortical collecting duct

Transmembrane sodium transport pathways were studied in principal and intercalated cells of the isolated perfused rabbit cortical collecting duct. Intracellular electrolyte concentrations in individual collecting duct cells were measured by electron microprobe analysis during blockage of basolateral Na-K-ATPase by ouabain and simultaneous inhibition of sodium entry across the apical and/or basolateral cell membrane. In principal cells the ouabain-induced rise in cell sodium concentration could only partially be blocked by amiloride (10(-4) mol/l) in the perfusion fluid. Amiloride (10(-3) mol/l) added to the bathing solution produced a further, significant reduction of sodium influx. In principal cells the ouabain-induced increase in sodium concentration was completely prevented by amiloride in the perfusion solution in combination with omission of sodium from the peritubular bathing solution. In intercalated cells ouabain caused a less pronounced increase in sodium concentration than in principal cells. Neither amiloride in the perfusate, nor amiloride in both bathing and perfusion solution, significantly reduced the ouabain-induced rise in intercalated cell sodium concentration. These results indicate that in principal cells amiloride-sensitive sodium channels constitute the predominant pathway for sodium entry across the apical cell membrane. In addition, substantial amounts of sodium enter principal cells across the basolateral cell membrane, probably via Na-H exchange. Finally, the data suggest that in intercalated cells sodium channels and the Na-H exchange are sparse or even absent.

Effect of ouabain on cellular free calcium and cellular cyclic AMP production in response to arginine vasopressin in rat renal papillary collecting tubule cells in culture

The effect of extracellular calcium (Ca2+) on the cellular action of arginine vasopressin (AVP) was examined using an Na+, K+-ATPase inhibitor in rat renal papillary collecting tubule cells in culture. The pretreatment of cells with ouabain enhanced basal and AVP-induced cAMP production in a dose-dependent manner. The augmentation by ouabain of cellular cAMP production in response to AVP was totally abolished by co-treatment with cobalt, lanthanum, verapamil or Ca2+-free medium containing 1 mmol EGTA/l, each blocking cellular Ca2+ uptake by different mechanisms. Two other findings indicated that ouabain directly stimulated cellular Ca2+ mobilization; namely, that ouabain significantly increased 45Ca2+ influx and cellular free Ca2+ concentration [( Ca2+]i) determined by Fura-2 fluorescence. The ouabain-induced increase in [Ca2+]i was completely blocked by either cobalt or Ca2+-free medium containing 1 mmol EGTA/l. AVP at 0.1 mumol/l increased [Ca2+]i to 177.1 +/- 26.2 nmol/l from 92.2 +/- 8.0 nmol/l (P less than 0.01) in renal papillary collecting tubule cells, and ouabain significantly enhanced the AVP-induced increase in [Ca2+]i. The increase of cellular free Ca2+ induced by ouabain probably binds to calmodulin to form an active complex of Ca2+-calmodulin in the cell, since two chemically dissimilar antagonists of calmodulin attenuated the enhancement by ouabain of cAMP production in response to AVP. These results therefore indicate that ouabain increases cellular Ca2+ uptake and enhances AVP-induced cellular free Ca2+ mobilization and its own second messenger cAMP production in renal papillary collecting tubule cells, and that extracellular Ca2+ is an important source for ouabain-mobilized cellular Ca2+.

Effect of ouabain applied by intrastriatal microdialysis on the in vivo release of dopamine, acetylcholine, and amino acids in the brain of conscious rats.

In the present study we have applied a brain microdialysis technique to investigate the effects of ouabain infusion on the release of dopamine, acetylcholine, and amino acids from striatal neurons in freely moving rats. Ouabain caused an increase in the dialysate levels of dopamine; its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC); and the amino acids glutamate, aspartate, taurine, glycine, alanine, serine, asparagine, and threonine. The ouabain-induced increase in dopamine was dose dependent and explosive (100-fold at an infusion concentration of 1 mmol/L) and contrasted strongly with the small effect of the glycoside on the output of DOPAC. We investigated the nature of ouabain-induced transmitter release by determining its sensitivity to coinfusion with tetrodotoxin or the calcium antagonist Mg2+. In the case of dopamine two mechanisms of ouabain-induced release could be established. At lower infusion concentrations ouabain induced an exocytotic type of release whereas at higher concentrations the release was probably carrier mediated. In the case of amino acids we noticed a calcium-independent release which was nerve impulse flow dependent in the case of glutamate and aspartate and impulse flow independent in the case of alanine, serine, glycine, threonine, and asparagine. Ouabain induced a decrease in the release of acetylcholine and glutamine.

Induction of system A amino acid transport through long-term treatment with ouabain: correlation with increased (Na+/K+)-ATPase activity.

Mouse embryo fibroblast cells (C3H-10T1/2) and the methylcholanthrene-transformed derivative (MCA-10T1/2) were treated with basal modified Eagle's medium (BME) containing 10% fetal bovine serum and varying concentrations of ouabain ranging from 0.05 mM to 0.7 mM for 16 h in culture. After replacing the ouabain-containing medium with Earl's balanced salts solution, System A amino acid transport activity increased from approximately 40 to 500 pmol AIB accumulated.mg protein-1.min-1 in the C3H-10T1/2 cells and from approximately 300 to 700 pmol AIB accumulated.mg protein-1.min-1 in the MCA-10T1/2 cells. The (Na+/K+)-ATPase pump activity also increased from approximately 12 to 46 nmol Rb+ accumulated.mg protein-1.min-1 in the normal cells and from approximately 20 to 42 nmol Rb+ accumulated.mg protein-1.min-1 in the transformed cells. System A and the (Na+/K+)ATPase activity were maximally increased at approximately 0.4-0.6 mM ouabain in the normal cells in contrast to the transformed cells which were maximally stimulated at a concentration of approximately 0.2 mM ouabain. This treatment with ouabain increased the [Na+]i/[K+]i as measured by atomic absorption spectroscopy, and thereby decreased the Na+ and K+ electrochemical gradients. Our data show that the internal ion gradients inverted at a lower concentration of ouabain in the transformed cells compared to the normal cells. The ouabain-induced increase in pump and System A activity shown here was used as a tool to further investigate the coordinated ion transport regulation in the control of cell growth.