Serum stimulation of potassium fluxes,ouabain binding,and sodium fluxes in quiescent chicken embryo fibroblasts

Growth-contingent alterations in potassium and sodium fluxes, ouabain binding, and potassium ion content were examined following serum stimulation of quiescent, density-inhibited chicken embryo fibroblasts. Serum stimulation resulted in very rapid 1.5- to 1.8-fold increases in ouabain-sensitive potassium influx and lesser 1.4- to 1.5-fold increases in potassium efflux and sodium influx. Potassium influx stimulation was maximal after addition of 5–20% calf serum and was unaffected by cycloheximide inhibition of protein synthesis. Reflecting the slightly greater stimulation of potassium influx versus potassium efflux, potassium ion levels were 10–15% higher in serum-stimulated compared to unstimulated cells. Specific ouabain binding levels in stimulated and unstimulated control cells were initially similar, however, by four hours after stimulation a 40–50% increase in specific ouabain binding was observed. Incubation with ouabain was found also to inhibit later serum-stimulated hexose uptake and thymidine incorporation; this blockage may be a consequence of subnormal potassium levels rather than ouabain inhibition of the serum-stimulated potassium influx.     

Isolation of ouabain-resistant human diploid fibroblasts

Seventeen clones resistant to the cytotoxic action of ouabain were isolated in culture by direct selection from 5 independent strains of diploid human fibroblasts. Resistant clones were recovered at frequencies on the order of 10^?7 per wild type cell selected from populations treated with the mutagen EMS, but no resistant cells were detected among 10⁸ unmutagenized cells. Most selected clones remained ouabain-resistant following further propagation in the absence of drug. The growth of wild type cells was inhibited by 50% at ouabain concentrations of 2–5 × 10^?8 M, while resistant clones required 15–180 fold higher drug concentrations to cause equivalent inhibition. Ouabain-resistant clones showed increased resistance of K+ transport function to ouabain inhibition that paralleled their increased resistance to growth inhibition. Initial experiments suggest that under selective conditions the resistant diploid fibroblasts differ significantly from wild type in binding of ³H-ouabain per unit surface area. The ouabain-resistant cells were similar to wild type in transport properties unrelated to ouabain inhibition. Resistant cells had normal karyotypes and senesced with a lifespan similar to control clones. The ouabain-resistant phenotypes of these diploid human fibroblast isolates apparently reflect point mutations that specifically affect the Na+/K+ transport ATPase with respect to ouabain-binding and/or response to bound ouabain.     

Ouabain-induced stimulation of sodium-hydrogen exchange in rat optic nerve astrocytes

Sodium-dependent transporters are inhibited indirectly by the Na-K-ATPase inhibitor ouabain. Here we report stimulation of sodium-hydrogen exchange (NHE) in ouabain-treated cells. BCECF was used to measure cytoplasmic pH in cultured rat optic nerve astrocytes. Ammonium chloride was applied to acid load the cells. On removal of ammonium chloride, cytoplasmic pH fell abruptly, then gradually recovered toward baseline. Ouabain (1 microM) did not change cell sodium content, but the rate of pH recovery increased by 68%. Ouabain speeded pH recovery both in the presence and absence of bicarbonate. In bicarbonate-free medium, dimethylamiloride, an NHE inhibitor, eliminated the effect of 1 microM ouabain on pH recovery. Western blot analysis showed an NHE1 immunoreactive band but not NHE2, NHE3, or NHE4. Immunoprecipitation studies showed phosphorylation of NHE1 in cells treated with 1 microM ouabain. Ouabain evoked an increase of cAMP, and the effect of 1 microM ouabain on pH recovery was abolished by H-89, a protein kinase A inhibitor. 8-Bromoadenosine-cAMP increased the pH recovery rate, and this recovery was not further increased by ouabain. Although 1 microM ouabain did not alter cytoplasmic calcium concentration, it stimulated calcium entry after store depletion, a response abolished by 2-APB. Ouabain-induced stimulation of pH recovery was suppressed by inhibitors of capacitative calcium entry, SKF-96365, and 2-APB, as well as the cytoplasmic calcium chelator BAPTA. The cAMP increase in ouabain-treated cells was abolished by BAPTA and 2-APB. Taken together, the results are consistent with increased capacitative calcium entry and subsequent cAMP-PKA-dependent stimulation of NHE1 in ouabain-treated cells.     

Regulation of the sodium permeability of the luminal border of toad bladder by intracellular sodium and calcium: role of sodium-calcium exchange in the basolateral membrane

Sodium movement across the luminal membrane of the toad bladder is the rate-limiting step for active transepithelial transport. Recent studies suggest that changes in intracellular sodium regulate the Na permeability of the luminal border, either directly or indirectly via increases in cell calcium induced by the high intracellular sodium. To test these proposals, we measured Na movement across the luminal membrane (th Na influx) and found that it is reduced when intracellular Na is increased by ouabain or by removal of external potassium. Removal of serosal sodium also reduced the influx, suggesting that the Na gradient across the serosal border rather than the cell Na concentration is the critical factor. Because in tissues such as muscle and nerve a steep transmembrane sodium gradient is necessary to maintain low cytosolic calcium, it is possible that a reduction in the sodium gradient in the toad bladder reduces luminal permeability by increasing the cell calcium activity. We found that the inhibition of the influx by ouabain or low serosal Na was prevented, in part, by removal of serosal calcium. To test for the existence of a sodium- calcium exchanger, we studied calcium transport in isolated basolateral membrane vesicles and found that calcium uptake was proportional to the outward directed sodium gradient. Uptake was not the result of a sodium diffusion potential. Calcium efflux from preloaded vesicles was accelerated by an inward directed sodium gradient. Preliminary kinetic analysis showed that the sodium gradient changes the Vmax but not the Km of calcium transport. These results suggest that the effect of intracellular sodium on the luminal sodium permeability is due to changes in intracellular calcium.     

Expression of a Cx43 Deletion Mutant in 3T3 A31 Fibroblasts Prevents PDGF-Induced Inhibition of Cell Communication and Suppresses Cell Growth

Communication through gap junctions was first suggested to have a role in the social control of cell growth over 30 years ago. However, despite extensive experimentation, the importance of gap junctions as a general mechanism of growth control remains to be established. A number of different studies have shown that a common early response of cells in culture to polypeptide growth factors such as PDGF is a rapid and transient inhibition of cell communication suggesting that a cell may have to lose communication with its neighbors before it can undergo cell division. Here we show that 3T3 A31 fibroblasts exposed to PDGF exhibit a 50% decrease in cell communication as measured by dye transfer in the absence of significant changes in the cellular content and distribution of Cx43. Likewise, PDGF inhibited cell communication in cells transfected either with a vector which did not contain a cDNA or with an expression vector encoding full-length Cx43 fused to a c-myc tag (Cx43-M). In contrast, 3T3 A31 fibroblasts transfected with an expression construct encoding a deletion mutant of Cx43 (Cx43-256M) consisting of amino acids 1-256 of Cx43 fused to a c-myc tag maintain high levels of gap junction activity following exposure to PDGF. These results suggest that sites which trigger loss of cell communication in response to PDGF are located within amino acids 257 to 382 of the Cx43 molecule. Cells transfected with an expression vector encoding full-length Cx43 fused to a c-myc tail exhibited a reduced basal growth rate compared to both parent cells and cells transfected with a control vector but maintained a strong mitogenic response to PDGF. In contrast, both the basal growth rate and the mitogenic response to PDGF was markedly reduced in cells which expressed Cx43-256M consistent with the hypothesis that loss of cell communication is required before a cell can respond to mitogenic stimuli.     

Ouabain stimulates protein kinase B (Akt) phosphorylation in opossum kidney proximal tubule cells through an ERK-dependent pathway

Endogenous cardiotonic glycosides bind to the inhibitory binding site of the plasma membrane sodium pump (Na⁺/K⁺-ATPase). Plasma levels of endogenous cardiotonic glycosides increase in several disease states, such as essential hypertension and uremia. Low concentrations of ouabain, which do not inhibit Na⁺/K⁺-ATPase, induce cell proliferation. The mechanisms of ouabain-mediated response remain unclear. Recently, we demonstrated that in opossum kidney (OK) proximal tubular cells, low concentrations of ouabain induce cell proliferation through phosphorylation of protein kinase B (Akt) in a calcium-dependent manner. In the present study, we identified ERK as an upstream kinase regulating Akt activation in ouabain-stimulated cells. Furthermore, we provide evidence that low concentrations of ouabain stimulate Na⁺/K⁺-ATPase-mediated ⁸⁶Rb uptake in an Akt-, ERK-, and Src kinase-dependent manner. Ouabain-mediated ERK phosphorylation was inhibited by blockade of intracellular calcium release, calcium entry, tyrosine kinases, and phospholipase C. Pharmacological inhibition of phosphoinositide-3 kinase and Akt failed to inhibit ouabain-stimulated ERK phosphorylation. Ouabain-mediated Akt phosphorylation was inhibited by U0126, a MEK/ERK inhibitor, suggesting that ouabain-mediated Akt phosphorylation is dependent on ERK. In an in vitro kinase assay, active recombinant ERK phosphorylated recombinant Akt on Ser⁴⁷³. Moreover, transient transfection with constitutively active MEK1, an upstream regulator of ERK, increased Akt phosphorylation and activation, whereas overexpression of constitutively active Akt failed to stimulate ERK phosphorylation. Ouabain at low concentrations also promoted cell proliferation in an ERK-dependent manner. These findings suggest that ouabain-stimulated ERK phosphorylation is required for Akt phosphorylation on Ser⁴⁷³, cell proliferation, and stimulation of Na⁺/K⁺-ATPase-mediated ⁸⁶Rb uptake in OK cells. opossum kidney cells; sodium/potassium adenosine triphosphatase; extracellular signal-regulated kinase; cell proliferation     

Ouabain induces cell proliferation through calcium-dependent phosphorylation of Akt (protein kinase B) in opossum kidney proximal tubule cells

Cardiotonic glycosides, like ouabain, inhibit Na⁺-K⁺-ATPase. Recent evidence suggests that low molar concentrations of ouabain alter cell growth. Studies were conducted to examine the effect of ouabain on Akt phosphorylation and rate of cell proliferation in opossum kidney (OK) proximal tubule cells. Cells exposed to 10 nM ouabain displayed increased Akt Ser⁴⁷³ phosphorylation, as evidenced by an increase in phospho-Akt Ser⁴⁷³ band density. Ouabain-stimulated Akt Ser⁴⁷³ phosphorylation was inhibited by pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 and wortmannin), a PLC inhibitor (edelfosine), and an Akt inhibitor. Moreover, ouabain-mediated Akt Ser⁴⁷³ phosphorylation was suppressed by reduction of extracellular calcium (EGTA) or when intracellular calcium was buffered by BAPTA-AM. An inhibitor of calcium store release (TMB-8) and an inhibitor of calcium entry via store-operated calcium channels (SKF96365) also suppressed ouabain-mediated Akt Ser⁴⁷³ phosphorylation. In fura-2 AM-loaded cells, 10 nM ouabain increased capacitative calcium entry (CCE). Ouabain at 10 nM did not significantly alter baseline cytoplasmic calcium concentration in control cells. However, treatment with 10 nM ouabain caused a significantly higher ATP-mediated calcium store release. After 24 h, 10 nM ouabain increased the rate of cell proliferation. The Akt inhibitor, BAPTA-AM, SKF96365, and cyclopiazonic acid suppressed the increase in the rate of cell proliferation caused by 10 nM ouabain. Ouabain at 10 nM caused a detectable increase in ⁸⁶Rb uptake but did not significantly alter Na⁺-K⁺-ATPase (ouabain-sensitive pNPPase) activity in crude membranes or cell sodium content. Taken together, the results point to a role for CCE and Akt phosphorylation, in response to low concentrations of ouabain, that increase the rate of cell proliferation without inhibiting Na⁺-K⁺-ATPase-mediated ion transport. Na⁺-K⁺-ATPase; opossum kidney cells     

Ouabain-induced enhancement of rat mast cells response. Modulation by protein phosphorylation and intracellular pH

The digitalic glicoside ouabain induces potentiation of rat mast cell histamine release in response to several stimuli, which is mediated by Na+/Ca2+ exchanger. In this work, we studied the effect of ouabain on cytosolic calcium, intracellular pH and histamine release with Ca2+ ionophore A23187 in conditions designed to maximize ouabain-induced potentiation of rat mast cells response. The effect of protein kinase C (PKC), cAMP and phosphatase inhibition was also tested. Ouabain induced an enhancement in histamine release, cytosolic calcium and intracellular pH. The adenylate cyclase activator forskolin reduced the effect of ouabain on histamine release and intracellular pH, but enhanced the effect on cytosolic calcium. PKC activator PMA enhanced the effect of ouabain on histamine release and cytosolic calcium, without affecting intracellular pH. A PKC inhibitor, GF-109203X, reduced ouabain-induced enhancement of histamine release and intracellular pH, but increased the enhancement on cytosolic calcium. Finally, inhibition of protein phosphatases 1 and 2A with okadaic acid, increased the effect of ouabain on histamine release and intracellular pH, but reduced cytosolic calcium in presence of ouabain. This result suggest that ouabain-induced potentiation of rat mast cell histamine release with A23187 is modulated by kinases, and this modulation may be carried out by changes in intracellular alkalinization. However, the mechanism underlying cellular alkalinization remains to be elucidated.     

Three correlations between extracellular calcium concentration and myocardial cell injury induced by drugs

Primary cultures of newborn rat myocardial cells were treated in various extracellular calcium concentrations (0, 1.35, 2.7, 4.05, and 5.4 mM) with three different drugs; namely, ouabain, sulmazole, and chlorpromazine. Lactate dehydrogenase (LDH) release was used as an indicator of damage. The results showed 10(-3) M ouabain caused apparent damage of the cells and the damage was increased by an increased extracellular calcium concentration. Sulmazole (10(-3)M) caused damage of the cells in the absence of calcium; but it did not cause damage of the cells in the presence of calcium; it protected the cells from damage caused by high calcium concentrations (4.05 and 5.4 mM). Chlorpromazine (1.6 X 10(-4)M) caused severe damage of the cells. The various calcium concentrations had no influence on the degree of the damage. Correlation coefficients showed that correlations between the calcium concentrations and the cell damage caused by ouabain, sulmazole and chlorpromazine were positive correlation, negative correlation, and no correlation, respectively. It is suggested that influx of extracellular calcium is not a final common pathway of drug-induced myocardial cell injury, although it plays an important role in cell injury.     

ATP Splitting and Calcium Binding by Brain Microsomes Measured with a Rapid Perfusion Method

Rat brain microsomes, immobilized on a filter, were perfused with ATP-containing solutions in a device which made possible rapid change of perfusion media and frequent sampling of effluent. Inorganic phosphate production could be measured 10 times per sec. When ATP, sodium, or potassium was absent from the first perfusion medium and present in a second, and introduced without interrupting flow, phosphate output rose within a few tenths of a second. Inhibition by ouabain began within 0.3 sec but did not become maximal for at least 10 sec. Rapid binding of ouabain was minimal or absent, as was rapid release of ouabain on introducing potassium abruptly. Although the preparation bound some calcium reversibly, no measurable uptake of calcium occurred coincident with activation by ATP or by potassium, and no measurable release of calcium occurred coincident with the onset of ouabain inhibition. However, activation by sodium was consistently associated with simultaneous release (within < 1 sec) of calcium, averaging 46 pmole per mg of protein. Calcium release in response to sodium also occurred in the absence of ATP or in the presence of ouabain. At 0°C sodium produced neither activation nor calcium release. The results are consistent with the possibility that sodium and calcium are competitively bound, even in the absence of ATP, to an active site on the enzyme distinct from the sites of potassium activation or glycoside inhibition.     

Regulation of cellular growth by sodium pump activity.

Cellular growth has been found to be directly related to the amount of sodium pumping activity in mouse lymphoblasts (L5178-Y) cultured in varying concentrations of the cardiac glycoside, ouabain. No short-term adaptation (within one generation) occured; i.e., neither growth rate nor (Na+ + K+)-ATPase activity increased in cells cultured for 1-2 days in ouabain. Growth inhibition commenced after two hours, occurring concomitantly with decreased 3H-leucine incorporation into protein. The time course of this inhibition of protein synthesis, measured by leucine incorporation was similar to, but slightly slower than the time course or the dissipation of the sodium gradient. On the other hand, 3H-thymidine incorporation is unaffected by ouabain treatment over the same period. The uptake of 3H-alanine, a neutral amino acid thought to be transported via a Na+-dependent carri-r, was depressed concurrently with the sodium gradient dissipation. It is suggested, therefore, that ouabain inhibition of cellular growth results primarily from the dissipation of the sodium gradient leading to decreased Na+-dependent transport of amino acids (e.g., alanine) and, therefore, decreased protein synthesis, as observed by leucine incorporation. A sensitive and rapid method for determining ouabain inhibition of cell volume regulation is also described, which may prove potentially useful for assaying Na pump activity.     

Autoregulation of the electrogenic sodium pump

The dependence of electrogenic sodium pump activity on changes in the cell volume of Helix pomatia neurons with different levels of intracellular sodium ion concentration was studied. Hypertonic solutions caused hyperpolarization of the membrane and increased membrane resistance in cells with a low sodium content (low-sodium cells; LSC). The activity of the electrogenic sodium pump in hypertonic solutions was increased compared to the activity in hypotonic solutions in LSC and decreased in cells with a high sodium content (high-sodium cells; HSC). The concentration of ouabain which led to maximal inhibition of active 22Na efflux from the neurons was 10(-4) M. Lower concentrations of ouabain (10(-8) M and lower) did not inhibit the sodium pump but stimulated it. The swelling of neurons in hypotonic solutions was accompanied by an increase in the number of binding sites for ouabain, while shrinking in hypertonic solutions led to the opposite effect--a decrease in binding sites. An increase in the number of binding sites also took place in normal isotonic potassium-free solutions compared with normal Ringer's solution. Two saturable components of ouabain binding were detectable in all solutions examined. gamma-Aminobutyric acid (GABA) and acetylcholine (ACh) increased the number of ouabain binding sites on the membrane. The results suggest that there are two opposite mechanisms by which cell volume changes can modulate the pump activity. One of them depends on the intracellular sodium ion concentration and causes pump activation in hypertonic solutions in LSC and saturation in HSC, while a second mechanism mediates the activating effect of cell swelling on the sodium pump in HSC. In addition, there may be a negative feedback between the pump activity and the number of functioning pump units in the membrane.     

Na,K-ATPase Activity Is Required for Formation of Tight Junctions, Desmosomes, and Induction of Polarity in Epithelial Cells

Na,K-ATPase is a key enzyme that regulates a variety of transport functions in epithelial cells. In this study, we demonstrate a role for Na,K-ATPase in the formation of tight junctions, desmosomes, and epithelial polarity with the use of the calcium switch model in Madin-Darby canine kidney cells. Inhibition of Na,K-ATPase either by ouabain or potassium depletion prevented the formation of tight junctions and desmosomes and the cells remained nonpolarized. The formation of bundled stress fibers that appeared transiently in control cells was largely inhibited in ouabain-treated or potassium-depleted cells. Failure to form stress fibers correlated with a large reduction of RhoA GTPase activity in Na,K-ATPase-inhibited cells. In cells overexpressing wild-type RhoA GTPase, Na,K-ATPase inhibition did not affect the formation of stress fibers, tight junctions, or desmosomes, and epithelial polarity developed normally, suggesting that RhoA GTPase is an essential component downstream of Na,K-ATPase-mediated regulation of these junctions. The effects of Na,K-ATPase inhibition were mimicked by treatment with the sodium ionophore gramicidin and were correlated with the increased intracellular sodium levels. Furthermore, ouabain treatment under sodium-free condition did not affect the formation of junctions and epithelial polarity, suggesting that the intracellular Na(+) homeostasis plays a crucial role in generation of the polarized phenotype of epithelial cells. These results thus demonstrate that the Na,K-ATPase activity plays an important role in regulating both the structure and function of polarized epithelial cells.     

He—Ne激光照射对成纤维细胞内[Ca^2＋]i浓度影响的流式细胞计测定

目的：Ｈｅ－Ｎｅ激光照射治疗的机理不明,激光照射引起细胞内Ｃａ＾２＋水平变化,为治疗机理提供理论依据。方法：Ｈｅ－Ｎｅ激光照射引起鼠成纤维细胞Ｌ９２９内［Ｃａ＾２＋］ｉ的变化,用ＨＯ３４２对细胞ＤＮＡ活性染色,Ｆｌｕｏ－３ＡＭ对细胞内Ｃａ＾２＋染色,利用ＦＣＭ同时定量分析细胞ＤＮＡ和细胞内Ｃａ＾２＋的变化。结果：激光照射１５ｍｉｎ（光剂量１１．８１Ｊ／ｃｍ＾２后,ＦＣＭ分析可见ＤＮＡ分布直方图右移     

Feedback inhibition of NaCl entry inNecturus gallbladder epithelial cells

Summary WhenNecturus gallbladder epithelium is treated with ouabain the cells swell rapidly for 20–30 minutes then stabilize at a cell volume 30% greater than control. The cells then begin to shrink slowly to below control size. During the initial rapid swelling phase cell Na activity, measured with microelectrodes, rises rapidly. Calculations of the quantity of intracellular Na suggest that the volume increase is due to NaCl entry. Once the peak cell volume is achieved, the quantity of Na in the cell does not increase, suggesting that NaCl entry has been inhibited. We tested for inhibition of apical NaCl entry during ouabain treatment either by suddenly reducing the NaCl concentration in the mucosal bath or by adding bumetanide to the perfusate. Both maneuvers caused rapid cell shrinkage during the initial phase of the ouabain experiment, but had no effect on cell volume if performed during the slow shrinkage period. The lack of sensitivity to the composition of the mucosal bath during the shrinkage period occurred because of apparent feedback inhibition of NaCl entry. Another maneuver, reduction of the Na in the serosal bath to 10mm, also resulted in inhibition of apical NaCl uptake. The slow shrinkage which occurred after one or more hours of ouabain treatment was sensitive to the transmembrane gradients for K and Cl across the basolateral membrane and could be inhibited by bumetanide. Thus during pump inhibition inNecturus gallbladder epithelium cell Na and volume first increase due to continuing NaCl entry and then cell volume slowly decreases due to inhibition of the apical NaCl entry and activation of basolateral KCl exit.     

Exocytotic Release of [3H]-Acetylcholine by Ouabain Involves Intracellular Ca2+ Stores in Rat Brain Cortical Slices

1. The effect of ouabain on the release of [3H]acetylcholine ([3H]ACh) in rat brain cortical slices was investigated. 2. The ouabain-induced release of [3H]ACh was calcium-independent and not blocked by EGTA. 3. BAPTA-AM, a chelator of intracellular calcium, inhibited the ouabain effect suggesting the involvement of intracellular calcium stores. 4. Vesamicol, a drug that blocks the storage of acetylcholine in synaptic vesicles inhibited by 73% the ouabain-induced release of [3H] ACh, suggesting exocytotic release of the neurotransmitter. 5. Dantrolene and tetracaine, inhibitors of ryanodine and InP3 receptors, inhibited by 57 and 66% respectively, the ouabain-elicited release of [3H]ACh in brain cortical slices. 6. Confocal microscopy and calcium imaging showed that ouabain increased the levels of [Ca2+]i in cholinergic SN56 cells and that this increase was concentrated in the cell soma. 7. In conclusion, we suggested that ouabain causes Ca2+ release from intracellular stores that can increase [3H] ACh exocytosis from rat brain cortical slices.     

Ouabain evokes exocytosis dependent on ryanodine and mitochondrial calcium stores that is not followed by compensatory endocytosis at the neuromuscular junction

Ouabain is a cardiotonic glycoside that inhibits the sodium potassium ATPase pump leading to sodium accumulation in nerve terminals. At the frog neuromuscular junction, ouabain induces acetylcholine release and a rapid depletion of synaptic vesicles. In the present work, we used FM1–43 vital labeling to dissect the effect of ouabain on synaptic vesicles recycling. We first examined images of nerve-muscle preparations that were stained with FM1–43 by electrical stimulation of the nerve and destained with ouabain. We observed that ouabain induced exocytosis of synaptic vesicles independently of extracellular calcium, implying a mechanism of exocytosis that can bypass the requirement for extracellular calcium. We therefore tested the hypothesis that ouabain induces exocytosis by mobilizing intracellular calcium and we report that calcium release from endoplasmic reticulum through ryanodine receptors is necessary for ouabain-evoked exocytosis. In addition, the ouabain-evoked exocytosis was dependent on calcium released from mitochondria. We also investigated if exocytosis evoked by ouabain is followed by compensatory endocytosis. We observed that muscles incubated with FM1–43 in the presence of ouabain did not present significant staining. In conclusion, our data demonstrate that exocytosis evoked by ouabain is independent on extracellular calcium but dependent on calcium release from endoplasmic reticulum and mitochondrial stores. In addition, we suggest that ouabain can be used as a pharmacological tool to uncouple synaptic vesicles exocytosis from endocytosis at the neuromuscular junction.     

The alpha-2 isomer of the sodium pump is inhibited by calcium at physiological levels

The inhibition of the (Na,K)ATPase by calcium was investigated in plasma membrane preparations of rat axolemma, skeletal muscle and kidney outer medulla. Ouabain titration curves demonstrated that physiological calcium (0.08-5 microM) inhibited mainly the high affinity alpha 2 isomer. In axolemma all the (Na,K)ATPase had high ouabain affinity and calcium inhibited 40-50% of the activity with a Ki of 1.9 +/- 0.9 x 10(-7) M. In skeletal muscle high and low ouabain affinity components were present in equal amounts and calcium inhibited only the high affinity component with a Ki of 1.3 +/- 0.3 x 10(-7) M. Kidney enzyme had a low affinity for ouabain and showed very little sensitivity to calcium in the physiological range. It was demonstrated that high calcium levels inhibit the enzyme in a general sense, irrespective of the isomer, with a Ki of 6.5 +/- 6 x 10(-4) M for the kidney and 5.9 +/- 4 x 10(-4) M for the axolemma enzymes. In axolemma, enzyme activity was studied as a function of sodium concentration. Physiological calcium reduced Vmax while not significantly changing K 0.5 for sodium binding.     

Ouabain stimulates endothelin release and expression in human endothelial cells without inhibiting the sodium pump.

Ouabain, a sodium pump (Na+/ K+-ATPase) inhibitor, has been shown to act as a hormone and is possibly involved in the pathogenesis of hypertension. The mechanism by which ouabain may act was investigated using primary cultures of human umbilical artery endothelial cells (HUAECs), which are known to express and release the vasoconstrictive hormone endothelin (ET-1). Five minutes after application, low concentrations of ouabain induced Ca2+ oscillations and stimulated ET-1 release from endothelial cells into the medium. To investigate whether the observed effects were due to inhibition of the sodium pump, the effects of ouabain on the uptake of 86Rb+ by HUAECs were examined. Unexpectedly, ouabain concentrations below 10 nm stimulated 86Rb+ uptake by 15-20%, and in some experiments by 50%, results that are consistent with a stimulation of the pump. Within the concentration range 1-10 nm, ouabain induced a 2.5-fold stimulation (phosphorylation) of mitogen-activated protein kinase (MAP kinase). After incubation of HUAECs with ouabain for 12 h, the glycoside stimulated cell growth by 49 +/- 4%, as measured by cell number, with a maximum response at 5 nm. At similar concentrations, ouabain also increased ET-1 mRNA abundance by 19.5 +/- 3.1%. The results indicate that, by influencing ET-1 expression and release, ouabain may contribute to the regulation of vascular tone. The data also confirm that it is not a global inhibition of the sodium pump that is involved in the mechanism of action of this cardiac glycoside.     

Calcium transport in intact human erthrocytes

Intact human erythrocytes can be readily loaded with calcium by incubation in hypersomotic media at alkaline pH. Erythrocyte calcium content increases from 15-20 to 120-150 nmol/g hemoglobin after incubation for 2 h at 20 degree C in a 400 mosmol/kg, pH 7.8 solution containing 100 mM sodium chloride, 90 mM tetramethylammonium chloride, 1 mM potassium chloride, and 10 mM calcium chloride. Calcium uptake is a time-dependent process that is associated with an augmented efflux of potassium. The ATP content in these cells remains at more than 60% of normal and is not affected by calcium. Calcium uptake is influenced by the cationic composition of the external media. The response to potassium is diphasic. With increasing potassium concentrations, the net accumulation of calcium initially increases, becoming maximal at 1 mM potassium, then diminishes, falling below basal levels at concentrations above 3 mM potassium. Ouabain inhibits the stimulatory effect of low concentrations of potassium. The inhibitory effects of higher concentrations of potassium are ouabain insensitive and independent of the external calcium concentration. Sodium also inhibits calcium uptake but this inhibition can be modified by altering the external concentration of calcium. The effux of calcium from loaded erythrocytes is not significantly altered by changes in osmolality, medium ion composition, or ouabain. It is concluded that hypertonicity increases the net uptake of calcium by increasing the influx of calcium and that some part of the sodium potassium transport system is involved in this influx process.