Refined evaluation of the exponential curve parameters and initial exchange rate constant for 22Na+ washout in cultured human skin fibroblasts

A technique is proposed to evaluate the exponential curve parameters and the initial exchange rate constant (kie) for 22Na+ washout from cultured human skin fibroblasts. After loading with the isotope, the cells were subjected to cold washing and warming steps. A desaturation curve for 22Na+ washout was developed including the activity in the warming medium that corresponded to t = 0 min. Using nonlinear regression analysis, a general three exponential function adequately described the 22Na+ washout in the time interval of 0-70 min. A back extrapolation was performed to estimate the initial time (ti; a negative number) when the total activity was present in the cells. The ti was substituted into the first derivative function of the three exponents to yield the kie. Calculated from the equilibrium distribution of 22Na+ and the specific activity of the medium, the concentration of Na+ (in mM; mean +/- SD) for fibroblasts of two individuals were 13.3 +/- 2.3, n = 3, and 19.0 +/- 5.2, n = 4. This indicates that the washout originated mainly or exclusively from the cellular milieu. Therefore, the kie represents the equilibrium exchange rate constant for Na+ washout from an inhomogeneous cell-related space. Multiple experiments demonstrated that the kie value for the two subjects were significantly higher than the initial slopes of the washout curves (kA), a commonly used parameter to characterize Na+ washout, and significantly lower than the slopes of the fastest exponential components (k3): kie = 0.531 +/- 0.017, kA = 0.502 +/- 0.019, and k3 = 0.557 +/- 0.017 min-1 (n = 3) for one subject, and kie = 0.567 +/- 0.065, kA = 0.479 +/- 0.031, and k3 = 0.667 +/- 0.094 min-1 (n = 6) for the other subject. The respective equilibrium exchange rates for these cells, namely the products of kie and cellular Na+ contents, were 1.10 +/- 0.16 and 1.19 +/- 0.24 nmole/10(5) cells. Using the exponential curve parameters, analytical solutions of a serial model and a parallel model with three compartments were performed. According to these analyses the major portion of the cellular Na+ comprises a fast exchangeable cellular compartment. The relative size of this compartment (expressed as a fraction of total cellular Na+ content) for fibroblasts of the two subjects was 96.2 and 89.2% for the serial model and 96.1 and 89.3% according to the parallel model.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of acute serum depletion on Na+-K+ homeostasis in cultured human skin fibroblasts

In order to elucidate changes in cell transport behavior of cultured human skin fibroblasts in response to acute serum depletion, we performed uptake and washout of 22Na+ and 86Rb+ as well as measurements of the intracellular Na+ and K+ levels in the presence and absence of ouabain. Pronounced and lasting increase in cellular Na+ and decrease in K+ were observed after removal of fetal bovine serum (FBS) from the medium. The sum of the Na+ and K+ contents (nEq/10(5) cells) was lower in FBS-free medium (mean +/- SD; 17.3 +/- 2.2) than in FBS-containing medium (26.2 +/- 3.8; P less than .02). Simultaneously, a decrease in cellular water volume was detected in the FBS-free medium. The cation uptake and washout data suggest that FBS removal primarily renders the cells more permeable to Na+ and K+ with a secondary stimulation of the ouabain-sensitive Na+ extrusion mechanism. FBS at a concentration of 0.2% prevented approximately 50% of the maximal increase in the 86Rb+ washout rate constant associated with FBS depletion. Ouabain (2 microM) produced an increase in the 86Rb+ washout rate constant. This effect was substantially larger in cells subjected to medium without FBS (from 0.0303 to 0.2500 min-1) than in fibroblasts incubated in medium with FBS (from 0.0107 to 0.0487 min-1). The cellular K+ content was drastically reduced by ouabain to a level not different in medium with or without FBS (33.9 +/- 4.5 to 1.75 +/- 0.38 and 16.7 +/- 1.4 to 1.4 +/- 0.13 nEq/10(5) cells, respectively). The 22Na+ washout data exhibited a three-exponential pattern. Analytical solutions of the washout data by means of two models (serial and parallel) with three compartments showed that FBS depletion resulted in increase of the size of all three compartments. It is concluded that in cultured human skin fibroblasts, FBS is essential to the maintenance of a normal Na+ and K+ homeostasis. The removal of FBS results in dramatic permutation of this homeostasis that develops within minutes and lasts for hours.     

Angiotensin II effect on cytosolic pH in cultured rat vascular smooth muscle cells

This study investigated fluctuations of cytosolic pH (pHi) of cultured rat vascular smooth muscle cells (VSMCs) in reaction to metabolic alterations induced by angiotensin II (AII). Serially passed VSMCs from Wistar rat aortae were grown on coverslips and loaded with the pH-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. A biphasic reaction was seen after exposure of these cells to AII (1 nM to 1 microM); an initial and relatively brief phase of acidification was followed by sustained alkalinization. The rate of acidification and magnitude of alkalinization were dose-dependent. This biphasic effect of AII was also demonstrated in Ca2+-free medium and was mimicked by subjecting VSMCs to the calcium ionophore A23187 (5 microM) in Ca2+-containing medium but not in Ca2+-free medium. Verapamil (10 microM) almost entirely eliminated the AII-induced acidification, whereas amiloride analogues 5-(N-methyl-N-isobutyl)amiloride and 5-(N-ethyl-N-isopropyl)amiloride (100 microM) as well as Na+-deficient medium abolished the subsequent (alkalinization) phase produced by the hormone. Activation of the Na+/H+ antiport by subjecting VSMCs to phorbol 12-myristate 13-acetate (100 nM) prevented a subsequent effect of AII on the pHi profile. This resistance to a further action of the hormone was not mediated via cytoplasmic alkalinization. AII produced a dramatic redistribution in the cellular compartments of 45Ca2+ associated with accelerated 45Ca2+ washout. These findings suggest that the AII-induced acidification phase may relate to activation of the Ca2+ pump (Ca2+/H+ exchange) and that this process can take place in the presence and absence of extracellular Ca2+. The alkalinization phase is the consequence of stimulation of the Na+/H+ antiport, which in cultured VSMCs can be activated by a rise in cytosolic free Ca2+ as well as other mechanisms.     

Differences of Ca2+ regulation in skin fibroblasts from blacks and whites

Black people have a higher propensity than caucasians toward essential hypertension. To explore the possibility that this racial difference relates to cellular Ca2+ metabolism, we measured 45Ca2+ washout and uptake and cytosolic free concentration of Ca2+ [Ca2+]i in serially passed skin fibroblasts from normotensive black and white males. Depending on the experimental conditions, 45Ca2+ washout in these cells was described by either two or three exponential functions, whereas 45Ca2+ uptake was described only by a two-exponent function. There were no racial differences in 45Ca2+ uptake and washout of unstimulated fibroblasts. However, stimulation by human serum resulted in an increase in the 45Ca2+ washout that was higher in fibroblasts from blacks than from whites. The racial differences were expressed primarily by higher values of the apparent washout rate constant (k1) of 45Ca2+ from the largest and most rapidly exchangeable cellular pool. The effect of human serum was not related to its origin (blacks vs. whites). In 2 mM Ca2+ medium and 10% serum from blacks, the respective k1 (mean +/- SEM; x 10(-2)/min) values for fibroblasts from blacks and whites were 89.68 +/- 5.23 and 73.29 +/- 4.0; in the presence of 10% serum from whites, the k1 values for cells from blacks and whites were 84.14 +/- 2.80 and 76.36 +/- 3.23 (overall significance of P less than .01). In Ca2+-deficient medium in the presence of 10% human serum, the k1 for fibroblasts from blacks and whites were 115.57 +/- 3.76 and 102.15 +/- 3.30 (P less than .05). Serum substantially increased the 45Ca2+ uptake in fibroblasts from both blacks and whites; however, racial differences were not observed. Basal levels of [Ca2+]i were not different in fibroblasts of blacks vs. whites (46.8 +/- 6.8 and 43.2 +/- 7.1 nM for blacks and whites, respectively). However, the peak response of Cai2+ transients for cell stimulated by 5% human serum was significantly higher in blacks than whites (blacks = 963 +/- 213, whites = 481 +/- 162 nM; P = .0286). We conclude that Ca2+ regulation is different in serum-stimulated fibroblasts from blacks and whites and that, at least in part, this difference may relate to a greater agonist-induced mobilization of Ca2+ in fibroblasts from blacks.     

Inotropic effects and changes in sodium and calcium contents associated with inhibition of monovalent cation active transport by ouabain in cultured myocardial cells

Cultured monolayers of spontaneously contracting chick embryo ventricular cells were perfused with culture medium containing ouabain. Contractile state was monitored by an optical-video system recording amplitude and velocity of cell wall motion. Positive inotropic effects of 2.5 x 10(-7) to 10(-6) M ouabain were manifest within 1.5-2 min, and reached a stable plateau within 5-6 min. The inotropic effect was fully reversed within 5 min after washout of ouabain. Inhibition of uptake of 42K+ (or the K+ analog 86Rb+) and efflux of 24Na+ occurred 1.5-2 min after exposure to ouabain. The degree of inhibition of transport was closely related to the magnitude of the positive inotropic effect throughout the ouabain concentration range 10(-7) to 10(-6) M. After washout of ouabain from monolayers, the monovalent cation active transport rate returned to normal within 1 min. Thus, both the onset and offset of inotropic action of ouabain were closely related temporally to inhibition of the sodium pump. Exposure to ouabain caused significant increases in exchangeable Na and Ca contents that appeared to be developed within 5 min. These data support the hypothesis that inhibition of monovalent cation active transport by ouabain is causally related to the development of positive inotropy and are consistent with modulation of Ca content by intracellular Na+ via the Na+-Ca2+ exchange carrier mechanism.     

Localization of the Na+/K+-ATPase and of an amiloride sensitive Na+ uptake on thyroid epithelial cells

The Na+/K+-ATPase was localized using purified specific antibodies, on the basolateral membranes of rat thyroid epithelial cells and of cultured porcine thyroid cells, by immunofluorescence and immunoelectron microscopy. No staining was observed on the apical membranes. When cultured cells formed monolayers, with their apical pole in contact with the culture medium, 22Na+ uptake was inhibited by amiloride. Inhibition was dependent upon extracellular Na+ concentration, half maximal inhibition was obtained with 0.7 microM amiloride in the presence of 5 mM Na+. Ouabain was ineffective on Na+ uptake into intact monolayers. A brief treatment of the monolayers with ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) opened the tight junctions and allowed the access of ouabain to the basal pole of the cells. In this condition ouabain increased Na+ uptake. When cells were reorganized into follicle-like structures, with their basal pole in contact with the culture medium, Na+ uptake was not modified by amiloride but was increased by ouabain. We conclude that in thyroid cells, the Na+/K+-ATPase is present on the basolateral domain of the plasma membrane whereas an amiloride sensitive sodium uptake occurs at the apical surface.     

Role of sodium ions and their uptake by cells of cultured blue-green algae, Spirulina platensis and Spirulina maxima

The growth of the blue-green algae Spirulina platensis and Spirulina maxima, cultured in complete mineral Zarouk medium containing Na+ or Na+-deficient medium, was studied over a period of 24 h. The optical densities of S. platensis and S. maxima cells, determined during the last hour of exposure to sodium deficiency, amounted to 55.6 and 32.6%, respectively, of the optical densities of the same cells grown in complete Zarouk medium. Moreover, the cultures grown in Na+-deficient medium exhibited increased ability to take up sodium (which was low in S. platensis and S. maxima cells cultured in complete mineral medium). It is concluded that the two species studied are characterized by periodic, on the order of minutes, changes in the cellular uptake and release of sodium.     

Synergistic Effect of Prostaglandin E2 and Ouabain on Catecholamine Release from Cultured Bovine Adrenal Chromaffin Cells

We recently reported that prostaglandin E2 (PGE2) stimulated phosphoinositide metabolism in cultured bovine adrenal chromaffin cells and that PGE2 and ouabain, an inhibitor of Na+,K+-ATPase, synergistically induced a gradual secretion of catecholamines from the cells. The effect on catecholamine release was specific for prostaglandin E1 (PGE1) and PGE2 among prostaglandins tested (E1 = E2 greater than F2 alpha greater than D2). The release evoked by PGE2 plus ouabain was greatly reduced in Na+-depleted medium and not observed in Ca2+-free medium. Here we examined the synergistic effect of PGE2 and ouabain on the release with specific reference to ion fluxes. Regardless of the presence of PGE2, ouabain stimulated the release in a dose-dependent manner with half-maximal stimulation at 1 microM, and omission of K+ from the medium, a condition which suppresses the Na+,K+-ATPase activity, also enhanced the release from chromaffin cells exposed to PGE2. Ouabain induced a continuous accumulation of 22Na+ and 45Ca2+, as well as secretion of catecholamines. Although PGE2 itself showed hardly any effects on these cellular responses, PGE2 potentiated all of them induced by ouabain. The time course of catecholamine release was correlated with that of accumulation of 45Ca2+ rather than with that of 22Na+. The release evoked by PGE2 and ouabain was inhibited in a dose-dependent manner by amiloride and the analogue ethylisopropylamiloride, inhibitors of the Na+,H+-antiport, but not by the Na+-channel inhibitor tetrodotoxin nor by the nicotinic receptor antagonist hexamethonium. Ethylisopropylamiloride at 1 microM inhibited PGE2-enhanced accumulation of 22Na+ and 45Ca2+ and release of catecholamine by 40, 83, and 71%, respectively. Activation of the Na+,H+-antiport by elevation of the extracellular pH from 6.6 to 8.0 increased the release of catecholamines linearly. Furthermore, PGE2 induced a sustained increase in intracellular pH by about 0.1 pH unit above the resting value, which was abolished by amiloride or in Na+-free medium. These results taken together indicate that PGE2 activates the Na+,H+-antiport by stimulating phosphoinositide metabolism and that the increase in intracellular Na+ by both inhibition of Na+,K+-ATPase and activation of Na+,H+-antiport may lead to the redistribution of Ca2+, which is the initial trigger of catecholamine release.     

Autoradiographic localisation of [3H]ouabain bound to cultured epithelial cell monolayers of MDCK cells

[3H]Ouabain binding to intact MDCK (cultured monolayers of dog kidney) cells of 60 serial passages is dependent upon ouabain concentration, time and medium K+. By utilising high K+ incubations to estimate non-specific [3H]ouabain-binding, the concentration of ouabain giving half maximal specific binding was estimated to be 1.0 . 10(-7) M and the total maximum binding to be 2.33 . 10(5) sites/cell. Ouabain inhibition of (Na+, K+)-pump function was monitored by the cellular uptake of 86Rb over 5 min. The larger fraction of 86Rb uptake was ouabain sensitive and the ouabain concentration giving half-maximal inhibition was 2 . 10(-7) M. The cellular distribution of the (Na+ + K+)-ATPase was investigated using [3H]ouabain autoradiography of intact freeze-dried epithelial monolayers of MDCK cells grown upon millipore filter supports. Binding of [3H]ouabain is localised over the lateral cellular membranes. Autoradiographic silver grain density is close to background levels over both the apical and basal (attachment) membranes.     

Characterization of Na(+)-K+ homeostasis of cultured human skin fibroblasts in the presence and absence of fetal bovine serum.

Previously, we demonstrated that removal of fetal bovine serum (FBS) from the medium of human skin fibroblasts resulted in an accelerated 86Rb+ washout, decreased cellular K+, and increased Na+ contents. In the present study we examined the mechanism underlying these changes. The efflux rate constant for 86Rb+, and the cellular contents of Na+ and K+ were measured. Verapamil (K1/2 = 15 microM) and chlorpromazine (K1/2 = 1 microM) reduced by approximately 70% the increased 86Rb+ washout evoked by FBS removal. The effect of the two drugs was additive at low, but not high, concentrations. Verapamil and chlorpromazine also attenuated the decrease in cellular K+ content and prevented the increase in cellular Na+ content associated with FBS depletion. Bumetanide (50 microM) was only partially effective in offsetting the enhanced 86Rb+ efflux and was completely without any effect on the cellular Na+ and K+ changes induced by FBS removal. In the presence of FBS, A-23187 produced a slight and transient increase of the 86Rb+ washout. The protein kinase C activator phorbol 12-myristate 13-acetate enhanced the 86Rb+ efflux in FBS-containing medium for a prolonged period but this increase was only a fraction of that caused by serum removal. Cellular Na+ and K+ contents were not changed by the phorbol ester. We conclude that FBS removal raises the cellular Na+ content, and enhances 86Rb+ efflux, through a calmodulin-dependent pathway activated by calcium influx.     

Effect of inhibition of Na+/K(+)-ATPase on the vasopressin-induced increase in intracellular Na+ concentration in vascular smooth muscle cells.

The effect of inhibition of Na+/K(+)-ATPase by ouabain on the arginine vasopressin (AVP)-induced increase in intracellular Na+ concentration [( Na+]i) was examined in cultured rat vascular smooth muscle cells (VSMC) by the direct measurement of [Na+]i using a fluorescent indicator dye. AVP at a concentration of 1 x 10(-9) M or higher increased [Na+]i in a dose-dependent manner in cultured rat VSMC. The preincubation of cells with 1 x 10(-4) M ouabain for 1 hr at 37 degrees C did not affect the basal [Na+]i but enhanced the 1 x 10(-6) M AVP-induced increase in [Na+]i. The preincubation was not necessary because similar results were obtained after the simultaneous administration of AVP and ouabain. The treatment with ouabain did not affect the intracellular pH changes induced by AVP. These results therefore indicate that the inhibition of Na+/K(+)-ATPase enhances the AVP-induced increase in [Na+]i by decreasing cellular Na+ efflux in cultured rat VSMC.     

Energetic cooperation via ion-permeable junctions in mixed animal cell cultures

Low ouabain concentration (1 x 10(-6) M) is shown to decrease intracellular K+ (K+in) and to increase intracellular Na+ (Na+in) in human fibroblast cell cultures. The same ouabain concentration was without effect upon K+in ad Na+in in rodent cultures such as BHK-21, mouse fibroblasts and rat glyoma C6 cells. K+in and Na+in in the mixed cultures of human and BHK-21 fibroblasts or human and mouse fibroblasts were found to be resistant to 1 x 10(-6) M ouabain whereas that of the mixtures of human and rat glyoma C6 cells proved to be ouabain-sensitive. The gap-junction-mediated dye transfer was revealed between human and BHK-21 cells. Such an effect was very small in the human-C6 cell mixed culture. It is concluded that cells with active ion pumps can support the maintenance of K+ and Na+ gradients in cells with inactive pumps, provided that effective ion transport via gap junctions takes place.     

Clearance of store-released Ca2+ by the Na+-Ca2+ exchanger is diminished in aortic smooth muscle from Na+-K+-ATPase alpha 2-isoform gene-ablated mice

Two alpha-isoforms of the Na+-K+-ATPase are expressed in vascular smooth muscle cells (VSMCs). The alpha 1-isoform is proposed to serve a cytosolic housekeeping role, whereas the alpha 2-isoform modulates Ca2+ storage via coupling to the Na+-Ca2+ exchanger (NCX) in a subsarcolemmal compartment. To evaluate the ramifications of this proposed interaction, Ca2+-store load and the contributions of the primary Ca2+ transporters to Ca2+ clearance were studied in aortic VSMCs from embryonic wild-type (WT) and Na+-K+-ATPase alpha 2-isoform gene-ablated, homozygous null knockout (alpha 2-KO) mice. Ca2+ stores were unloaded by inhibiting the sarco(endo)plasmic reticulum Ca2+-ATPase with cyclopiazonic acid (CPA) in Ca2+-free media to limit Ca2+ influx. Ca2+ clearance by the plasma membrane Ca2+-ATPase (PMCA), NCX, or mitochondria was selectively inhibited. In WT VSMCs, NCX accounted for 90% of the Ca2+ efflux. In alpha 2-KO VSMCs, preferential clearance of store-released Ca2+ by NCX was lost, whereas PMCA activity was increased. Selective inhibition of the alpha 2-isoform (0.5 microM ouabain for 20 min), before treatment with CPA enhanced the store load in VSMCs from WT, but not alpha 2-KO mice. A subsequent analysis of capacitative Ca2+ entry (CCE) indicated that the magnitude of Ca2+ influx was significantly greater in alpha 2-KO cells. Our findings support the concept of a subsarcolemmal space where the alpha 2-isoform coupled with NCX modulates Ca2+-store function and, thereby, CCE.     

Na+ participates in loop diuretic-sensitive Cl(-)-cation co-transport in the pancreatic beta-cells

In order to investigate whether Na+ participates in loop diuretic-sensitive Cl(-)-cation co-transport in the beta-cells, we tested the interaction between the effects of Na+ deficiency, furosemide and D-glucose on 86Rb+ fluxes in beta-cell-rich mouse pancreatic islets. Removal of extracellular Na+ slightly reduced the ouabain-resistant 86Rb+ influx and the specific effect of 1 mM furosemide on this influx was significantly smaller in Na(+)-deficient medium. The capacity of 20 mM D-glucose to reduce the ouabain-resistant 86Rb+ influx was not changed by removal of extracellular Na+. The 86Rb+ efflux from preloaded islets was rapidly and reversibly reduced by Na+ deficiency. Furosemide (1 mM) reduced the 86Rb+ efflux and the effect of the combination of Na+ deficiency and 1 mM furosemide was not stronger than the effect of furosemide alone. 22Na+ efflux was reduced by both ouabain and furosemide and the effects appeared to be additive. The data suggest that Na+ participates in loop diuretic-sensitive Cl(-)-cation co-transport in the pancreatic beta-cells. This adds further support to the idea that beta-cells exhibit a Na+, K+, Cl- co-transport system. Since some of the furosemide effect on 86Rb+ efflux persisted in the Na(+)-deficient medium, it is likely that also loop diuretic-sensitive K+, Cl- co-transport exists in this cell type.     

Influence of sodium pump and Na(+), K(+), CL(-)-cotransport on the resting membrane potential of somatic muscle cells of the earthworm Lumbricus terrestris]

Tetrodotoxin and acidic pH do not change the resting membrane potential (RMP), whereas Na+ or Cl- free solutions or ouabain and furosemide equally depolarize the membrane of the earthworm somatic muscle cells. The findings of the RMP depending on extracellular K+ concentration corroborate theoretical model by Goldman-Hodgkin-Katz only in Na(+)-free medium or in presence of ouabain. The data suggest that the RMP is the sum of potassium and chlorine diffusion potentials as well as of the potential produced by electrogenic component of Na+ pump and, probably, by furosemide-sensitive Na+,K+Cl- co-transport.     

Ouabain-resistant cells cultured in a synthetic medium

Several strains of kidney and liver cells cultured in a synthetic medium were found to be resistant to ouabain. These cell strains were characterized because this resistance may serve as a good marker in genetic studies on somatic cells in chemically defined conditions in the absence of Na+ related growth factors and hormones. The phenotype was stable in the absence of selection for at least two years, and the original strains before adaptation to the synthetic medium were found to have ouabain sensitivity equal to the corresponding cells in the synthetic medium. The resting membrane potential, Na+,K+-ATPase activity, and growth rate of the resistant cells were similar to those of ouabain-sensitive cells. The resistance of the cells was not affected by serum or antibodies against some cytoskeletal proteins and the sensitivity of the Na+,K+-ATPase was not restored by partial purification of the membranes. Western blotting of the Na+,K+-ATPase of the ouabain-resistant cells showed that the molecular weights of its two subunits and its immunoreactivity were similar to those of the enzyme from the ouabain-sensitive strain. Thus the ouabain resistance is caused not by ouabain-like hormone produced by the cells or change in the cytoskeletal system, but by a mutation resulting in expression of an ouabain-resistant ATPase gene.     

Monovalent cation metabolism and cytopathic effects of poliovirus-infected HeLa cells.

To better understand the significance of 22Na+ accumulation by poliovirus-infected HeLa cells (C. N. Nair, J. W. Stowers, and B. Singfield, J. Virol. 31:184, 1979), measurements of cellular Na+, K+, and Cl- contents, volume, and density were carried out at intervals after infection. In addition, the rates of 22Na+ washout from infected and control cells were determined. Starting at around 3 h postinfection, the Na+ content of infected cells increased, whereas the K+ content decreased progressively, resulting in a net loss in the monovalent cation content decreased progressively, resulting in a net loss in the monovalent cation content per cell. The loss in cellular chloride content exceeded that in monovalent cation content. The kinetics of 22Na+ washout from infected and control cells revealed the presence of an extra Na+ compartment in infected cells. A net loss in the monovalent cation activity of infected cells was indicated by the loss of cell water as reflected in a decrease in cell volume and an increase in cell density. In spite of a net loss in monovalent cation content per cell, Na+ accumulation coupled with cell shrinkage resulted in substantial increases in the concentrations of not only Na+ but also K+. The results suggested a possible role for tonicity change in the morphological lesions of poliovirus cytotoxicity.     

Two classes of ouabain receptors in chick ventricular cardiac cells and their relation to (Na+,K+)-ATPase inhibition, intracellular Na+ accumulation, Ca2+ influx, and cardiotonic effect

The biochemical and pharmacological properties of the (Na+,K+)-ATPase have been studied at different stages of chick embryonic heart development in ovo and under cell culture conditions. The results show the existence of two families of ouabain binding sites: a low affinity binding site with a dissociation constant (Kd) of 2-6 microM for the ouabain-receptor complex and a high affinity binding site with a Kd of 26-48 nM. Levels of high affinity sites gradually decrease during cardiac ontogenesis to reach a plateau near 14 days of development. Conversely the number of low affinity binding sites is essentially invariant between 5 days and hatching. Cultured cardiac cells display the same binding characteristics as those found in intact ventricles. Inhibition of 86Rb+ uptake in cultured cardiac cells and an increase in intracellular Na+ concentration, due to (Na+,K+)-ATPase blockade, occur in a ouabain concentration range corresponding to the saturation of the low affinity ouabain site. Ouabain-stimulated 45Ca2+ uptake increases in parallel with the increase in the intracellular Na+ concentration. It is suppressed in Na+-free medium or when Na+ is replaced by Li+ suggesting that the increase is due to the indirect activation of the Na+/Ca2+ exchange system in the plasma membrane. Dose-response curves for the inotropic effects of ouabain on papillary muscle and on ventricular cells in culture indicate that the development of the cardiotonic properties is parallel to the saturation of the low affinity binding site for ouabain. Therefore, inhibition of the cardiac (Na+,K+)-ATPase corresponding to low affinity ouabain binding sites seems to be responsible for both the cardiotonic and cardiotoxic effects of the drug.     

Role of the Na+/K+/Cl- transporter in the positive inotropic effect of ouabain in cardiac myocytes

In this study we have characterized the bumetanide-sensitive K+/Na+/Cl- cotransport in cultured rat cardiac myocytes. 1) It carries about 10% of the total K+ influx. 2) It is sensitive to furosemide (Ki0.5 = 10(-6)M) and bumetanide (Ki0.5 = 10(-7)M). 3) It is strongly dependent on the extracellular concentrations of Na+ and Cl-. 4) It carries out influx of both ions, K+ and Na+. A therapeutic concentration of ouabain (10(-7) M) stimulated the bumetanide-sensitive K+ influx (as measured by 86Rb+), in the cultured myocytes, with no effect on the bumetanide-resistant K+ influx, which was mediated mostly by the Na+/K+ pump. Stimulation of the bumetanide-sensitive Rb+ influx by a low ouabain concentration was strongly dependent on Na+ and Cl- in the extracellular medium. A low concentration of ouabain (10(-7) M) was found to increase the steady-state level of cytosolic Na+ by 15%. This increase was abolished by the addition of bumetanide or furosemide. These findings suggest that ouabain, at a low (10(-7) M) concentration, induced its positive inotropic effect in rat cardiac myocytes by increasing Na+ influx into the cells through the bumetanide-sensitive Na+/K+/Cl- cotransporter. In order to examine this hypothesis, we measured the effect of bumetanide on the increased amplitude of systolic cell motion induced by ouabain. Bumetanide or furosemide, added to cultured cardiac myocytes, inhibited the increased amplitude of systolic cell motion induced by ouabain. Neither bumetanide nor furosemide alone has any significant effect on the basal amplitude of systolic cell motion. We propose that stimulation of bumetanide-sensitive Na+ influx plays an essential role in the positive inotropic effect in rat cardiac myocytes induced by low concentration of ouabain.     

Erythroid differentiation and the Na+,K+-pump in ouabain-sensitive and ouabain-resistant Friend erythroleukemia cell lines

The selection and biochemical characterization of ouabain-resistant erythroleukemia cell lines are described. Treatment of ouabain-resistant Friend erythroleukemia cell (FLC) lines with 1 mM ouabain demonstrated a reduced ouabain-sensitive 86Rb+-uptake after Na+-preloading in comparison with ouabain-sensitive cells. The ouabain- and diuretic (piretanide)-insensitive component of the 86Rb+-uptake (residual influx) was significantly enhanced in the ouabain-resistant FLC clones. Measurements of the Na+,K+-ATPase activity (E.C. 3.6.1.3) in plasma membrane preparations of the ouabain-resistant FLC clone B6/2 indicated that a ouabain-resistant Na+,K+-ATPase activity of about 20% of the total enzyme activity existed in the presence of 1 mM ouabain. Further experiments showed that the Na+,K+-ion-gradient in ouabain-resistant B6/2 cells was unaffected by ouabain exposure whereas the gradient collapsed in wild type 12 N cells. Another property of the ouabain-resistant cell lines was a decrease of the 86Rb+-uptake due to the Na+,K+, 2Cl(-)-cotransport system measured as piretanide-sensitive 86Rb+-uptake. The data on ion transport mechanisms in QuaR and QuaS FLC are discussed with respect to mutagen-induced and spontaneous cellular ouabain resistance. In addition, the role of altered ion transport mechanisms is considered for induced erythroid differentiation.