Na+-ATPase is a different entity from the (Na+ + K+)-ATPase in rat kidney basolateral plasma membranes

In this work, we present evidence in agreement with the hypothesis that there exist two Na+-stimulated ATPase activities in basolateral plasma membranes from rat kidney proximal tubular cells: (1) (Na+ + K+)-ATPase activity, which is inhibited by ouabain and by treating the membranes with trypsin, is insensitive to furosemide and reaches maximal activity upon treatment with SDS at an SDS/protein ratio of 1.6; (2) the Na+-ATPase activity, which is insensitive to ouabain and to trypsin treatment, is inhibited by furosemide and reaches maximal activity upon treatment with SDS at an SDS/protein ratio of 0.4.     

Backdoor phosphorylation of basolateral plasma membranes of small intestinal epithelial cells: characterization of a furosemide-induced phosphoprotein related to the second sodium pump

Enterocyte has two different Na+-stimulated ATPases, the ouabain-sensitive Na+/K+ ATPase and a furosemide-inhibitable Na+ ATPase. To identify the polypeptide associated with the Na+-ATPase, 32Pi phosphorylation into basolateral membranes of enterocyte was investigated. Both, ouabain and furosemide induced Mg2+-dependent, vanadate-sensitive 32Pi incorporation into a 100kDa polypeptide. K(m) for Pi was 17.7+/-1.82 microM and 16.8+/-0.69 microM for ouabain-induced and furosemide-induced phosphorylation, respectively. K(m) for furosemide was 1.3+/-0.21 mM. Furosemide-induced 32Pi incorporation was sensitive to alkaline pH and hydroxylamine suggesting an acyl-phosphate bond. Na+ and K+ inhibited 32Pi incorporation induced by ouabain. In contrast, Na+ stimulated furosemide-induced phosphorylation with a K(m) of 16.5+/-5.59 mM while K+ had no effect. Purified Na+/K+ ATPase only presented ouabain-induced phosphoprotein, indicating that furosemide-induced phosphorylation is not related to this enzyme and appears to correspond to a new member of P-type ATPases associated with the second Na+ pump.     

Na+-stimulated ATPase activities in basolateral plasma membranes from guinea-pig small intestinal epithelial cells

Two ATPase activities, a Na+-ATPase and a (Na+ + K+)-ATPase, have been found associated with sheets of basolateral plasma membranes from guinea-pig small intestinal epithelial cells. The specific activity of the former is 10-15% of the latter. The two ATPase activities differ in their affinity for Na+, their optimal pH, their K+ requirement and particularly in their behaviour in the presence of some inhibitors and of Ca2+. Thus the Na+-ATPase is refractory to ouabain but it is strongly inhibited by ethacrynic acid and furosemide, whilst the (Na+ + K+)-ATPase is totally suppressed by ouabain, partially by ethacrynic acid and refractory to furosemide. In addition, the Na+-ATPase is activated by micromolar concentrations of calcium and by resuspension of the membrane preparation at pH 7.8. The Na+-ATPase is only stimulated by sodium and to a lesser extent by lithium; however, this stimulation is independent of the anion accompanying Na+. The latter rules out the participation of an anionic ATPase. The relation between the characteristics of the sodium transport mechanism in basolateral membrane vesicles (Del Castillo, J.R. and Robinson, J.W.L. (1983) Experientia 39,631) and those of the two ATPase activities present in the same membranes, allow us to postulate the existence of two separate sodium pumps in this membranes. Each pump would derive the necessary energy for active ion transport from the hydrolysis of ATP, catalyzed by different ATPase systems.     

Effect of Ca2+ on the ouabain-insensitive, active Na+ uptake in inside-out basolateral plasma membrane vesicles from rat kidney proximal tubular cells

The ouabain-insensitive, active Na+ uptake of inside-out vesicles prepared with basolateral plasma membranes from rat kidney proximal tubular cells can be increased by the presence of micromolar concentrations of Ca2+ in the assay medium. The concomitant ATP hydrolysis associated with the Na+ uptake is also increased by the presence of Ca2+. The Na+ uptake and the concomitant ATP hydrolysis are inhibited by 2 mM furosemide. The effect of Ca2+ is not due to the activity of an Na+-Ca2+ exchanger. The present results are in accordance with our previous model (Proverbio, F., Proverbio, T. and Marín, R. (1982) Biochim. Biophys. Acta 688, 757-763) in which we proposed that Ca2+ seems to modulate the activity of the ouabain-insensitive Na+ pump, in two different ways: (1) in a strong association with the membranes in which Ca2+ (stable component) is essential for the pump activity and (2) in a weak association with the membranes in which Ca2+ (labile component) can be quickly and easily removed by reducing the free Ca2+ concentration of the assay medium to values lower than 1 microM. The Ka for Ca2+ (for the labile component) is around 5 microM. The Ca2+ modulation of the ouabain-insensitive Na+ pump is an indication that Ca2+ could regulate the magnitude of the Na+ extrusion accompanied by Cl- and water present in rat kidney proximal tubular cells.     

Detection of a Na+-activated, furosemide-inhibited ATPase activity in rat intestinal mucosa

An ouabain-insensitive, Mg++-dependent, Na+-stimulated ATPase activity which is inhibited by furosemide was found in mucosal homogenate of rat small intestine. The subcellular localization of this ATPase activity was studied by means of isolated purified brush borders and basolateral plasma membranes. The results suggest a nearly identical distribution of Na+-activated and (Na+K+)-activated ATPase within the epithelial cells. Under conditions of alloxan and streptozotocin diabetes an increase of both ATPase activities can be found only in the basolateral plasma membranes. These observations agree well with the convective model of intestinal absorption.     

The colonic H+,K+-ATPase functions as a Na+-dependent K+(NH4+)-ATPase in apical membranes from rat distal colon.

Recent studies have suggested that the colonic H+,K+-ATPase (HKalpha2) can secrete either Na+ or H+ in exchange for K+. If correct, this view would indicate that the transporter could function as either a Na+ or a H+ pump. To investigate this possibility a series of experiments was performed using apical membranes from rat colon which were enriched in colonic H+,K+-ATPase protein. An antibody specific for HKalpha2 was employed to determine whether HKalpha2 functions under physiological conditions as a Na+-dependent or Na+-independent K+-ATPase in this same membrane fraction. K+-ATPase activity was measured as [gamma-32P]ATP hydrolysis. The Na+-dependent K+-ATPase accounted for approximately 80% of overall K+-ATPase activity and was characterized by insensitivity to Sch-28080 but partial sensitivity to ouabain. The Na+-independent K+-ATPase activity was insensitive to both Sch-28080 and ouabain. Both types of K+-ATPase activity substituted NH4+ for K+ in a similar manner. Furthermore, our results demonstrate that when incubated with native distal colon membranes, the blocking antibody inhibited dramatically Na+-dependent K+-ATPase activity. Therefore, these data demonstrate that HKalpha2 can function in native distal colon apical membranes as a Na+-dependent K+-ATPase. Elucidation of the role of the pump as a transporter of Na+ versus H+ or NH4+ versus K+ in vivo will require additional studies.     

Identification and reconstitution of a Na+/K+/Cl- cotransporter and K+ channel from luminal membranes of renal red outer medulla

Electrophysiological studies on renal thick ascending limb segments indicate the involvement of a luminal Na+/K+/Cl- cotransport system and a K+ channel in transepithelial salt transport. Sodium reabsorption across this segment is blocked by the diuretics furosemide and bumetanide. The object of our study has been to identify in intact membranes and reconstitute into phospholipid vesicles the Na+/K+/Cl- cotransporter and K+ channel, as an essential first step towards purification of the proteins involved and characterization of their roles in the regulation of transepithelial salt transport. Measurements of 86Rb+ uptake into membrane vesicles against large opposing KCl gradients greatly magnify the ratio of specific compared to non-specific isotope flux pathways. Using this sensitive procedure, it has proved possible to demonstrate in crude microsomal vesicle preparations from rabbit renal outer medulla two 86Rb+ fluxes. (A) A furosemide-inhibited 86Rb+ flux in the absence of Na+ (K+-K+ exchange). This flux is stimulated by an inward Na+ gradient (Na+/K+ cotransport) and is inhibited also by bumetanide. (B) A Ba2+-inhibited 86Rb+ flux, through the K+ channel. Luminal membranes containing the Na+/K+/Cl- cotransporter and K+ channels, and basolateral membranes containing the Na+/K+ pumps were separated from the bulk of contaminant protein by metrizamide density gradient centrifugation. The Na+/K+/Cl- cotransporter and K+ channel were reconstituted in a functional state by solubilizing both luminal membranes and soybean phospholipid with octyl glucoside, and then removing detergent on a Sephadex column.     

Temperature sensitivity of potassium flux into red blood cells in the familial pseudohyperkalaemia syndrome

The temperature dependence of potassium flux into the red cells of normal and pseudohyperkalaemic individuals over the range 4-40 degrees C was measured using 86RbCl as tracer. Flux through the pump was measured as the ouabain-sensitive component (0.2 mM ouabain) and flux via Na+,K+-cotransport was measured as the decrease in the rate of K+ influx in the presence of 1 mM furosemide. The residual passive permeability of the red cell plasma membranes to K+ was that influx which was unaffected by either inhibitor. When Na+ influxes were measured, the ratio of Na+ to K+ transported via the furosemide-sensitive component was 1 over the full temperature range studied. The temperature sensitivity of K+ influx via the pump was normal as was the enzymic activity of the Na+,K+-ATPase. In contrast, the activity of the Na+,K+-cotransport system in pseudohyperkalaemics was more temperature sensitive than that of controls and affected individuals also showed a greater passive permeability to K+ at low temperatures. Red cell membranes from affected individuals have significantly increased amounts of phosphatidylcholine which are balanced, to a degree, by a decreased content of phosphatidylethanolamiane. It is proposed that in this example of familial pseudohyperkalaemia there is an alteration in the structure of the red cell plasma membrane which influences the temperature sensitivity of both its cotransport and passive permeability properties.     

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.     

Na+, K+ and Cl- transport in isolated small intestinal cells from guinea pig. Evidences for the existence of a second Na+ pump

Isolated small intestinal epithelial cells, after incubation at 4 degrees C for 30 min, reach ion concentrations (36 mM K+, 113 mM Na+ and 110 mM Cl-) very similar to those of the incubation medium. Upon rewarming to 37 degrees C, cells are able to extrude Na+, Cl- and water and to gain K+. Na+ extrusion is performed by two active mechanisms. The first mechanism, transporting Na+ by exchanging it for K+, is inhibited by ouabain and is insensitive to ethacrynic acid. It is the classical Na+ pump. The second mechanism transports Na+ with Cl- and water, is insensitive to ouabain but is inhibited by ethacrynic acid. Both mechanisms are inhibited by dinitrophenol and anoxia. The second Na+ extruding mechanism could be the Na+/K+/2Cl- cotransport system. However, this possibility can be ruled out because the force driving cotransport would work inwards, and because Na+ extrusion with water loss continues after substitution of Cl- by NO3-. We propose that enterocytes have a second Na+ pump, similar to that proposed in proximal tubular cells.     

Cation flux in the ehrlich ascites tumor cell. Evidence for Na+-for-Na+ and K+-for-K+ exchange diffusion.

In a previous study, evidence was presented for an external Na+-dependent, ouabain-insensitive component of Na+ efflux and an external K+-dependent component of K+ efflux in the Ehrlich ascites tumor cell. Evidence is now presented that these components are inhibited by the diuretic furosemide and that under conditions of normal extracellular Na+ and K+ they represent Na+-for-Na+ and K-+for-K+ exchange mechanisms. Using 86Rb to monitor K+ movements, furosemide is shown to inhibit an ouabain-insensitive component of Rb+ influx and a component of Rb+ efflux, both representing approx. 30 percent of the total flux. Inhibition of Rb+ efflux is greatly reduced by removal of extracellular K+. Furosemide does not alter steady-state levels of intracellular K+ and it does not prevent cells depleted of K+ by incubation in the cold from regaining K+ upon warming. Using 22Na to monitor Na+ movements, furosemide is shown to inhibit an ouabain-insensitive component of unidirectional Na+ efflux which represents approx. 22 percent of total Na+ efflux. Furosemide does not alter steady-state levels of intracellular Na+ and does not prevent removal of intracellular Na+ upon warming from cells loaded with Na+ by preincubation in the cold. The ability of furosemide to affect unidirectional Na+ and K+ fluxes but not net fluxes is consistent with the conclusion that these components of cation movement across the cell membrane represent one-for-one exchange mechanisms. Data are also presented which demonstrate that the uptake of alpha-aminoisobutyrate is not affected by furosemide. This indicates that these components of cation flux are not directly involved in the Na+-dependent amino acid transport system A.     

Partial characterization of the ouabain-insensitive, Na+-stimulated ATPase activity of kidney basal-lateral plasma membranes

The present paper characterizes the Na+-stimulated ATPase activity present in basal-lateral plasma membranes from guinea-pig kidney proximal tubular cells. These characteristics are compared with those of the (Na+ + K+)-stimulated ATPase activity, and they are: (A) Na+-ATPase activity: (1) requires Mg2+; (2) may be activated by mu molar quantities of Ca2+; (3) optimal ratio Mg:ATP = 5:1-2 and Ka for Mg:ATP = 3:0.60 mM; (4) Ka for Na+:8 mM; (5) does not require K+; (6) is only stimulated by Na+ and Li+ (in a lower extent); (7) is similarly stimulated by the Na+ salt of different anions; (8) hydrolyzes only ATP; (9) optimal temperature: 47 degrees C; (10) optimal pH: 6.9; (11) is ouabain insensitive; (12) is totally inhibited by 1.5 mM ethacrynic acid, 2 mM furosemide and 0.75 mM triflocin. (B) (Na+ + K+)-ATPase activity: (1) also requires Mg2+; (2) is inhibited by Ca2+; (3) optimal ratio Mg:ATP = 1.25:1 and Ka for Mg:ATP = 0.50: 0.40 mM; (4) Ka for Na+: 14 mM (data not shown); (5) needs K+ together with Na+; (6) K+ may be substituted by: Rb+ greater than NH+4 greater than Cs+; (7) is anion insensitive; (8) hydrolyzes mostly ATP and to a lesser extent GTP, ITP, UTP, ADP, CTP; (9) optimal temperature: 52 degrees C; (10) optimal pH: 7.2; (11) 100% inhibited by 1 mM ouabain; (12) 63% inhibited by 1.5 mM ethacrynic acid, 10% inhibited by 2 mM furosemide and insensitive to 0.75 mM triflocin.     

Na+ and K+ transport at basolateral membranes of epithelial cells. I. Stoichiometry of the Na,K-ATPase

The stoichiometry of pump-mediated Na/K exchange was studied in isolated epithelial sheets of frog skin. 42K influx across basolateral membranes was measured with tissues in a steady state and incubated in either beakers or in chambers. The short-circuit current provided estimates of Na+ influx at the apical membranes of the cells. 42K influx of tissues bathed in Cl- or SO4-Ringer solution averaged approximately 8 microA/cm2. Ouabain inhibited 94% of the 42K influx. Furosemide was without effect on pre-ouabain-treated tissues but inhibited a ouabain-induced and Cl--dependent component of 42K influx. After taking into account the contribution of the Na+ load to the pump by way of basolateral membrane recycling of Na+, the stoichiometry was found to increase from approximately 2 to 6 as the pump-mediated Na+ transport rate increased from 10 to 70 microA/cm2. Extrapolation of the data to low rates of Na+ transport (less than 10 microA/cm2) indicated that the stoichiometry would be in the vicinity of 3:2. As pump-mediated K+ influx saturates with increasing rates of Na+ transport, Na+ efflux cannot be obligatorily coupled to K+ influx at all rates of transepithelial Na+ transport. These results are similar to those of Mullins and Brinley (1969. Journal of General Physiology. 53:504-740) in studies of the squid axon.     

Na+ and K+ transport at basolateral membranes of epithelial cells. II. K+ efflux and stoichiometry of the Na,K-ATPase

Changes of 42K efflux (J23K) caused by ouabain and/or furosemide were measured in isolated epithelia of frog skin. From the kinetics of 42K influx (J32K) studied first over 8-9 h, K+ appeared to be distributed into readily and poorly exchangeable cellular pools of K+. The readily exchangeable pool of K+ was increased by amiloride and decreased by ouabain and/or K+-free extracellular Ringer solution. 42K efflux studies were carried out with tissues shortcircuited in chambers. Ouabain caused an immediate (less than 1 min) increase of the 42K efflux to approximately 174% of control in tissues incubated either in SO4-Ringer solution or in Cl-Ringer solution containing furosemide. Whereas furosemide had no effect on J23K in control tissues bathed in Cl-rich or Cl-free solutions, ouabain induced a furosemide-inhibitable and time-dependent increase of a neutral Cl-dependent component of the J23K. Electroconductive K+ transport occurred via a single-filing K+ channel with an n' of 2.9 K+ efflux before ouabain, normalized to post-ouabain (+/- furosemide) values of short-circuit current, averaged 8-10 microA/cm2. In agreement with the conclusions of the preceding article, the macroscopic stoichiometry of ouabain-inhibitable Na+/K+ exchange by the pump was variable, ranging between 1.7 and 7.2. With increasing rates of transepithelial Na+ transport, pump-mediated K+ influx saturated, whereas Na+ efflux continued to increase with increases of pump current. In the usual range of transepithelial Na+ transport, regulation of Na+ transport occurs via changes of pump-mediated Na+ efflux, with no obligatory coupling to pump-mediated K+ influx.     

Ouabain-insensitive active sodium transport in rat jejunum: Evidence from ATPase activities,Na uptake by basolateral membrane vesicles and in vitro transintestinal transport

The basolateral membrane of the jejunal enterocyte of the rat was separated by self-orienting Percoll-gradient centrifugation and further purified from brush border contamination. Pellets were analysed for Mg-, Na- and (Na, K)-ATPase activities. The uptake of 0·02 M NaCl was also followed by the rapid micro-filtration technique. Transintestinal transport of fluid and electrolytes, and cell water, Na and K were determined in the in vitro everted and incubated jejunum. There is ouabain-insensitive Na-ATPase in addition to the well-known (Na, K)-ATPase in the basolateral membrane. These are differently inhibited by furosemide and ethacrynate. Na uptake by osmotically active basolateral membrane vesicles is enhanced by ATP and a further enhancement is obtained if there is intravesicular K. The ATP effect is inhibited differently by strophanthidin, furosemide and ethacrynate. In the everted sac preparation, transintestinal transport of Na and fluid still occurs when the Na/K pump is totally inhibited by ouabain. These experimental results suggest that there is also a ouabain-insensitive Na pump, different from the Na/K pump, in the basolateral membrane.     

The hydrogen ion-pumping adenosine triphosphatase of platelet dense granule membrane. Differences from F1F0- and phosphoenzyme-type ATPases

Using a coupled transport assay which detects only those ATPase molecules functionally inserted into the platelet dense granule membrane, we have characterized the inhibitor sensitivity, substrate specificity, and divalent cation requirements of the granule H+ pump. Under identical assay conditions, the granule ATPase was insensitive to concentrations of NaN3, oligomycin, and efrapeptin which almost completely inhibit ATP hydrolysis by mitochondrial membranes. The granule ATPase was inhibited by dicyclohexylcarbodiimide but only at concentrations much higher than those needed to maximally inhibit mitochondrial ATPase. Vanadate (VO3-) ion and ouabain also failed to inhibit granule ATPase activity at concentrations which maximally inhibited purified Na+,K+-ATPase. Two alkylating agents, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole and N-ethylmaleimide both completely inhibited H+ pumping by the granule ATPase under conditions where ATP hydrolysis by mitochondrial membranes or Na+,K+-ATPase was hardly affected. These results suggest that the H+-pumping ATPase of platelet granule membrane may belong to a class of ion-translocating ATPases distinct from both the phosphoenzyme-type ATPases present in plasma membrane and the F1F0-ATPases of energy-transducing membranes.     

Role of separate K+ and Cl- channels and of Na+/Cl- cotransport in volume regulation in Ehrlich cells

Cells resuspended in hypotonic medium initially swell as nearly perfect osmometers, but later recover their volume with an associated KCl loss. This regulatory volume decrease (RVD) is unaffected when nitrate is substituted for Cl- or if bumetanide or 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) is added. It is inhibited by quinine, Ba2+, low pH, anticalmodulin drugs, and depletion of intracellular Ca2+. It is accelerated by the Ca2+ ionophore A23187, or by a sudden increase in external Ca2+ and at high pH. A net KCl loss is also seen after addition of ionophore A23187 in isotonic medium. Similarities are demonstrated between the KCl loss seen after addition of A23187 and the KCl loss seen during RVD. It is proposed that separate conductive K+ and Cl- channels are activated during RVD by release of Ca2+ from internal stores, and that the effect is mediated by calmodulin. After restoration of tonicity the cells shrink initially, but recover their volume with an associated KCl uptake. This regulatory volume increase (RVI) is inhibited when NO3- is substituted for Cl-, and is also inhibited by furosemide or bumetanide, but it is unaffected by DIDS. The unidirectional Cl-flux ratio is compatible with either a coupled uptake of Na+ and Cl-, or an uptake via a K+/Na+/2Cl- cotransport system. No K+ uptake was found, however, in ouabain-poisoned cells where a bumetanide-sensitive uptake of Na+ and Cl- in nearly equimolar amounts was demonstrated. Therefore, it is proposed that the primary process during RVI is an activation of an otherwise quiescent Na+/Cl- cotransport system with subsequent replacement of Na+ by K+ via the Na+/K+ pump. There is a marked increase in the rate of pump activity in the absence of a detectable increase in intracellular Na+ concentration.     

CV-1 cell recipients of the mouse ouabain resistance gene express a ouabain-insensitive Na,K-ATPase after growth in cardioactive steroids

The cation-transporting activity and Na,K-ATPase activity of CV-1 cell recipients of the mouse ouabain resistance gene (ouaR6, or OR6 cells; see Levenson, R., Racaniello, V., Albritton, L., and Housman, D. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1489-1493) have been further characterized. OR6 cells grown in strophanthidin (a cardiac aglycon which may be removed rapidly from the Na,K-ATPase) possess both ouabain-sensitive and -insensitive 86Rb+ uptake activities. The ouabain-sensitive 86Rb+ uptake activity of these cells (OR6-S cells) exhibits the same Ki for ouabain as that of the CV-1 parent cells (Ki(app) = 3 x 10(-7) M ouabain), but accounts for only approximately 30% of total 86Rb+ uptake into Na+-loaded OR6-S cells, compared to 80% for CV-1 cells. Most of the ouabain-resistant 86Rb+ uptake in OR6-S cells is dependent on internal Na+ and is insensitive to furosemide, suggesting that it is due to an ouabain-resistant Na,K pump. In OR6-S cell lysates, 50% of Na+-dependent ATPase activity is insensitive to 1 mM ouabain, compared to less than 5% in CV-1 cell lysates. In addition, purified plasma membranes from OR6-S cells contain a 100-kDa protein which is transiently phosphorylated by ATP in an Na+-dependent, K+-sensitive manner, like the alpha subunit of the CV-1 Na,K-ATPase and the canine renal Na,K-ATPase, but which is unaffected by preincubation in 1 mM ouabain. All of these data suggest that OR6-S cells possess a ouabain-insensitive Na,K pump with characteristics similar to the ouabain-sensitive pump of CV-1 parent cells. Since the mouse ouabain resistance gene does not encode either subunit of the Na,K-ATPase, these results suggest that the ouabain resistance gene product may modify the ouabain sensitivity of the endogenous CV-1 Na,K pump.     

The mechanism underlying the laxative properties of Parsley extract

Parsley has been claimed in folk medicine to possess laxative properties attributed to the presence therein of some volatile oils that are more concentrated in seeds than in stems or leaves. The advocated physiological effect of parsley, does not have, however, any proven scientific background and relies mainly on simple observations and empirical information. This work aims at providing the scientific evidence that would confirm or reject the claimed laxative role of parsley, and at determining its mechanism of action if present. A perfusion technique was used to measure the net fluid absorption from the rat colon. The addition of an aqueous extract of parsley seeds to the perfusion buffer, and the omission of sodium, both reduced significantly net water absorption from the colon, as compared to the control. Parsley, added to a sodium-free buffer did not lead to any further significant change in water absorption as compared to parsley alone inferring that with parsley sodium absorption was already inhibited. Since K+ and Cl- secretion depends on the activity of the NaKCl2 transporter, the latter was inhibited with furosemide which increased significantly net water absorption. When parsley and furosemide were added together, net water absorption was significantly higher than with parsley alone and significantly lower than with furosemide alone. In addition, parsley extract was shown to inhibit the in vitro activity of the Na+-K+ATPase in a colon homogenate and the activity of a partially purified dog kidney ATPase. The results suggest that parsley acts by, inhibiting sodium and consequently water absorption through an inhibition of the Na+-K+ pump, and by stimulating the NaKCl, transporter and increasing electrolyte and water secretion.