Effect of inhibition of Na+-K+ ATPase on the prostacyclin generation of cultured human vascular endothelial cells

Prostacyclin (PGI2) generation of cultured human vascular endothelial cells (VEC) was observed coincidentally with the increase of 45Ca net influx. Ca ionophore A23187 enhanced not only PGI2 generation and 45Ca net influx but also 45Ca efflux. PGI2 generation was completely abolished by the pretreatment with Ca++ immobilizer, TMB-8. A Na+-K+ ATPase inhibitor, ouabain increased 45Ca net influx, but decreased 45Ca efflux, and enhanced PGI2 generation. These observation indicate that PGI2 generation of VEC may be regulated by not only Ca++ but also Na+, and it was suggested that enhanced PGI2 generation by ouabain might be derived from the increased cytosolic Ca++concentration by the decreased Ca++ efflux, and it was considered to be originated from the suppression of Na+-Ca++ exchange systems by the increased intracellular Na+ concentration via inhibition of Na+-K+ ATPase activity by ouabain. Enhancement of PGI2 generation of VEC by the increased ouabain like substances (OLS) in hypertension is suspected to be beneficial on the maintenance of vascular homeostasis.     

Rapid changes in bidirectional K+ fluxes preceding DMSO-induced granulocytic differentiation of HL-60 human leukemic cells

When grown in medium containing 5 mM potassium and 140 mM sodium, HL-60, a human promyelocytic cell line, maintained a steady-state intracellular K+ concentration of 145 mmol/L cells and a steady-state intracellular Na+ concentration of 30 mmol/L cells. Nearly 90% of the unidirectional 42K+ influx could be inhibited by the cardiac glycoside ouabain with a Ki of 5 X 10(-8) M. This ouabain-sensitive component of influx rose as a saturating function of the extracellular K+ concentration with a K1/2 of 0.85 mM. The component of 42K+ influx resistant to ouabain inhibition was a linear function of the extracellular K+ concentration and was insensitive to inhibition by the diuretic furosemide. Unidirectional K+ efflux followed first order kinetics with a half-time of 55 min. Addition of 1.5% dimethyl sulfoxide (DMSO) to a culture of HL-60 cells allowed two population doublings followed by the cessation of growth without an impairment of cell viability. Beginning 2 to 3 days after DMSO addition, the cells underwent a dramatic reduction in volume (from 925 microns 3 to 500 microns 3) and began to take on the morphological features of mature granulocytes. Throughout this process of differentiation there was no change in the intracellular sodium or potassium concentration. However, immediately following the addition of DMSO to a culture of cells, there began an immediate, coordinated reduction in bidirectional K+ flux. The initial rate of the ouabain-sensitive component of K+ influx fell with a half-time of 11 h to a final rate, at 6 days induction, equal to one ninth that of the uninduced control, and over the same period, the rate constant for K+ efflux fell with a half-time of 14 h to a final value one fourth that of the uninduced control. The rapidity with which these flux changes occur raises the possibility that they play some role in the control of subsequent events in the process of differentiation.     

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.     

Modulation of functional and optimal (Na+-K+)ATPase activity during the cell cycle of neuroblastoma cells

Functional and optimal activities of the (Na+-K+)ATPase, as determined by ouabain-sensitive K+ influx in intact cells and ATP hydrolysis in cell homogenates respectively, have been measured during the cell cycle of neuroblastoma (clone Neuro-2A) cells. The cells were synchronized by selective detachment of mitotic cells. The ouabain-sensitive K+ influx decreased more than fourfold from 1.62 +/- 0.11 nmoles/min/10(6) cells to 0.36 +/- 0.25 nmoles/min/10(6) cells on passing from mitosis to early G1 phase. On entry into S phase a transient sixfold increase to 2.07 +/- 0.30 nmoles/min/10(6) cells was observed, followed by a rapid decline, after which the active K+ influx rose again steadily from 1.03 +/- 0.25 nmoles/min/10(6) cells in early S phase to 2.10 +/- 0.92 nmoles/min/10(6) cells just prior to the next mitosis. The ouabain-insensitive component rose linearly through the cycle in the same manner as the protein content/cell. Combining total K+ influx values with efflux data obtained previously showed that net loss of K+ occurred with transition from mitosis to G1 phase while net accumulation occurred with entry into S. Throughout mid-S phase net K+ flux was virtually zero, but a large net influx occurred again just before the next mitosis. The (Na+-K+)ATPase activity measured in cell homogenates decreased rapidly from mitosis to G1 phase and increased steadily throughout S phase, but the transient activation on entry into S phase was not observed. Complete inhibition of the (Na+-K+)ATPase mediated K+ influx by ouabain (5 mM) prevents the cells from entering S phase, while partial inhibition by lower concentrations of ouabain (0.2 and 0.5 mM; km = 0.17 mM) causes partial blockage in G1 and, to a lesser extent, a reduced rate of progression through the rest of the cell cycle. We conclude that the transient increase in (Na+-K+)ATPase mediated K+ influx at the G1/S transition is a prerequisite for entry into S phase, while maintenance of adequate levels of K+ influx is necessary for normal rate of progression through the rest of the cell cycle.     

Acetylcholine Incorporation by Cholinergic Synaptic Vesicles from Torpedo marmorata

[3H]Acetylcholine efflux and Na+-K+ ATPase ion pump activity were measured concomitantly in rat cortical synaptosomes. Ouabain (500 microM), strophanthidin (500 microM), and parachloromercuribenzene sulfonate (500 microM) each inhibited ouabain-sensitive 86Rb uptake and elevated [3H]acetylcholine release independently of the external calcium concentration. Veratridine (10 microM), electrical field stimulation (60 V, 60 Hz, 5-ms pulse duration), or the calcium ionophore A23187 (10 micrograms/ml) also inhibited ouabain-sensitive 86Rb uptake and released [3H]acetylcholine, but via a calcium-dependent process. Veratridine-induced [3H]acetylcholine release and ion pump inhibition were correlated over a wide range of drug concentrations and both effects were blocked by pre-treatment with tetrodotoxin (1 microM). The rate of [3H]acetylcholine efflux from superfused synaptosomes was increased within 15 s of exposure to ouabain, strophanthidin, veratridine, A23187, or field stimulation, while ouabain-sensitive 86Rb uptake was significantly decreased within a similar interval. These results suggest that [3H]acetylcholine release is due at least in part to inhibition of Na+-K+ ATPase.     

Effect of extracellular volume expansion on erythrocyte sodium transport in rats.

The effects of extracellular volume expansion (EVE) on the major sodium transport systems and sodium and potassium contents in rat erythrocytes have been examined in the present study. Study has been performed in anesthetized Wistar rat weighing about 300 g. Acute extracellular volume expansion (EVE) was induced by a constant intravenous saline infusion (3% body wt, 3 hours). Rats anaesthetized and catheterized but not expanded were used as controls. Arterial blood samples from control and expanded rats were obtained at the same time, and assayed immediately. Intracellular sodium and potassium concentration and ouabain sensitive (Na(+)-K(+)-pump) and bumetanide sensitive (Na(+)-K(+)-cotransport system) outward Na+ fluxes in erythrocytes were measured. The effect of plasma on erythrocyte transport was also analyzed by measuring 86Rb uptake. Neither of two plasma cations (Na+ and K+) were modified by the EVE. Also intracellular Na+ and K+ levels remained unvariable. Total Na+ efflux was not modified by EVE, but pump-mediated Na+ efflux was smaller after than before EVE. The ouabain-inhibible Na+ efflux rate constant decreased after EVE (from 687 +/- 81 to 525 +/- 29 h-1 x 10(-3); P less than 0.05). Both Na(+)-K(+)cotransport-mediated Na+ efflux and passive permeability increased significantly after EVE. The incubation with plasma from saline-infused animals induced a significant decrease in Rb uptake rate constant, that was not observed after incubation with plasma from non-expanded rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

An altered response of virally transformed 3T3 cells to ouabain.

The effect of ouabain on K+ transport was examined in 3T3 and virally transformed 3T3 cells. A 10 min exposure to ouabain (10(-3) M) produced approximately 40% inhibition of the unidirectional K+ influx in all cell lines. In 3T3 cells the response was not significantly altered by up to 70 min exposure to the drug. In contrast, the continued exposure of transformed cells to ouabain produced a time-dependent increase in the K+ influx. This increased influx was shown to be accompanied by an increase in the K+ efflux. The results suggest that, in transformed cells, ouabain produces both an inhibition of Na+-K+ exchange and a stimulation of K+-K+ exchange. The latter was shown to be more readily reversible than the former.     

Lys691 and Asp714 of the Na+/K+-ATPase alpha subunit are essential for phosphorylation, dephosphorylation, and enzyme turnover

P-type ATPases such as the sodium pump appear to be members of a superfamily of hydrolases structurally typified by the L-2-haloacid dehalogenases. In the dehalogenase L-DEX-ps, Lys151 serves to stabilize the excess negative charge in the substrate/reaction intermediates and Asp180 coordinates a water molecule that is directly involved in ester intermediate hydrolysis. To investigate the importance of the corresponding Lys691 and Asp714 of the sodium pump alpha subunit, sodium pump mutants were expressed in yeast and analyzed for their properties. Lys691Ala, Lys691Asp, Asp714Ala, and Asp714Arg mutants were inactive, not only with respect to ATPase activity but also to interaction with the highly sodium pump-specific inhibitors ouabain or palytoxin (PTX). In contrast, conservative mutants Lys691Arg and Asp714Glu retained some of the partial activities of the wild-type enzyme, although they completely failed to display any ATPase activity. Yeast cells expressing Lys691Arg and Asp714Glu mutants are sensitive to the sodium pump-specific inhibitor PTX and lose intracellular K+. Their sensitivity to PTX, with EC50 values of 118 +/- 24 and 76.5 +/- 3.6 nM, respectively, was clearly reduced by almost 7- or 4-fold below that of the native sodium pump (17.8 +/- 2.7 nM). Ouabain was recognized under these conditions with low affinity by the mutants and inhibited the PTX-induced K+ efflux from the yeast cells. The EC50 for the ouabain effect was 183 +/- 20 microM for Lys691Arg and 2.3 +/- 0.08 mM for the Asp714Glu mutant. The corresponding value obtained with cells expressing the native sodium pump was 69 +/- 18 microM. In the presence of Pi and Mg2+, none of the mutant sodium pumps were able to bind ouabain. When Mg2+ was omitted, however, both Lys691Asp and Asp714Glu mutants displayed ouabain binding that was reduced by Mg2+ with an EC50 of 0.76 +/- 0.11 and 2.3 +/- 0.2 mM, respectively. In the absence of Mg2+, ouabain binding was also reduced by K+. The EC50 values were 1.33 +/- 0.23 mM for the wild-type enzyme, 0.93 +/- 0.2 mM for the Lys691Arg mutant, and 1.02 +/- 0.24 mM for the Asp714Glu enzyme. None of the neutral or nonconservative mutants displayed any ouabain-sensitive ATPase activity. Ouabain-sensitive phosphatase activity, however, was present in membranes containing either the wild-type (1105 +/- 100 micromol of p-nitrophenol phosphate hydrolyzed min(-1) mg of protein(-1)) or the Asp714Glu mutant (575 +/- 75 micromol min(-1) mg(-1)) sodium pump. Some phosphatase activity was also associated with the Lys691Arg mutant (195 +/- 63 micromol min(-1) mg(-1)). The results are consistent with Lys691 and Asp714 being essential for the phosphorylation/dephosphorylation process that allows the sodium pump to accomplish the catalytic cycle.     

An Analysis of the Leakages of Sodium Ions into and Potassium Ions out of Striated Muscle Cells

Net sodium influx under K-free conditions was independent of the intracellular sodium ion concentration, [Na]_i, and was increased by ouabain. Unidirectional sodium influx was the sum of a component independent of [Na]_i and a component that increased linearly with increasing [Na]_i. Net influx of sodium ions in K-free solutions varied with the external sodium ion concentration, [Na]_o, and a steady-state balance of the sodium ion fluxes occurred at [Na]_o = 40 mM. When solutions were K-free and contained 10^-4 M ouabain, net sodium influx varied linearly with [Na]_o and a steady state for the intracellular sodium was observed at [Na]_o = 13 mM. The steady state observed in the presence of ouabain was the result of a pump-leak balance as the external sodium ion concentration with which the muscle sodium would be in equilibrium, under these conditions, was 0.11 mM. The rate constant for total potassium loss to K-free Ringer solution was independent of [Na]_i but dependent on [Na]_o. Replacing external NaCl with MgCl₂ brought about reductions in net potassium efflux. Ouabain was without effect on net potassium efflux in K-free Ringer solution with [Na]_o = 120 mM, but increased potassium efflux in a medium with NaCl replaced by MgCl₂. When muscles were enriched with sodium ions, potassium efflux into K-free, Mg⁺⁺-substituted Ringer solution fell to around 0.1 pmol/cm²·s and was increased 14-fold by addition of ouabain.     

Sodium and potassium fluxes and membrane potential of human neutrophils: evidence for an electrogenic sodium pump

Sodium and potassium ion contents and fluxes of isolated resting human peripheral polymorphonuclear leukocytes were measured. In cells kept at 37 degrees C, [Na]i was 25 mM and [K]i was 120 mM; both ions were completely exchangeable with extracellular isotopes. One-way Na and K fluxes, measured with 22Na and 42K, were all approximately 0.9 meq/liter cell water . min. Ouabain had no effect on Na influx or K efflux, but inhibited 95 +/- 7% of Na efflux and 63% of K influx. Cells kept at 0 degree C gained sodium in exchange for potassium ([Na]i nearly tripled in 3 h); upon rewarming, ouabain-sensitive K influx into such cells was strongly enhanced. External K stimulated Na efflux (Km approximately 1.5 mM in 140-mM Na medium). The PNa/PK permeability ratio, estimated from ouabain insensitive fluxes, was 0.10. Valinomycin (1 microM) approximately doubled PK. Membrane potential (Vm) was estimated using the potentiometric indicator diS-C3(5); calibration was based on the assumption of constant-field behavior. External K, but not Cl, affected Vm. Ouabain caused a depolarization whose magnitude dependent on [Na]i. Sodium-depleted cells became hyperpolarized when exposed to the neutral exchange carrier monensin; this hyperpolarization was abolished by ouabain. We conclude that the sodium pump of human peripheral neutrophils is electrogenic, and that the size of the pump-induced hyperpolarization is consistent with the membrane conductance (3.7-4.0 microseconds/cm2) computed from the individual K and Na conductances.     

Na+ channel and Na+-K+ ATPase involvement in norepinephrine- and veratridine-stimulated metabolism in perfused rat hind limb.

In the constant flow perfused rat hind limb, norepinephrine (NE) evoked increases in oxygen uptake (VO2) and lactate efflux (LE) were inhibited by the cardiac glycoside ouabain (1 mM), without interrupting the NE-mediated vasoconstriction. The membrane labilizer veratridine, previously shown to increase VO2 and LE, without increasing perfusion pressure, was also shown to be inhibited by the cardiac glycoside ouabain, as well as by the ouabain analogues digitoxin and digoxin. The stimulatory actions of veratridine on VO2 were inhibitable by low doses of the specific sodium channel blocker tetrodotoxin (TTX), while NE effects were unaffected, suggesting that NE may be acting via a TTX-insensitive sodium channel. It is concluded that agents such as NE (a vasoconstrictor) or veratridine (a membrane labilizer), which stimulate VO2 in the perfused rat hind limb, do so by increasing Na+ influx. The observed increases in oxygen consumption and LE are due to Na+-K+ ATPase activity to pump Na+ out of the cell at the expense of ATP turnover. Energy dissipation due to Na+ cycling may be a form of facultative thermogenesis attributable to NE that can be stimulated by membrane labilizers such as veratridine in the constant flow perfused rat hind limb.     

Effect of ouabain upon diuretic-sensitive K+ transport in cultured cells. Evidence for separate modes of operation of the transporter

(1) Unidirectional K+ (86Rb) influx and efflux were measured in subconfluent layers of MDCK renal epithelial cells and HeLa carcinoma cells. (2) In both MDCK and HeLa cells, the furosemide-inhibitable and chloride-dependent component of K+ influx/efflux was stimulated 2-fold by a 30 min incubation in 1 . 10(-3) M ouabain. (3) Measurements of net K+ loss and Na+ gain in ouabain-treated cells at 1 h failed to show any diuretic sensitive component, confirming the exchange character of the diuretic-sensitive fluxes. (4) Prolonged incubations for 2.5 h in ouabain revealed a furosemide- and anion-dependent K+ (Cl-) outward net flux uncoupled from net Na+ movement. Net K+ (Cl-) outward flux was half-maximally inhibited by 2 microM furosemide. (5) After 2.5 h ouabain treatment, the anion and cation dependence of the diuretic-sensitive K+ influx/efflux were essentially unchanged when compared to untreated controls.     

Effects of dehydroabietic acid on the erythrocyte membrane

The effects of dehydroabietic acid (DHAA), a dominant resin acid in pulp and paper mill effluents, on membrane-connected events were studied in human erythrocytes. Fifty percent haemolysis was achieved by 252 microM DHAA after 1 h of incubation at +37 degrees C. At sublytic concentrations, DHAA protected erythrocytes against hypotonic haemolysis, with maximum protection occurring at 125 microM. In the lower range of sublytic concentrations, DHAA induced a slight echinocytosis; at higher sublytic concentrations erythrocytes were transformed to sphero-echinocytes and a release of acetylcholinesterase (exovesicles) occurred. Furthermore, at sublytic concentrations DHAA increased potassium efflux and passive potassium influx, while active potassium influx ((Na(+)-K+)-pump activity) and phosphate efflux were decreased. Our study indicates that DHAA acts on human erythrocytes in a way typical for amphiphilic compounds. It is proposed that DHAA by intercalating into the lipid bilayer of the membrane, affects the dynamics of the bilayer which in turn alters the permeability of the bilayer and the function of ion transporting membrane proteins.     

Assay of muscarinic acetylcholine receptor function in cultured cardiac cells by stimulation of 86Rb+ efflux

An assay for the increase in potassium permeability mediated by muscarinic acetylcholine receptors (mAChR) in cultured cardiac cells is described, using the K+ ion substitute 86Rb+ as the tracer ion. Cardiac cells accumulate 86Rb+ from the extracellular medium in a Na+/K+ ATPase-dependent manner. Subsequent efflux of 86Rb+ in the absence and presence of muscarinic agonists follows kinetics similar to those previously reported for 42K+. The mAChR agonist carbamylcholine (carbachol) stimulated 86Rb+ efflux with an EC50 of 50 nM. The half-time for efflux is reduced by greater than 40% at maximally effective concentrations of agonist. Stimulation of 86Rb+ efflux by carbachol is blocked by the mAChR antagonist atropine with an IC50 of 15 nM. The stimulation of 86Rb+ efflux by carbachol is not affected by the presence of the Na+/K+ ATPase inhibitor ouabain. This assay provides a method for quantitating the mAChR-mediated increase in K+ permeability in cardiac cells without the use of 42K+.     

Exogeneous diacylglycerols downregulate the activity of Na(+)-K+ pump in Xenopus laevis oocytes.

In this study, cell permeable diacylglycerols, sn-1,2-dioctanoglycerol (DiC8), and sn-1-oleoyl-2-acetylglycerol (OAG) were found to downregulate the activity of Na(+)-K+ pump in Xenopus laevis oocytes. Both DiC8 and OAG decreased the binding of [3H]ouabain to intact oocytes while phorbol esters did not appreciably influence the same. These diacylglycerols inhibited the amiloride-sensitive 22Na+ influx and ouabain-sensitive 86Rb+ uptake in the oocytes. Furthermore, DiC8 prevented the 22Na+ efflux from the oocytes preloaded with 22Na+. Addition of H-7 to DiC8- and OAG-treated oocytes stimulated the pump activity curtailed by the two latters. The impairment of Na(+)-K+ pump activity by diacylglycerols suggests that protein kinase C activators may stimulate endocytosis of membrane-coupled Na(+)-K+ ATPase.     

Class I antiarrhythmic drug effects on ouabain binding to guinea pig cardiac Na+ -K+ ATPase

The notion that the inhibition of the Mg2+ -dependent ATP-hydrolytic function of the myocardial Na+ -K+ ATPase by class I antiarrhythmic agents occurs as a result of their binding to the same receptor sites as the digitalis glycosides was tested by performing competitive binding assays of [3H]ouabain (OUA) with eight drugs: disopyramide, encainide, lidocaine, lorcainide, phenytoin, procainamide, quinidine, and tocainide in guinea pig heart microsomal preparations. In the first set of experiments, 10-200 microM concentrations of the drugs were preincubated with the enzyme and displacement assays performed with 250 nM OUA. The drugs showed receptor occupancy of 19-32% at 50 microM, 25-44% at 100 microM, and 37-56% at 200 microM. Then, 10-500 nM concentrations of OUA were preincubated with the enzyme, and competitive assays were performed using 200 microM concentrations of the drugs. OUA occupied 39-51% of the receptor sites at 100 nM, 44-67% at 250 nM, and 62-82% at 500 nM, displacing the drugs in a concentration-dependent fashion. The results show that antiarrhythmic drugs interact with the same or similar receptor sites as ouabain on the Na+ -K+ ATPase, pointing to a possible contribution of these interactions to the mechanism for their inhibitory actions on the enzyme, and perhaps their arrhythmogenic effects.     

Cation transport and content in serum-stimulated CHO-773 cells. II. Late changes in rubidium influx and intracellular potassium content

Serum stimulation of stationary cultures of Chinese hamster ovary cells CHO-K1 (clone 773) is accompanied by sustained increase in ouabain-sensitive rubidium (potassium) influx which results in the elevation of intracellular potassium content from 0.5-0.6 to 0.7-0.8 mmole per gram of protein. Cytofluorometric studies of serum-stimulated CHO-773 cultures have shown that the intracellular potassium increase is necessary for successful G1----S progression. The elevation of intracellular potassium was found to occur simultaneously with the cellular protein growth. Cycloheximide (10 micrograms/ml) does not influence the early Na,K-ATPase activation induced by serum; however, it abolishes the sustained increase of both rubidium influx and intracellular potassium content. In serum stimulated cells ouabain increases the potassium efflux; this ouabain effect is not observed after S phase, when rubidium (potassium) influx decreases and intracellular potassium content stops growing.     

Alteration of 86Rb+ influx and efflux following depletion of membrane sterol in L-cells

Ouabain-sensitive uptake of 86Rb+ (an analogue of K+) was enhanced in L-cells that had been treated with 25-hydroxycholesterol or 7-ketocholesterol in order to deplete their sterol concentration. Ouabain-insensitive Rb+ efflux also increased in the sterol-depleted cells and the intracellular concentration of K+ diminished while the concentration of Na+ increased. All of these effects of 25-hydroxycholesterol were counteracted by the addition of mevalonate to the culture medium. Despite the evidence for increased active Rb+ transport in the 25-hydroxycholesterol-treated cells, the level of sodium and potassium ion-activated adenosine triphosphatase ((Na+ + K+)-activated ATPase) activity measured in homogenates and plasma membrane preparations from the treated cells was not significantly different from the control values. Rb+ uptake was more sensitive to ouabain inhibition in sterol-depleted cells than in control cells, although ATPase activity in plasma membrane fractions isolated from treated cells was not more sensitive to ouabain inhibition than was that from control cells. It is possible that the ability of the oxygenated sterols to inhibit DNA synthesis and cell division (Kandutsch, A. A., and Chen, H. W. (1977) J. Biol. Chem. 252, 409-415) is related to their effects upon cellular ion transport.     

Content of K+ and Na+ in seminiferous tubule and rete testis fluids from Sertoli cell-enriched testes

Seminiferous tubules of rats exposed to x-irradiation before birth were subjected to micropuncture in situ at 50 days of age to obtain samples of fluid 4 h after ligation of efferent ducts. The concentrations of cations in this fluid were: potassium, 39.7 +/- 1.2 mM, and sodium, 136.3 +/- 1.2 mM (means and standard errors, n = 5). Histologic examination revealed that germ cells constitute less than 1% of the cell population within the seminiferous tubules of these rats; the remaining cells were all Sertoli cells. Sertoli cells showed efflux of 86Rb+ with t1/2 of approximately 11 min and an active ATPase in plasma membranes. These activities were similar to those of Sertoli cells from normal rats. Germ cells from normal rats showed less rapid efflux of 86Rb+ (t1/2 greater than 60 min) and less active Na+/K+ ATPase in plasma membranes. It is concluded that Sertoli cells are responsible for the high concentration of potassium in seminiferous tubule fluid and that plasma membranes of these cells contain an active K+ pump that is not inhibited by ouabain (1 mM).     

Epidermal growth factor accelerates recovery of LLC-PK1 cells following oxidant injury

Summary In renal tubular epithelial cells, oxidant injury results in several metabolic alterations including ATP depletion, decreased Na⁺K⁺ ATPase activity, and altered intracellular sodium and potassium content. To investigate the recovery of LLC-PK1 cells following oxidant injury and to determine if recovery can be accelerated, we induced oxidant stress in LLC-PK1 cells with 500 μM hydrogen peroxide for 60 min. Identical cohorts of oxidant-stressed cells were incubated in recovery medium without epidermal growth factor (EGF) or recovery medium containing 25 ng EGF per ml. ATP levels, Na⁺K⁺ ATPase activity in whole cells, Na⁺K⁺ ATPase activity in disrupted cells, and intracellular sodium and potassium ion content were determined at 0, 5, 24, 48, and 72 h following oxidant injury in each cohort of cells. In oxidant-stressed cells recovering in medium without EGF, ATP levels, Na⁺K⁺ ATPase activity, and intracellular ion content improved but continued to remain substantially lower than control values at all time points following oxidant stress. In cells recovering in medium with EGF, ATP levels, Na⁺K⁺ ATPase activity, and the intracellular potassium-to-sodium ratio were significantly higher at nearly all time points than values in cells recovering in medium alone. In cells recovering with added EGF, Na⁺K⁺ ATPase activity had improved to control levels, whereas ATP levels and intracellular ion content approached control values by 72 h following oxidant stress. We conclude that oxidant-mediated ATP depletion, altered Na⁺K⁺ ATPase activity, and intracellular ion content remain depressed for several d following oxidant stress and that EGF accelerated recovery of LLC-PK1 cells from oxidant injury.