Regulation of the Na,K-ATPase activity of Madin-Darby canine kidney cells in defined medium by prostaglandin E1 and 8-bromocyclic AMP.

The role of PGE1 in regulating the activity of the Na+, K(+)-ATPase in Madin Darby Canine Kidney (MDCK) cells has been examined. PGE1 increased the initial rate of ouabain-sensitive Rb+ uptake by MDCK cells, a process that continued to occur over a 5-day period. The increase in the initial rate of ouabain-sensitive Rb+ uptake in MDCK cells treated with PGE1 could be explained by a 1.6-fold increase in the Vmax for ouabain-sensitive Rb+ uptake. The increase in the Vmax for ouabain-sensitive Rb+ uptake observed in MDCK cells under these conditions can be explained either by an increase in the number of active Na+ pumps, or by an increase in the efficiency of the Na+ pumps. Consistent with the former possibility is the observed increase in the number of ouabain binding sites, as well as the increase in Na+, K(+)-ATPase activity in cell lysates obtained from MDCK monolayers treated with PGE1. The involvement of cyclic AMP in mediating these effects of PGE1 on the Na+, K(+)-ATPase in MDCK cells is supported by: (1) the observation of similar effects in 8-bromocyclic AMP treated MDCK monolayers, and (2) a dramatic reduction of the stimulatory effects of PGE1 and 8-bromocyclic AMP on the Vmax for ouabain-sensitive Rb+ uptake, and on the number of ouabain binding sites in dibutyryl cyclic AMP resistant clone 3 (DBr3) (which is defective in cyclic AMP dependent protein kinase activity). PGE1 independent MDCK monolayers exhibit both an increase in the Vmax for ouabain-sensitive Rb+ uptake and an increase in the number of ouabain binding sites in response to 8-bromocyclic AMP. Apparently, the cyclic AMP phosphodiesterase defect in these PGE1 independent cells did not cause cellular cyclic AMP levels to be elevated to a sufficient extent to maximally increase the Na+, K(+)-ATPase activity in these variant cells.     

Increases in Na/K pump numbers in isolated human lymphocytes exposed to lithium in vitro. Reversal by myo-inositol and by inhibitors of protein kinase C and the Na/H antiport

Lithium (1-8 mM) caused a dose-dependent increase in the number of [3H]ouabain binding sites and in sodium/potassium (Na/K) pump activity in normal lymphocytes after incubation for 72 h. The increase in Na/K pump activity was due to an increase in the Vmax of the pump, with no change in the apparent affinity (Km) for potassium (rubidium). There was no change in the turnover number of the pump and the intracellular sodium concentration fell. The increase in [3H]ouabain binding sites was prevented by the addition of myo-inositol (10 mM), by inhibition of the protein kinase C with staurosporine (100 nM) and by inhibition of the Na/H antiport with dimethylamiloride (50 microM). These results suggest that the increase in Na/K pump activity caused by lithium is due to an increase in pump numbers and not due to increased activity of individual pumps or to an alteration in the affinity of the pumps for potassium. The increase in Na/K pump numbers and activity in lymphocytes exposed to lithium for 72 h may be related to altered Na/H antiport activity secondary to inhibition of phosphoinositol breakdown by lithium.     

Two functionally different Na/K pumps in cardiac ventricular myocytes

The whole-cell patch-clamp technique was used to voltage clamp acutely isolated myocytes at -60 mV and study effects of ionic environment on Na/K pump activity. In quiescent guinea pig myocytes, normal intracellular Na+ is approximately 6 mM, which gives a total pump current of 0.25 +/- 0.09 pA/pF, and an inward background sodium current of 0.75 +/- 0.26 pA/pF. The average capacitance of a cell is 189 +/- 61 pF. Our main conclusion is the total Na/K pump current comprises currents from two different types of pumps, whose functional responses to the extracellular environment are different. Pump current was reversibly blocked with two affinities by extracellular dihydro-ouabain (DHO). We determined dissociation constants of 72 microM for low affinity (type-1) pumps and 0.75 microM for high affinity (type-h) pumps. These dissociation constants did not detectably change with two intracellular Na+ concentrations, one saturating and one near half- saturating, and with two extracellular K+ concentrations of 4.6 and 1.0 mM. Ion effects on type-h pumps were therefore measured using 5 microM DHO and on total pump current using 1 mM DHO. Extracellular K+ half- maximally activated the type-h pumps at 0.4 mM and the type-1 at 3.7 mM. Extracellular H+ blocked the type-1 pumps with half-maximal blockade at a pH of 7.71 whereas the type-h pumps were insensitive to extracellular pH. Both types of pumps responded similarly to changes in intracellular-Na+, with 9.6 mM causing half-maximal activation. Neither changes in intracellular pH between 6.0 and 7.2, nor concentrations of intracellular K+ of 140 mM or below, had any effect on either type of pump. The lack of any effect of intracellular K+ suggests the dissociation constants are in the molar range so this step in the pump cycle is not rate limiting under normal physiological conditions. Changes in intracellular-Na+ did not affect the half-maximal activation by extracellular K+, and vice versa. We found DHO-blockade of Na/K pump current in canine ventricular myocytes also occurred with two affinities, which are very similar to those from guinea pig myocytes or rat ventricular myocytes. In contrast, isolated canine Purkinje myocytes have predominantly the type-h pumps, insofar as DHO-blockade and extracellular K+ activation are much closer to our type-h results than type-1. These observations suggest for mammalian ventricular myocytes: (a) the presence of two types of Na/K pumps may be a general property. (b) Normal physiological variations in extracellular pH and K+ are important determinants of Na/K pump current. (c) Normal physiological variations in the intracellular environment affect Na/K pump current primarily via the Na+ concentration. Lastly, Na/K pump current appears to be specifically tailored for a tissue by expression of a mix of functionally different types of pumps.     

Synchronization Modulation Increases Transepithelial Potentials in MDCK Monolayers through Na/K Pumps

Transepithelial potential (TEP) is the voltage across a polarized epithelium. In epithelia that have active transport functions, the force for transmembrane flux of an ion is dictated by the electrochemical gradient in which TEP plays an essential role. In epithelial injury, disruption of the epithelial barrier collapses the TEP at the wound edge, resulting in the establishment of an endogenous wound electric field (∼100 mV/mm) that is directed towards the center of the wound. This endogenous electric field is implicated to enhance wound healing by guiding cell migration. We thus seek techniques to enhance the TEP, which may increase the wound electric fields and enhance wound healing. We report a novel technique, termed synchronization modulation (SM) using a train of electric pulses to synchronize the Na/K pump activity, and then modulating the pumping cycles to increase the efficiency of the Na/K pumps. Kidney epithelial monolayers (MDCK cells) maintain a stable TEP and transepithelial resistance (TER). SM significantly increased TEP over four fold. Either ouabain or digoxin, which block Na/K pump, abolished SM-induced TEP increases. In addition to the pump activity, basolateral distribution of Na/K pumps is essential for an increase in TEP. Our study for the first time developed an electrical approach to significantly increase the TEP. This technique targeting the Na/K pump may be used to modulate TEP, and may have implication in wound healing and in diseases where TEP needs to be modulated.     

Transport by the (Na+,K+) ATPase: modulation by differentiation inducers and inhibition of protein synthesis in the MDCK kidney epithelial cell line

MDCK kidney epithelial cell cultures exposed to the differentiation inducer hexamethylene bisacetamide (HMBA) for 24 hours exhibited a 50% decrease in transport activity per (Na+,K+)-ATPase molecule (turnover number) but an unchanged number of pump sites (Kennedy and Lever, 1984). Inhibition of protein synthesis by either 10 microM cycloheximide or 2 microM emetine blocked the inhibitory effects of HMBA on Na+/K+ pump efficiency assessed by measurements of [3H]-ouabain binding to intact cells, (Na+,K+) ATPase activity of detergent-activated cell extracts, and ouabain-sensitive Rb+ uptake. In the absence of inducer treatment, inhibition of protein synthesis increased Na+/K+ pump turnover number by twofold while maintaining Na+/K+ pump activity per cell at a constant level. Intracellular Na+ levels were decreased after cycloheximide treatment; therefore, pump stimulation was not due to substrate effects. Furthermore, cycloheximide effects of Rb+ uptake could be dissociated from effects on tight junctions. These observations suggest that the transport activity of the (Na+,K+) ATPase is tightly regulated by factors dependent on protein synthesis.     

Calcium-mobilizing hormones and phorbol myristate acetate mediate heterologous desensitization of the hormone-sensitive hepatic Na+/K+ pump.

The Na+/K+ pump in rat hepatocytes is stimulated in response to Ca2+-mobilizing hormones such as [arginine]vasopressin (AVP), angiotensin II and adrenaline, as well as tumour promoters such as 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). The ability of these agents to increase cellular contents of diacylglycerol and activate protein kinase C may be necessary to observe this response. In the present work, ouabain-sensitive 86Rb+ uptake was studied in isolated rat hepatocytes to help to explain why stimulation of the Na+/K+ pump by Ca2+-mobilizing hormones and tumour promoters is not temporally sustained relative to other hormone responses. A transient stimulation (3-4 min) of the Na+/K+ pump was observed in hepatocytes exposed to high (10 nM), but not low (0.1 nM), concentrations of AVP. Experiments with the Ca2+ chelator EGTA and the Na+ ionophore monensin indicate that the rapid secondary decrease in Na+/K+-pump activity which occurs after AVP stimulation is not due to changes in cytosolic Ca2+ and Na+ concentrations. When added after the stimulation and rapid decrease in Na+/K+-pump activity induced in hepatocytes by a high concentration of AVP, a second challenge with AVP or PMA failed to stimulate the pump. Similarly, previous exposure of hepatocytes to angiotensin, adrenaline or PMA attenuated the subsequent Na+/K+-pump responses to AVP and PMA. In contrast, previous exposure to AVP had no significant effect on subsequent stimulation of the Na+/K+-pump by monensin, glucagon, forskolin or 8-p-chlorophenylthio cyclic AMP. In addition, exposure to monensin had no effect on subsequent responses to AVP and PMA. These data indicate that high concentrations of Ca2+-mobilizing hormones and PMA result in heterologous desensitization of the hepatic Na+/K+ pump to subsequent stimulation by Ca2+-mobilizing hormones and PMA, but not by cyclic-AMP-dependent agonists or monensin.     

Sodium ion influx in proliferating lymphocytes: an early component of the mitogenic signal

Stimulation of pig peripheral blood lymphocytes with concanavalin A (Con A) provoked a rapid increase (two- to threefold) in the rate of ouabain-inhibitable K+ uptake observable within 3-10 min of stimulation with mitogen. At least two phases can be distinguished in the activation of the Na+/K+ pump: the early phase (till 3 h) is characterized by an unaltered number of ouabain binding sites and the later phase (noted at 5 h) by an increased number of such sites. Both K+ efflux and influx increased to the same extent, thereby maintaining [K+]i at the same level as in resting cells (120 mM). Within 3 min of addition of mitogen, the rates of total and amiloride-inhibitable Na+ uptake went up two- and fourfold, respectively, thus resulting in rapid increase in [Na+]i from 20 to about 50 mM. Activation of the Na+/K+ pump was not observed when the cells were stimulated with Con A in low Na+ medium (9 mM), nor did the usual rise in [Na+]i occur. When monensin (30 microM), a Na+/H+ ionophore, was added to resting cells, an increase in both [Na+]i and active K+ uptake occurred in normal medium but not when cells were suspended in low Na+ isotonic buffer. Amiloride (500 microM), on the other hand, prevented both the Con A-induced increase in [Na+]i and the activation of the Na+/K+ pump. Despite complete inhibition of the Na+,K+-ATPase in the presence of ouabain (1 mM), Con A activated the amiloride-inhibitable Na+ uptake in the usual way. In mouse splenocytes stimulated with Con A, there was also a parallel rise in both [Na+]i and active K+ uptake but this took considerably longer to occur than was the case in pig peripheral blood lymphocytes. Increase in both ionic fluxes, the former passive and the latter active, is essential to the entry and maintenance of the cells in proliferative cycle.     

Active sodium and potassium transport in high potassium and low potassium sheep red cells

The kinetic characteristics of the ouabain-sensitive (Na + K) transport system (pump) of high potassium (HK) and low potassium (LK) sheep red cells have been investigated. In sodium medium, the curve relating pump rate to external K is sigmoid with half maximal stimulation (K_1/2) occurring at 3 mM for both cell types, the maximum pump rate in HK cells being about four times that in LK cells. In sodium-free media, both HK and LK pumps are adequately described by the Michaelis-Menten equation, but the K_1/2 for HK cells is 0.6 ± 0.1 mM K, while that for LK is 0.2 ± 0.05 mM K. When the internal Na and K content of the cells was varied by the PCMBS method, it was found that the pump rate of HK cells showed a gradual increase from zero at very low internal Na to a maximum when internal K was reduced to nearly zero (100% Na). In LK cells, on the other hand, no pump activity was detected if Na constituted less than 70% of the total (Na + K) in the cell. Increasing Na from 70 to nearly 100% of the internal cation composition, however, resulted in an exponential increase in pump rate in these cells to about ⅙ the maximum rate observed in HK cells. While changes in internal composition altered the pump rate at saturating concentrations of external K, it had no effect on the apparent affinity of the pumps for external K. These results lead us to conclude that the individual pump sites in the HK and LK sheep red cell membranes must be different. Moreover, we believe that these data contribute significantly to defining the types of mechanism which can account for the kinetic characteristics of (Na + K) transport in sheep red cells and perhaps in other systems.     

Heterogeneity of the Na(+)-H+ antiport systems in renal cells.

This study analyzes the differential characteristics of the Na(+)-H+ antiport systems observed in several epithelial and non-epithelial renal cell lines. Confluent monolayers of LLC-PK1A cells have a Na(+)-H+ antiport system located in the apical membrane of the cell. This system, however, is not expressed during cell proliferation or after incubation in the presence of different mitogenic agents. In contrast, confluent monolayers of MDCK4 express minimal Na(+)-H+ antiport activity in the confluent monolayer state but reach maximal antiport activity during cell proliferation or after activation of the cells by different mitogenic agents. Similar results were obtained with the renal fibroblastic cell line BHK. The system present in MDCK4 cells is localized in the basolateral membrane of the epithelial cell. In LLC-PK1A cells, an increase in the extracellular Na+ concentration produces a hyperbolic increase in the activity of the Na(+)-H+ antiporter. In MDCK4 and BHK cells, however, an increase in external Na+ produces a sigmoid activation of the system. Maximal activation of the system occur at a pHo 7.5 in LLC-PK1A cells and pHo 7.0 in MDCK4 cells. The Na(+)-H+ antiporter of LLC-PK1A cells is more sensitive to the inhibitory effect of amiloride (Ki 1.8 x 10(-7) M) than is the antiporter of MDCK4 cells (Ki 7.0 x 10(-6) M). Moreover, 5-(N-methyl-N-isobutyl)amiloride is the most effective inhibitor of Na(+)-H+ exchange in LLC-PK1A cells, but the least effective inhibitor in MDCK4 cells. Conversely, the analog, 5-(N,N-dimethyl)amiloride, is the most effective inhibitor of Na(+)-H+ exchange in MDCK4 cells, but is the least effective inhibitor in LLC-PK1A cells. These results support the hypothesis that Na(+)-H+ exchange observed in LLC-PK1A and other cell lines may represent the activity of different Na(+)-H+ antiporters.     

Expression profiles of Na+,K+-ATPase during acute and chronic hypo-osmotic stress in the blue crab Callinectes sapidus

During acclimation to dilute seawater, the specific activity of Na+,K+-ATPase increases substantially in the posterior gills of the blue crab Callinectes sapidus. To determine whether this increase occurs through regulation of pre-existing enzyme or synthesis of new enzyme, mRNA and protein levels were measured over short (<24 h) and long (18 days) time courses. Na+,K+-ATPase expression, both mRNA and protein, did not change during the initial 24-h exposure to dilute seawater (10 ppt salinity). Thus, osmoregulation in C. sapidus during acute exposure to low salinity likely involves either modulation of existing enzyme or mechanisms other than an increase in the amount of Na+,K+-ATPase enzyme. However, crabs exposed to dilute seawater over 18 days showed a 300% increase in Na+,K+-ATPase specific activity as well as a 200% increase in Na+,K+-ATPase protein levels. Thus, it appears that the increase in Na+,K+-ATPase activity during chronic exposure results from the synthesis of new enzyme. The relative amounts of mRNA for the alpha-subunit increased substantially (by 150%) during the acclimation process, but once the crabs had fully acclimated to low salinity, the mRNA levels had decreased and were not different from levels in crabs fully acclimated to high salinity. Thus, there is transient induction of the Na+,K+-ATPase mRNA levels during acclimation to dilute seawater.     

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.     

Amino acid transport in kidney epithelial cell line (MDCK): characteristics of Na+/amino acid symport in membrane vesicles and basolateral localization in cell monolayers

Na+-stimulated amino acid transport was investigated in MDCK kidney epithelial cell monolayers and in isolated membrane vesicles. When transport polarity was assessed in confluent polarized epithelial cell monolayers cultured on Nucleopore filters and mounted between two lucite chambers, Na+-stimulated transport of 2-(methylamino)isobutyric acid (MeAIB), a substrate specific for the A system, was predominantly localized on the basolateral membrane. Na+-stimulated amino acid transport activity was maximal in subconfluent cultures, and was substantially reduced after confluence. A membrane vesicle preparation was isolated from confluent MDCK cell cultures which was enriched in Na+-stimulated MeAIB transport activity and Na+,K+,ATPase activity, a basolateral marker, but was not enriched in apical marker enzyme activities or significantly contaminated by mitochondria. Na+-coupled amino acid transport activity assayed in vesicles exhibited a marked dependence on external pH, with an optimum at pH 7.4. The pattern of competitive interactions among neutral amino acids was characteristic of A system transport. Na+-coupled MeAIB and AIB transport in vesicles was electrogenic, stimulated by creation of an interior-negative membrane potential. The Na+ dependence of amino acid transport in vesicles suggested a Na+ symport mechanism with a 1:1 stoichiometry between Na+ and amino acid.     

The occurrence of thermotolerance in non-proliferating rat hepatocytes in primary culture

The occurrence of thermotolerance, induced by an initial heat treatment at 42 degrees C for 30 min, was studied in adult non-proliferating rat hepatocytes in primary culture. Heat treatment at 42 degrees C for 30 min did not affect cell morphology, cell attachment, Na+, K+ pump activity, K+ content and lactate dehydrogenase accumulation into the medium. In contrast, after exposure to 44 degrees C for 30 min a dramatic change in all these parameters was observed. However, of the cells, which remained attached to the substratum 24 h after treatment, Na+, K+ pump activity and K+ content appeared to be normal compared with untreated cells. Cells, pre-treated at 42 degrees C for 30 min, followed by incubation at 37 degrees C for 16 h, were found to be completely thermal resistant against heat treatment at 44 degrees C, as judged by cell morphology, detachment from the substratum, lactate dehydrogenase accumulation, Na+, K+ pump activity and K+ content. These results show that induction and development of thermotolerance can be studied in non-proliferating cells in primary culture.     

Palytoxin induces an increase in the cation conductance of red cells

Palytoxin (PTX), isolated from the marine soft coral Palythoa tuberculosa, increases the cation conductance of human red cell membranes. In the presence of 10(-10) M PTX and 10(-5) M DIDS, the membrane potential approximates the equilibrium potential for Na+ or K+ rather than Cl-. Even in the absence of DIDS, the Na+ and K+ conductances were greater than the Cl- conductance. The selectivity of the PTX-induced cation conductance is K+ greater than Rb+ greater than Cs+ greater than Na+ greater than Li+ much greater than choline+ greater than TEA+ much greater than Mg2+. Measurements of K+ efflux revealed two apparent sites for activation by PTX, one with a Kal of 0.05 nM and a maximum flux, nu max1, of 1.4 mol/liter of cells per h and another with a Ka2 of 98 nM and a nu max2 of 24 mol/liter of cells per h. These effects of PTX are completely blocked by external ouabain (300 microM) and prevented by internal vanadate (100 microM). When the PTX channels are open, the Na,K pumps do not catalyze ATP hydrolysis. Upon thorough washout of cells exposed to about five molecules of PTX/pump, the Na,K pump of these cells operates normally. Blockage of the positively charged NH2 terminus of PTX with a p-bromobenzoyl group reduces the potency of the compound to induce Na and K fluxes by at least a factor of 100, and to compete with the binding of [3H]ouabain by at least a factor of 10. These data are consistent with the conclusion that PTX binds reversibly to the Na,K pumps in the red cell membrane and opens a (10-pS) channel equally permeable to Na and K at or near each pump site.     

Inhibition of Na(+)-independent H+ pump by Na(+)-induced changes in cell Ca2+

Apical membrane H+ extrusion in the renal outer medullary collecting duct, inner stripe, is mediated by a Na(+)-independent H+ pump. To examine the regulation of this transporter, cell pH and cell Ca2+ were measured microfluorometrically in in vitro perfused tubules using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and fura-2, respectively. Apical membrane H+ pump activity, assayed as cell pH recovery from a series of acid loads (NH3/NH+4 prepulse) in the total absence of ambient Na+, initially occurred at a slow rate (0.06 +/- 0.02 pH units/min), which was not sufficient to account for physiologic rates of H+ extrusion. Over 15-20 min after the initial acid load, the rate of Na(+)-independent cell pH recovery increased to 0.63 +/- 0.09 pH units/min, associated with a steady-state cell pH greater than the initial pre-acid load cell pH. This pattern suggested an initial suppression followed by a delayed activation of the apical membrane H+ pump. Replacement of peritubular Na+ with choline or N-methyl-D-glucosamine resulted in an initial spike increase in cell Ca2+ followed by a sustained increase in cell Ca2+. The initial rate of Na(+)-independent cell pH recovery could be increased by elimination of the Na+ removal-induced sustained cell Ca2+ elevation by: (a) performing studies in the presence of 135 mM peritubular Na+ (1 mM peritubular amiloride used to inhibit basolateral membrane Na+/H+ antiport); (b) clamping cell Ca2+ low with dimethyl-BAPTA, an intracellular Ca2+ chelating agent; or (c) removal of extracellular Ca2+. Cell acidification induced a spike increase in cell Ca2+. The late acceleration of Na(+)-independent cell pH recovery was independent of Na+ removal and of the method used to acidify the cell, but was eliminated by prevention of the cell Ca2+ spike and markedly delayed by the microfilament-disrupting agent, cytochalasin B. This study demonstrates that peritubular Na+ removal results in a sustained elevation in cell Ca2+, which inhibits the apical membrane H+ pump. In addition, rapid cell acidification associated with a spike increase in cell Ca2+ leads to a delayed activation of the H+ pump. Thus, cell Ca2+ per se, or a Ca(2+)-activated pathway, can modulate H+ pump activity.     

The Basis of Higher Na+ Transport by Inner Medullary Collecting Duct Cells from Dahl Salt-Sensitive Rats: Implicating the Apical Membrane Na+ Channel

The present experiments were designed to examine the function of Na/K pumps from Dahl salt-sensitive (S) and salt-resistant (R) rats. Previous reports have suggested that there is a difference in primary sequence in the α₁ subunit, the major Na/K pump isoform in the kidney. This sequence difference might contribute to differences in NaCl excretion in these two strains which in turn could influence the systemic blood pressure. Using ``back-door' phosphorylation of pumps isolated from basolateral membranes of kidney cortex, we found no differences between S and R strains. We also examined the Na/K pumps from cultured inner medullary collecting duct (IMCD) cells. This approach takes advantage of the fact that monolayers cultured from S rats transport about twice as much Na⁺ as monolayers cultured from R rats. In cells whose apical membrane was made permeable with amphotericin B, comparison of the affinities for ouabain, Na⁺, and K⁺, respectively, showed only small or no differences between S and R monolayers. Ouabain binding showed no difference in the number of Na/K pumps on the basolateral membrane of cultured cells, despite a 2-fold difference in Na⁺ transport rates. The analysis of the steady-state Na⁺ transport indicates that Na/K pumps in IMCD monolayers from S rats operate at a higher fraction of their maximum capacity than do pumps in monolayers from R rats. The results, taken together, suggest that the major reason for the higher rate of Na⁺ transport in S monolayers is because of a primary increase in the conductive permeability of the apical membrane to Na⁺. They suggest that the epithelial Na⁺ channel is intrinsically different or differently regulated in S and R rats. Received: 6 May 1996/Revised: 16 October 1996     

Changes in sodium pump transport activity and Na+, K+-ATPase expression level following lymphocytes activation in humans

Ouabain-inhibitable rubidium influxes, intracellular sodium content (Nai), and alpha 1-subunit abundance have been studied in human blood lymphocytes, stimulated by phytohemagglutinin (PHA) or by the phorbol 12,13-dibutyrate (PDBu), and calcium ionophore--ionomycin. It is shown that at early stages of PHA-induced activation, the Na/K pump expression (as determined by Wesrn blots of alpha 1 protein in membrane fractions of total cell lysates) does not change, and the increase in Rb influx is due to the increase in Nai and results from the enhanced transport activity of Na/K pumps present in plasma membrane. During the late stages of G0-->G1-->S transit (16-48 h), the increase in Rb influx occurs without changes in Nai, and monensin increases both Nai, and the Rb influx via the Na/K pump. To the end of the first day of mitogen activation, the alpha 1 protein content was found to increase by 5-7 times. A correlation was revealed between changes in ouabain-inhibitable Rb influxes, alpha 1 protein abundance, and the proliferation rate. It is concluded that blasttransformathion of normal human lymphocytes is accompanied by the increase in membrane-associated pool of alpha 1-subunit of Na+,K(+)-ATPase, and the enhanced activity of sodium pump during the G0-->G1-->S progression is provided by increased number of Na+,K(+)-ATPase pumps in plasma membrane.