Peripheral nerve blocks improve analgesia after total knee replacement surgery

Using an in vitro cell system and Cs+ NMR techniques we were able to show that porcine aortic endothelial cells (PAEC) reduce their Na(+)-K(+)-ATPase activity upon an increase in intracellular cAMP. Reduction in the pump rate was due to phosphorylation of the alpha-subunit of the ATPase as shown by immunoprecipitation. Apart from a pump inhibiton using 8-Br-cAMP and IBMX, we were also able to show that changes in the Na(+)-K(+)-ATPase activity could be mediated by the adenosine-A2 and prostaglandin receptor agonists 5'-N-Ethylcarboxamidoadenosine and Iloprost, respectively. Parallel to a decrease in pump activity we also observed a decrease in intracellular Cs+, indicating opening of K+ channels.     

Effect of inhibitors of Na+/H+-exchange and gastric H+/K+ ATPase on cell volume, intracellular pH and migration of human polymorphonuclear leucocytes

1. Stimulation of chemotaxis of human polymorphonuclear leucocytes (PMNs) with the chemoattractive peptide fMLP (N-formyl-Met-Leu-Phe) is paralleled by profound morphological and metabolic alterations like changes of intracellular pH (pHi) and cell shape. The present study was performed to investigate the interrelation of cell volume (CV) regulatory ion transport, pHi and migration of fMLP stimulated PMNs. 2. Addition of fMLP to PMNs stimulated directed migration in Boyden chamber assays and was accompanied by rapid initial intracellular acidification and cell swelling. 3. Inhibition of the Na+/H+ exchanger suppressed fMLP stimulated cell migration, accelerated the intracellular acidification and inhibited the fMLP-induced cell swelling. 4. Step omission of extracellular Na+ caused intracellular acidification, which was accelerated by subsequent addition of gastric H+/K+ ATPase inhibitor SCH 28080, or by omission of extracellular K+ ions. In addition Na+ removal caused cell swelling, which was further enhanced by fMLP. 5. H+/K+ATPase inhibitors omeprazole and SCH 28080 inhibited stimulated migration and blunted the fMLP-induced increase in CV. 6. Increasing extracellular osmolarity by addition of mannitol to the extracellular solution caused cell shrinkage followed by regulatory volume increase, partially due to activation of the Na+/H+ exchanger. In fMLP-stimulated cells the CV increase was counteracted by simultaneous addition of mannitol. Under these conditions the fMLP stimulated migration was inhibited. 7. The antibacterial activity of PMNs was not modified by Hoe 694 or omeprazole. 8. Western analysis with a monoclonal anti gastric H+/K+ ATPase beta-subunit antibody detected a glycosylated 35 kD core protein in lysates of mouse and human gastric mucosa as well as in human PMNs. 9. The results indicate that fMLP leads to cell swelling of PMNs due to activation of the Na+/H+ exchanger and a K+-dependent H+-extruding mechanism, presumably an H+/K+ ATPase. Inhibition of these ion transporters suppresses the increase in CV and precludes PMNs from stimulated migration.     

Gastric oxyntic cell structure as related to secretory activity

The oxyntic, or parietal cell has two characteristic membrane systems. The mammalian intracellular canaliculi are specialized networks of narrow channels lined with numerous microvilli. The other common to all oxyntic cells is the tubulovesicles, a system of tubules and vesicles. The tubulovesicular compartment is drastically depleted during maximal gastric acid secretion and this is coincident with an increase in the cell surface membrane area. A plausible explanation of this process is the fusion and transfer of tubulovesicular membranes to the plasma membrane. However, for many years there was no convincing evidence of the connections between these two membrane systems. How the tubulovesicular membranes transform into plasma membrane without demonstrable connections has been an enigma to electron microscopists. Recent ultra-high resolution scanning electron microscopic observation on the rat oxyntic cell treated with aldehyde-osmium-aldehyde method revealed that in the resting stage, the tubulovesicles were isolated spherical vesicles. But after tetragastrin stimulation, they were interconnected by slender connecting tubules forming a tubulovesicular network. Then this network was fused to the intracellular canaliculus at relatively few points. These connections between the tubulovesicles and luminal surface membrane was also demonstrated in the frog oxynticopeptic cells. In this review, these membrane transformations as well as changes of the H+/K(+)-ATPase, the lectin binding glycocalyx and the cytoskeleton during secretion will be illustrated and discussed.     

Role of gastrin/CCK-B receptor in the regulation of gastric acid secretion in rat

The aim of this study was to investigate whether gastrin regulates morphological changes and alpha-subunit gene expression in parietal cells through the gastrin/CCK-B receptor on enterochromaffin-like cells by histamine release. Treatment with 100 mg/kg of YM022, a potent and selective gastrin/CCK-B receptor antagonist, for one week in rats did not alter mRNA levels of histidine decarboxylase or H+, K+-ATPase. However, parietal cell morphology predominantly changed to the resting form, although the serum gastrin concentration was significantly increased. Additional treatment with YM022 and oral omeprazole, 100 mg/kg, for one week markedly suppressed the increases of mRNA levels of histidine decarboxylase and H+, K+-ATPase and completely blocked the morphological transformation of the parietal cells to a stimulated form induced by treatment with omeprazole alone. This indicates that the morphological transformation of parietal cells to an activated form with a subsequent increase in H+, K+-ATPase synthesis caused by hypergastrinemia is mediated by increased histidine decarboxylase gene expression in enterochromaffin-like cells via gastrin/CCK-B receptors.     

Relationship between fluid transport and in situ inhibition of Na(+)-K+ adenosine triphosphatase in corneal endothelium

PURPOSE: To examine the relationship between the activity of the sodium pump of the corneal endothelium and corneal thickness. It was postulated that because inhibition pressure of the stroma decreases as thickness increases, a partially inhibited sodium pump would result in a new steady-state thickness of the cornea when reduced rates of fluid influx and efflux were equal. Measurements of physiologic behavior and biochemical activity were to be made in the same tissue and thus establish the relationship directly. METHODS: Rabbit corneas were superfused with a bicarbonate Ringer solution containing different concentrations of ouabain. Exposure to ouabain was either continuous for 4 hours or for an initial 10 minutes followed by ouabain-free superfusion. Thickness was measured, and, after superfusion, endothelium was removed from the corneas, sonicated, and assayed for Na(+)-K+ adenosine triphosphatase (ATPase) activity without further addition of ouabain to the assay medium. Thickness was also measured during superfusion with suboptimal concentrations of Na+ or HCO3- and with brefeldin A, an inhibitor of protein trafficking. RESULTS: Continuous exposure to ouabain caused corneas to swell, but no new steady-state thickness was reached. At low concentrations, swelling rates increased with time, as did the extent of inhibition of the Na(+)-K+ ATPase. With only a 10-minute exposure to ouabain, swelling rates with 10(-4) M to 10(-5) M decreased with the duration of ouabain-free superfusion. Similar swelling curves were obtained by reductions in Na+ or HCO3- concentrations in the superfusion medium, indicating that partial inhibition of the endothelial fluid transport processes, whether via the Na(+)-K+ ATPase or by suboptimal ionic conditions, led toward a new equilibrium thickness of the cornea. However, when superfusion was continued for more than 4 hours, the corneas exposed for 10 minutes to 3 x 10(-5) M or lower-concentration ouabain showed increasing Na(+)-K+ ATPase activity and began to thin, indicating a recovery of fluid transport capability. This recovery was blocked by addition of brefeldin A during the ouabain-free superfusion. CONCLUSIONS: Inhibition of Na(+)-K+ ATPase by low concentrations of ouabain increases with time. Temporary exposure to ouabain causes swelling at rates that decline with time as ouabain dissociates from enzyme sites. This dissociation, together with the turnover of Na(+)-K+ ATPase in the plasma membrane, can lead to recovery of normal thickness in ouabain-exposed corneas. Twenty percent of Na(+)-K+ ATPase in the endothelium is estimated to be intracellular, and about 20% of the activity can be inhibited without inducing swelling.     

Noninvasive determination of language lateralization by functional transcranial Doppler sonography: a comparison with the Wada test

E.coli bacteria, grown in anaerobic conditions, in the medium with glucose, lactose or maltose, upon transferring into a fresh medium with the same sugar acidify it and accumulate potassium ions with a fixed stoichiometry for the N,N'-dicyclohexylcarbodiimide-inhibited cation fluxes, which is equal to 2H+ of the cytoplasm per one K+ of the medium. Such an H(+)-K(+)-exchange is sensitive to osmotic shock. In the medium with lactose or maltose cation fluxes are less and the intracellular K+ activity at the moment of maximal accumulation is lower. The H(+)-K(+)-exchange with a variable stoichiometry of cation fluxes is lost in bacteria, grown in a medium with lactose or maltose.     

Features of the parasitic system of Ixodid ticks--Borrelia--small mammals in the Russian Northwest

BACKGROUND AND AIMS: Long-term treatment with H(+)-K(+)-adenotriphosphatase (ATPase) inhibitors, such as omeprazole or lansoprazole, for severe gastroesophageal reflux disease is now widely used. Whether such treatment will result in vitamin B12 deficiency is controversial. We studied whether long-term treatment with omeprazole alters serum vitamin B12 levels in patients with Zollinger-Ellison syndrome. METHODS: In 131 consecutive patients treated with either omeprazole (n = 111) or histamine H2-receptor antagonists (n = 20), serum vitamin B12 and folate levels and complete blood counts were determined after acid secretion had been controlled for at least 6 months. These studies were repeated yearly. Serum vitamin B12 and folate levels were correlated with the type of antisecretory drug and the extent of inhibition of acid secretion. RESULTS: The mean duration of omeprazole treatment was 4.5 years, and for H2-receptor antagonists 10 years. Vitamin B12 levels, but not serum folate levels or any hematological parameter, were significantly (P = 0.03) lower in patients treated with omeprazole, especially those with omeprazole-induced sustained hyposecretion (P = 0.0014) or complete achlorhydria (P < 0.0001). In 68 patients with two determinations at least 5 years apart, vitamin B12 levels decreased significantly (30%; P = 0.001) only in patients rendered achlorhydric. The duration of omeprazole treatment was inversely correlated with vitamin B12 levels (P = 0.013), but not folate levels. Eight patients (6%) developed subnormal B12 levels during follow-up. CONCLUSIONS: Long-term omeprazole treatment leads to significant decreases in serum vitamin B12 but not folate levels. These results suggest patients with Zollinger-Ellison syndrome treated with H(+)-K(+)-ATPase inhibitors should have serum vitamin B12 levels monitored. Furthermore, these results raise the possibility that other patients treated chronically with H(+)-K(+)-ATPase inhibitors may develop B12 deficiency.     

A protocol to improve analgesia use in the accident and emergency department

In this experiment, intracellular K+ concentration ([K+]i) and ATPase activity of myocardiocytes were measured in early stage of burn injury. Comparing with control group, it was found that, 1. [K+]i were decreased after burn injury, [K+]i of 1st, 3rd, 8th and 24th hours were decreased to 96.2 +/- 1.3%, 85.8 +/- 1.3%, 65.9 +/- 1.0% and 73.7 +/- 1.1% of normal, respectively. 2. Cardiac sarcolemma total ATPase, Mg(2+)-ATPase and Na(+)-K(+)-ATPase activities were all reduced significantly at 8th hour after injury. These results suggest that, burn injury accelerates K+ efflux current, but inhibits K+ influx current, and the reduction of Na(+)-K(+)-ATPase activity is one reason of decrease of [K+]i after injury.     

The Na(+)-K(+)-ATPase beta 1 subunit is associated with the HK alpha 2 protein in the rat kidney

The Na-K-ATPase beta 1 subunit acts as the beta subunit for the HK alpha 2 protein in the rat kidney. The colonic H(+)-K(+)-ATPase is a member of the P-type ATPases, and has been shown to contribute to potassium transport by the mammalian kidney and colon. The P-type ATPases often consist of an alpha subunit that contains the catalytic site and a beta subunit that participates in regulation of enzyme activity and targeting of the enzyme to the plasma membrane. The cDNA of the alpha subunit (HK alpha 2) has been cloned and the HK alpha 2 protein has been isolated from the rat kidney and colon. However, a unique beta subunit for the colonic H(+)-K(+)-ATPase has not been described. To determine if one of the known beta subunits present in the kidney might act as the beta subunit for the colonic H(+)-K(+)-ATPase, microsomes enriched in the colonic H(+)-K(+)-ATPase were isolated using an HK alpha 2-specific antibody (AS 31.7) and the Minimac magnetic separation system. Immunoblots of rat kidney microsomal protein isolated with antibody AS 31.7 were probed with antibodies directed against the gastric HK beta subunit, Na(+)-K(+)-ATPase alpha 1, and Na(+)-K(+)-ATPase beta 1 subunits. A band of the appropriate size was detected with Na(+)-K(+)-ATPase beta 1-specific antibodies, but not those directed against HK beta 1. These data suggest that Na(+)-K(+)-ATPase beta 1 could be the beta subunit for the colonic H(+)-K(+)-ATPase in the kidney.     

An empirical study of object relations in adult children of depressed elderly mothers

The present report confirms the presence of Na(+)-K(+)-Mg2+ ATPase in the erythrocyte membranes of the frog Rana balcanica (previously Rana ridibunda) (Schneider et al. 1993; Sofianidou et al. 1994). The Na(+)-K(+)-Mg2+ ATPase activity was 60% reduced by the presence of ouabain. The pH optimum was 8.0, the optimum Mg2+:ATP concentration ratio was 2.2:1. The existence of an ATPase with a high K(m) for ATP (1.48 mmol/l) was postulated. At pH 7.4 and 8.0, the adenine nucleotide pattern of glucose-depleted erythrocytes showed a characteristic reduction in ATP contents. Adenine nucleotide concentrations were higher at pH 7.4 than at pH 8.0. Ouabain inhibited ATP breakdown at both pH values studied. The strongest inhibition was observed at pH 7.4. The decline of the total contents of adenine nucleotides appears to be determined by the rate of AMP breakdown.     

Effect of elevated glucose concentration on membrane voltage regulation in retinal capillary pericytes

The influence of elevated glucose concentration on resting membrane voltage, electrogenic Na(+)-K(+)-ATPase, and ATP-sensitive potassium channels (KATP channels) was studied in cultured bovine retinal capillary pericytes using conventional microelectrodes. The resting membrane voltage in cells grown in medium containing 5 mM glucose (control) averaged -27 +/- 1.2 mV (mean +/- SE, n = 26) and was not different from cells grown in medium containing 22.5 mM glucose (-26 1.2 mV, n = 26). Addition of ouabain (10(-4) M), a specific inhibitor of the Na(+)-K(+)-ATPase, depolarized the membrane potential by 3.6 +/- 0.4 mV (n = 10) in cells grown under control conditions and 0.7 +/- 0.2 mV (n = 6) in cells grown under elevated glucose conditions. Thus, electrogenic activity of the Na(+)-K(+)-ATPase was significantly (P < 0.0001) reduced to 19% compared with control conditions. Electrogenic Na(+)-K(+)-ATPase activity could be partially restored (ouabain-induced depolarization delta V = 2.0 +/- 0.2 mV, n = 6) in cells grown with high glucose in the presence of the aldose reductase inhibitor tolrestat (10(-5) M). The potassium channel opener Hoe 234 (10(-6) M) induced membrane potential hyperpolarization in control cells (delta V = 7.3 +/- 1.2 mV, n = 13), which could be completely inhibited by the KATP channel blocker glibenclamide (10(-7) M, n = 5). This indicates that pericytes possess KATP channels. The effect of KATP channels on membrane voltage was not significantly changed (P = 0.16) in cells cultured under high-glucose conditions (delta V = 9.6 +/- 2.0 mV, n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)     

Na(+)-K+ ATPase concentration in different adult rat skeletal muscles is related to oxidative potential

To investigate the relationship among fibre type, oxidative potential, and Na(+)-K+ ATPase concentration in skeletal muscle, adult male Wistar rats weighing 259 +/- 8 g (mean +/- SE) were sacrificed and the soleus (SOL), extensor digitorum longus (EDL), red vastus lateralis (RV), and white vastus lateralis (WV) removed. These muscles were chosen as being representative of the two major fibre type populations: slow twitch (SOL) and fast twitch (EDL, RV, WV) and exhibiting either a high (SOL, EDL, RV) or low (WV) oxidative potential. Na(+)-K+ ATPase concentration (pmol.g-1 wet weight), measured by the [3H]ouabain binding technique, differed (p < 0.01) only between the WV (238 +/- 7.9) and the SOL (359 +/- 9.6), EDL (365 +/- 10), and RV (403 +/- 12). Similarly, muscle oxidative potential as measured by the maximal activity of citrate synthase was different (p < 0.01) only between the WV and the other three muscles. Citrate synthase activity (mumol.min-1.g-1 wet weight) was 4.0 +/- 0.7, 12.3 +/- 0.9, 9.1 +/- 0.7, and 11.3 +/- 1.0 in the WV, SOL, EDL, and RV, respectively. These results indicate that Na(+)-K+ ATPase concentration is not related to the speed of contraction but to the oxidative potential of the muscle. Since chronic activity is a primary determinant of oxidative potential, it would be expected that increases in Na(+)-K+ ATPase would accompany increases in muscle utilization.     

Inhibition of acid secretion in gastric parietal cells by the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-93

A novel Ca2+/calmodulin-dependent protein kinase II (CaM Kinase II) inhibitor, KN-93 potently inhibits gastric acid secretion from parietal cells. As previously reported (1), treatment of parietal cells with a selective inhibitor of CaM kinase II, KN-62 resulted in the inhibition of cholinergic-stimulated rabbit parietal cell secretion, whereas it failed to inhibit the histamine and forskolin response. In contrast effects of carbachol, histamine and forskolin were significantly inhibited by KN-93 with an IC50 of 0.15, 0.3 and 1 microM, respectively; these effects occurred without any changes in intracellular cyclic AMP and Ca2+ levels. In the present study we investigated the mechanism by which KN-93 acts upon the acid-secreting machinery of gastric parietal cells. Neither redistribution of the proton pump activity nor the morphological transformation were affected by KN-93. The drug only weakly inhibited the H+, K(+)-ATPase activity but strongly dissipated the proton gradient formed in the gastric membrane vesicles and reduced the volume of luminal space. Thus KN-93 acts at pH gradient formation whereas KN-62 acts only at CaM Kinase II.     

Human gastric H,K-adenosine triphosphatase beta-subunit is a major autoantigen in atrophic corpus gastritis. Expression of the recombinant human glycoprotein in insect cells

BACKGROUND: Sera from patients with atrophic corpus gastritis with pernicious anemia frequently contain parietal cell autoantibodies. We have previously demonstrated that the human H,K-adenosine triphosphatase (H,K-ATPase) alpha-subunit constitutes a major autoantigen. The present study investigates whether the human H,K-ATPase beta-subunit is an autoantigen, too, METHODS: The gene of the human beta-subunit was expressed in insect cells by a baculovirus expression system. The reactivity of sera from 42 patients towards the recombinant glycoprotein was analyzed by means of an enzyme-linked immunosorbent assay. RESULTS: Thirty-nine of the 42 sera (93%) scored positive. Autoantibody binding in 41 sera (98%) was eliminated when unglycosylated beta-subunit was used as antigen, and antibody binding in the last serum was decreased by 30%. CONCLUSIONS: The results indicate that the beta-subunit is indeed a major autoantigen and that carbohydrates are involved in binding of the autoantibodies.     

Endothelin 1 stimulates Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport through ETA receptors and protein kinase C-dependent pathway in cerebral capillary endothelium

The effect of endothelins (ET-1 and ET-3) on 86Rb+ uptake as a measure of K+ uptake was investigated in cultured rat brain capillary endothelium. ET-1 or ET-3 dose-dependently enhanced K+ uptake (EC50 = 0.60 +/- 0.15 and 21.5 +/- 4.1 nM, respectively), which was inhibited by the selective ETA receptor antagonist BQ 123 (cyclo-D-Trp-D-Asp-Pro-D-Val-Leu). Neither the selective ETB agonists IRL 1620 [N-succinyl-(Glu9,-Ala11,15)-ET-1] and sarafotoxin S6c, nor the ETB receptor antagonist IRL 1038 [(Cys11,Cys15)-ET-1] had any effect on K+ uptake. Ouabain (inhibitor of Na+,K(+)-ATPase) and bumetanide (inhibitor of Na(+)-K(+)-Cl- cotransport) reduced (up to 40% and up to 70%, respectively) the ET-1-stimulated K+ uptake. Complete inhibition was seen with both agents. Phorbol 12-myristate 13-acetate (PMA), activator of protein kinase C (PKC), stimulated Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport. ET-1- but not PMA-stimulated K+ uptake was inhibited by 5-(N-ethyl-N-isopropyl)amiloride (inhibitor of Na+/H+ exchange system), suggesting a linkage of Na+/H+ exchange with ET-1-stimulated Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport activity that is not mediated by PKC.     

Endosomal recycling of the Na+/H+ exchanger NHE3 isoform is regulated by the phosphatidylinositol 3-kinase pathway

The NHE3 isoform of the Na+/H+ exchanger localizes to both the plasmalemmal and endosomal compartments in polarized epithelial and transfected Chinese hamster ovary (AP-1) cells. It is unclear how the distribution of NHE3 between these compartments is regulated. In this study, we examined the potential involvement of phosphatidylinositol 3'-kinase (PI3-K) in regulating the activity and distribution of NHE3, as this lipid kinase has been implicated in modulating vesicular traffic in the endosomal recycling pathway. Wortmannin and LY294002, both potent inhibitors of PI3-K, markedly inhibited NHE3-mediated H+ extrusion across the plasma membrane in a concentration- and time-dependent manner. The subcellular distribution of the antiporters was monitored by transfecting epitope-tagged NHE3 into AP-1 cells. In parallel with the inhibition of transport, PI3-K antagonists induced a pronounced loss of NHE3 from the cell surface and its accumulation in an intracellular compartment, as assessed by immunofluorescence microscopy and enzyme-linked immunosorbent assays. Further analysis using cells transfected with antiporters bearing an external epitope tag revealed that the redistribution reflected primarily a decrease in the rate of recycling of intracellular NHE3 to the cell surface. The wortmannin-induced inhibition and redistribution of NHE3 were prevented when cells were incubated at 4 degreesC, consistent with the known temperature dependence of the endocytic process. These observations demonstrate that NHE3 activity is controlled by dynamic endocytic and recycling events that are modulated by PI3-K.     

Calbindin D-28k and monoamine oxidase A immunoreactive neurons in the nucleus basalis of Meynert in senile dementia of the Alzheimer type and Parkinson''s disease

The lungs must be kept "dry" for efficient gas exchange. The mechanisms that contribute to clear alveoli from fetal lung fluid at birth are still present during adult life and allow recovery from alveolar flooding. It has recently been shown with the use of different approaches in vitro, as well as in vivo, that alveolar epithelium performs solute-coupled fluid transport. Fluid absorption from alveoli occurs chiefly as a result of active transepithelial Na+ transport. The mechanisms of Na+ transport have been partly elucidated; Na+ enters alveolar cells through apical Na+ channels and Na(+)-coupled solute transporters and is pumped out at the basolateral membrane by a Na(+)-K(+)-adenosinetriphosphatase (ATPase). Transepithelial Na+ transport and fluid absorption are stimulated by beta-adrenergic agonists, with adenosine 3',5'-cyclic monophosphate being the likely intracellular second messenger. K+ is probably secreted into alveoli because its concentration in the epithelial lining fluid is larger than expected for passive distribution. K+ channels have been described that, in conjunction with Na(+)-K(+)-ATP-ase, might provide pathways for active transport. Active proton secretion or bicarbonate absorption have been reported, which may explain the low pH of the alveolar epithelial lining fluid. It is probable that active solute transports are the main determinants of epithelial lining fluid depth and composition. A challenge for the future is to understand how this homeostasis is achieved.     

K+ fluxes mediated by Na(+)-K(+)-Cl- cotransport and Na(+)-K(+)-ATPase pumps in renal tubule cell lines transformed by wild-type and temperature-sensitive strains of Simian virus 40

The relative contributions of Na(+)-K(+)-ATPase pumps and Na(+)-K(+)-Cl- cotransport to total rubidium (Rb+) influx into primary cultures of renal tubule cells (PC.RC) and cells transformed either with the wild-type or a temperature-sensitive mutant of the simian virus 40 (SV40), were measured under various growth conditions. The Na(+)-K(+)-ATPase-mediated component represented 74% and 44-48% of total Rb+ influx into PC.RC and SV40-transformed cells, respectively. Proliferating transformed cells showed substantial ouabain-resistant bumetanide-sensitive (Or-Bs) Rb+ influx (41-45% of total) which indicated the presence of a Na(+)-K(+)-Cl- cotransport. The Or-Bs component of Rb+ influx was greatly reduced when temperature-sensitive transformed renal cells (RC.SVtsA58) grown in Petri dishes or on permeable filters were shifted from the permissive (33 degrees C) to the restrictive temperature (39.5 degrees C) to arrest cell growth. The ouabain-sensitive Rb+ influx mediated by the Na(+)-K(+)-ATPase, the total and amiloride-sensitive Na+ uptakes were not modified following inhibition of cell proliferation. A similar fall in the Or-Bs influx was obtained when renal tubule cells transformed by the wild-type SV40 (RC.SV) were incubated with the K+ channel blocker, tetraethylammonium (TEA) ion, which we had previously shown to arrest cell growth without affecting cell viability (Teulon et al.: J. Cell. Physiol., 151:113-125, 1992). Reinitiation of cell growth by removal of TEA or return to 33 degrees C of the temperature-sensitive cells restored the Or-Bs component of Rb influx. Taken together, these results indicate that the Na(+)-K(+)-Cl- cotransport activity is critically dependent on cell growth conditions.     

Effect of time in culture on modulation of Na(+)-K(+)-ATPase activity in rat alveolar type II cells

To determine the effect of time in culture on epithelial cell function, we evaluated the modulation of Na(+)-K(+)-ATPase activity in rat alveolar type II cells in culture. Ouabain sensitivity testing revealed that the alpha-1 predominance in the enzyme's isoforms was maintained over the 120 hours in culture. Basal Na(+)-K(+)-ATPase activity in the whole cell homogenate did not differ significantly between cells cultured for 48 hours and those cultured for 120 hours. Terbutaline (10 mM) did not activate Na(+)-K(+)-ATPase in the cells cultured for 48 hours, but, it significantly increased the activity of this enzyme in the cells cultured for 120 hours cells cultured for 48 hours, produced intracellular cyclic AMP after exposure to 10 mM of terbutaline. These results indicate that the coupling between Na(+)-K(+)-ATPase and the beta-adrenergic pathway in alveolar type II cells can be influenced by the time in cell culture.