Stabilisation of Na,K-ATPase structure by the cardiotonic steroid ouabain

Cardiotonic steroids such as ouabain bind with high affinity to the membrane-bound cation-transporting P-type Na,K-ATPase, leading to complete inhibition of the enzyme. Using synchrotron radiation circular dichroism we show that the enzyme-ouabain complex is less susceptible to thermal denaturation (unfolding) than the ouabain-free enzyme, and this protection is observed with Na,K-ATPase purified from pig kidney as well as from shark rectal glands. It is also shown that detergent-solubilised preparations of Na,K-ATPase are stabilised by ouabain, which could account for the successful crystallisation of Na,K-ATPase in the ouabain-bound form. The secondary structure is not significantly affected by the binding of ouabain. Ouabain appears however, to induce a reorganization of the tertiary structure towards a more compact protein structure which is less prone to unfolding; recent crystal structures of the two enzymes are consistent with this interpretation. These circular dichroism spectroscopic studies in solution therefore provide complementary information to that provided by crystallography

Localization of Na pumps in the tracheal epithelium of the dog.

Binding of [3H]ouabain by the dog's tracheal epithelium shows a nonspecific component depending linearly on ouabain concentration, and a specific saturable component with a Km of 10(-7) M. Control experiments showed that the tracer taken up was not trapped within the extracellular space nor bound to tissue collagen. Inhibition of the saturable uptake by high K, metabolic inhibition, low Na, and low temperature indicated that binding was to Na/K ATPase. One-sided exposures of tissue sheets to tracer showed that the submucosal side took up 10 X as much tracer as the luminal. Autoradiography localized tracer uptake under all conditions to the cells' basolateral membranes

Ouabain-induced cytoplasmic vesicles and their role in cell volume maintenance

Cellular swelling is controlled by an active mechanism of cell volume regulation driven by a Na+/K+-dependent ATPase and by aquaporins which translocate water along the osmotic gradient. Na+/K+-pump may be blocked by ouabain, a digitalic derivative, by inhibition of ATP, or by drastic ion alterations of extracellular fluid. However, it has been observed that some tissues are still able to control their volume despite the presence of ouabain, suggesting the existence of other mechanisms of cell volume control. In 1977, by correlating electron microscopy observation with ion and water composition of liver slices incubated in differentmetabolic conditions in the presence or absence of ouabain, we observed that hepatocytes were able to control their volume extruding water and recovering ion composition in the presence of ouabain. In particular, hepatocytes were able to sequester ions and water in intracellular vesicles and then secrete themat the bile canaliculus pole.We named this “vesicularmechanismof cell volume control.” Afterward, thismechanism has been confirmed by us and other laboratories in several mammalian tissues.This review summarizes evidences regarding this mechanism, problems that are still pending, and questions that need to be answered. Finally, we shortly review the importance of cell volume control in some human pathological conditions

Activity-dependent adenosine release may be linked to activation of Na+-K+ ATPase : an in vitro rat study

In the brain, extracellular adenosine increases as a result of neuronal activity. The mechanisms by which this occurs are only incompletely understood. Here we investigate the hypothesis that the Na+ influxes associated with neuronal signalling activate the Na+-K+ ATPase which, by consuming ATP, generates intracellular adenosine that is then released via transporters. By measuring adenosine release directly with microelectrode biosensors, we have demonstrated that AMPA-receptor evoked adenosine release in basal forebrain and cortex depends on extracellular Na+. We have simultaneously imaged intracellular Na+ and measured adenosine release. The accumulation of intracellular Na+ during AMPA receptor activation preceded adenosine release by some 90 s. By removing extracellular Ca2+, and thus preventing indiscriminate neuronal activation, we used ouabain to test the role of the Na+-K+ ATPase in the release of adenosine. Under conditions which caused a Na+ influx, brief applications of ouabain increased the accumulation of intracellular Na+ but conversely rapidly reduced extracellular adenosine levels. In addition, ouabain greatly reduced the amount of adenosine released during application of AMPA. Our data therefore suggest that activity of the Na+-K+ ATPase is directly linked to the efflux of adenosine and could provide a universal mechanism that couples adenosine release to neuronal activity. The Na+-K+ ATPase-dependent adenosine efflux is likely to provide adenosine-mediated activity-dependent negative feedback that will be important in many diverse functional contexts including the regulation of sleep

Synchronization modulation increases transepithelial potentials in MDCK monolayers through Na/K pumps

Peer reviewedPublisher PD

Different types of potassium transport linked to carbachol and γ-aminobutyric acid actions in rat sympathetic neurons

Carbachol and γ-aminobutyric acid depolarize mammalian sympathetic neurons and increase the free extracellular K+-concentration. We have used double-barrelled ion-sensitive microelectrodes to determine changes of the membrane potential and of the free intracellular Na+-, K+- and Cl−-concentrations ([Na+]i, [K+]iand [Cl−]i) during neurotransmitter application. Experiments were performed on isolated, desheathed superior cervical ganglia of the rat, maintained in Krebs solution at 30°C. Application of carbachol resulted in a membrane depolarization accompanied by an increase of [Na+]i, a decrease of [K+]i and no change in [Cl−]i. Application of γ-aminobutyric acid also induced a membrane depolarization which, however, was accompanied by a decrease of [K+]i and [Cl−]i, whereas [Na+]i remained constant. Blockade of the Na+/K+-pump by ouabain completely inhibited both the reuptake of K+ and the extrusion of Na+ after the action of carbachol, and also the post-carbachol undershoot of the free extracellular K+-concentration. On the other hand, in the presence of ouabain, no changes in the kinetics of the reuptake of K+ released during the action of γ-aminobutyric acid could be observed. Furosemide, a blocker of K+/Cl−-cotransport, inhibited the reuptake of Cl− and K+ after the action of γ-aminobutyric acid. In summary, the data reveal that rat sympathetic neurons possess, in addition to the Na+/K+-pump, another transport system to regulate free intracellular K+-concentration. This system is possibly a K+/Cl−-cotransport

Characterisation of the Na, K pump current in atrial cells from patients with and without chronic atrial fibrillation

<b>Objective:</b> To assess the contribution of the Na, K pump current (<i>I</i><sub>p</sub>) to the action potential duration (APD) and effective refractory period (ERP) in human atrial cells, and to investigate whether <i>I</i><sub>p</sub> contributes to the changes in APD and ERP associated with chronic atrial fibrillation (AF). <b>Methods:</b> Action potentials and ion currents were recorded by whole-cell patch clamp in atrial myocytes isolated from consenting patients undergoing cardiac surgery, who were in sinus rhythm (SR) or AF (>3 months). <b>Results:</b> In cells from patients in SR, the <i>I</i><sub>p</sub> blocker, ouabain (10 μM) significantly depolarised the membrane potential, Vm, from -80±2 (mean±S.E.) to -73±2 mV, and lengthened both the APD (174±17 vs. 197±23 ms at 90% repolarisation) and ERP (198±22 vs. 266±14 ms; P<0.05 for each, Student's t-test, <i>n</i>=7 cells, 5 patients). With an elevated pipette [Na<sup>+</sup>] of 30 mM, <i>I</i><sub>p</sub> was measured by increasing extracellular [K<sup>+</sup>] ([K<sup>+</sup>]o) from 0 to 5.4 mM. This produced an outward shift in holding current at -40 mV, abolished by 10 muM ouabain. K± and ouabain-sensitive current densities were similar, at 0.99±0.13 and 1.12±0.11 pA/pF, respectively (P>0.05; <i>n</i>=9 cells), confirming the K±induced current as <i>I</i><sub>p</sub>. <i>I</i><sub>p</sub> increased linearly with increasing Vm between -120 and +60 mV (<i>n</i>=25 cells). Stepwise increments in [K<sup>+</sup>]<sub>o</sub> (between 0 and 10 mM) increased Ip in a concentration-dependent manner (maximum response, <i>E</i><sub>max</sub>=1.19±0.09 pA/pF; EC50=1.71±0.15 mM; n=27 cells, 9 patients). In cells from patients in AF, the sensitivity of Ip to both Vm and [K+]o (<i>E</i><sub>max</sub>=1.02±0.05 pA/pF, EC50=1.54±0.11 mM; <i>n</i>=44 cells, 9 patients) was not significantly different from that in cells from patients in SR. Within the group of patients in AF, long-term digoxin therapy (<i>n</i>=5 patients) was associated with a small, but significant, reduction in <i>E</i><sub>max</sub> (0.92±0.07 pA/pF) and EC<sub>50</sub> (1.35±0.15 mM) compared with non-treatment (<i>E</i><sub>max</sub>=1.13±0.08 pA/pF, EC<sub>50</sub>=1.76±0.14 mM; P<0.05 for each, <i>n</i>=4 patients). In cells from non-digoxin-treated patients in AF, the voltage- and [K<sup>+</sup>]<sub>o</sub>-sensitivity (<i>E</i><sub>max</sub> and EC<sub>50</sub>) were similar to those in cells from patients in SR. <b>Conclusions:</b> The Na, K pump current contributes to the human atrial cell Vm, action potential shape and ERP. However, the similarity in Ip sensitivity to both [K<sup>+</sup>]<sub>o</sub> and <i>V</i><sub>m</sub> between atrial cells from patients with and without chronic AF indicates that <i>I</i><sub>p</sub> is not involved in AF-induced electrophysiological remodelling in patients

Thyroid thermogenesis in adult rat hepatocytes in primary monolayer culture: direct action of thyroid hormone in vitro.

We have studied the effect of 3,5,3'-triiodothyronine (T3) on the respiration of adult rat hepatocytes in primary monolayer culture prepared from hypothyroid rat liver. After addition of T3 to the culture medium at a concentration of 2 x 10(-7) M, oxygen consumption of the cultured cells increased detectably at 24 h and was maximal at 72--96 h, relative to control cultures (38.0 +/- 1.8 vs. 25.0 +/- 1.5 microliter/h.mg protein). The thyroid-responsive enzymes, Na+ + K+-activated adenosine triphosphatase (NaK-ATPase) and alpha-glycerophosphate dehydrogenase (GPD), each exhibited increased activity in response to T3, in parallel with the change in oxygen consumption, whereas the activity of Mg-dependent ATPase was unaffected. These responses to T3 were dose dependent over similar concentration ranges, the half-maximal response for each occurring at ca 8 x 10(-10) M. In thyroid-treated cells, the observed increase in respiration was almost completely (90%) inhibited after addition of ouabain (10(-3) M) to the culture medium. It was found also that a 4-h exposure of the cultured hepatocytes to T3 was sufficient to elicit a significant thermogenic response, measured at a time (48 h later) when T3 was no longer present in the medium. The response to T3 occurred in fully defined culture medium and was independent of the presence or absence of hypothyroid rat serum, corticosterone, or insulin, and cellular ATP was unaffected by T3 in concentrations up to 2 x 10(-7) M. The findings document that adult rat hepatocytes in primary monolayer culture respond directly to thyroid hormone; the increases in respiration and NaK-ATPase activity elicited by T3 were cotemporal and apparently coordinate