ATP-dependent Sodium Research Articles

Suppression of Na +K +-ATPase activity by reversible phosphorylation over the winter in a freeze-tolerant insect

Larvae of the gall fly, Eurosta solidaginis, use the cold hardiness strategy of freeze tolerance as well as entry into a hypometabolic state (diapause) to survive the winter. Cold hardiness strategies have been extensively explored in this species, but the metabolic features of winter hypometabolism have received little attention. A primary consumer of energy in cells is the ATP-dependent sodium–potassium ion pump (Na +K +-ATPase) so inhibitory controls over transmembrane ion movements could contribute substantially to energy savings over the winter months. Na +K +-ATPase activity was quantified in larvae sampled between October and April. Activity was high in October (0.56±0.13 nmol/min/mg) but fell by 85% in November, remained low through midwinter, and then increased strongly in April. To determine whether the seasonal change in Na +K +-ATPase activity was linked with posttranslational modification of the enzyme, extracts from 15 °C-acclimated larvae were incubated under conditions that stimulated protein kinases A, G, or C. The action of all three kinases suppressed Na +K +-ATPase activity to levels just 3–8% of control values whereas the opposite treatment with alkaline phosphatase had no effect. Hence, the seasonal suppression of Na +K +-ATPase activity may be linked to enzyme phosphorylation. Furthermore, acute cold (3 °C) or hypoxia exposures of 15 °C-acclimated larvae did not alter enzyme activity, and freezing at −16 °C increased activity, so environmental factors do not appear to directly influence enzyme activity. Rather, it appears that winter suppression of ion motive ATPase activity may be part of a program of winter metabolic suppression.

Tissue hydration in kidneys during preservation: a relaxometric analysis of time-dependent differences between cortex and medulla.

Cold preservation of donor organs induces hypothermia-related tissue edema as a result of a reduced activity of the ATP-dependent sodium pump at low temperatures. Hypothermia-induced tissue edema occurs in kidney preservation and is a significant risk factor for delayed graft function (DGF) after transplantation. DGF remains a major problem in kidney transplantation and is significantly associated with preservation injury. The state of hydration of cold-stored organs can be assessed from a biopsy for determination of the wet/dry weight ratio. As a non-invasive method to determine tissue hydration MRI T1 and T2 relaxometry can be used. In this study we have compared changes in tissue hydration in UW-preserved porcine kidneys with increasing cold ischemia times (CIT) using wet/dry weight ratio and MR ralaxometry. The results of the two techniques were correlated to evaluate the use of MR relaxometry. Wet/dry weight ratios of the renal cortex decreased with prolonged CIT (P < 0.01) whereas those of the medulla did not change significantly. T1 values of the cortex decreased with prolonged CIT (P < 0.01). T2 values of the cortex showed a non-significant decline with increased CIT. No significant changes in T1 and T2 were found in the medulla. The correlation between T1 and the wet/dry weight ratio of the cortex was significant (P = 0.05, linear correlation coefficient 0.8698). We conclude that MR relaxometry can be a valuable noninvasive technique to assess tissue hydration in cadaveric donor kidneys before transplantation.

The basolateral membrane of rat enterocyte: Its purification from brush border contamination

Basolateral membranes obtained by self-orienting Percoll-gradient centrifugation were treated with 5 m m CaCl 2 to minimize the cross-contamination by brush border membranes. From marker enzyme-specific activities it was calculated that in this preparation the basolateral/brush border membrane ratio was 22.6. A low l-glucose permeability across basolateral membrane vesicles together with ATP-dependent sodium uptake was observed.

Sodium pump-catalyzed sodium-sodium exchange associated with ATP hydrolysis.

Inside-out red cell membrane vesicles have been used to study sodium interactions with the ATP-dependent sodium pump at sites accessible to both membrane surfaces. ATP-dependent 22Na+ influx (equivalent to efflux from cells) shows sigmoid dependence on extravesicular Na+ concentration. This is observed both in the absence of intravesicular cations and in the presence of intravesicular K or Rb ions. The kinetic behavior is similar to that observed earlier with intact cells, (Garay, R. P., and Garrahan, P. J. (1973) J. Physiol. (Lond.) 231, 297-325) and is consistent with a ratio of close to three Na ions transported per molecule of ATP hydrolyzed. With vesicles having relatively high intravesicular sodium concentration, (approximately 50 mM NaCl), the sodium pump effects an ATP-dependent sodium efflux coupled to sodium influx and to strophanthidin-sensitive ATP hydrolysis. The influx:efflux stoichiometry is approximately 1:1, and the influx:ATP hydrolysis ratio is close to 3. This ATP-dependent exchange has a higher affinity for vanadate than ATP plus ADP-dependent sodium exchange. It is concluded that this sodium-sodium exchange mode resembles sodium-potassium exchange whereby intravesicular sodium, i.e. sodium at the extracellular surface, at relatively high concentration, behaves like potassium.

Sidedness of (sodium, potassium)-adenosine triphosphate of inside-out red cell membrane vesicles. Interactions with potassium.

Inside-out membrane vesicles of human red cells, prepared according to the method of Steck et al. (1970) Science 168, 255-257) have sufficiently low cation permeability to allow the examination of the side-specific interactions of ligands with the asymmetric sodium pump complex. In accordance with the known properties of the pump in intact cells the following results were observed: (a) ATP-dependent sodium influx and (b) maximal (sodium, potassium)-ATPase with K+ present inside the vesicles with larger than or equal to 20 micronM ATP. With much lower [ATP], K+ inhibited sodium-activated ATPase. K+ was inhibitory at either surface. Inhibition was different on the two sides since cytoplasmic (extravesicular) Na+ counteracted inhibition by cytoplasmic (extravesicular) K+ but not inhibition by K+ at the plasma or external membrane surface, i.e. intravesicular K+. A decrease in the steady state level of the phosphenzyme intermediate of sodium-activated ATPase was caused also by K+ at either surface. The effect of cytoplasmic K+ is compatible with its competitive inhibition of activation of phosphorylation of the enzyme by cytoplasmic Na+. At 37 degrees, the inhibitory effect of external K+ is due to interaction with the phosphoenzyme to form a stable complex of K+ with the dephosphenzyme resulting in a decreased overall reaction rate but increased turnover of the phosphoenzyme (E-P + K leads to EK + Pi). At 0 degree, external K+ inhibits by interacting with the unphosphorylated enzyme to form an occluded enzyme-K complex. This results in a decreased overall rate but relatively small change in apparent turnover of the phosphoenzyme. At 0 degree, but not at 37 degrees, external Na+ counteracted the inhibitory effects of external K+.

Reconstituction of an ATP-dependent sodium pump with an ATPase from electric eel and pure phospholipids

An ATP-dependent sodium pump was reconstituted with an ATPase preparation from the electric eel and purified phospholipids. Vesicles reconstituted by the sonication procedure catalyzed Na + translocation at a rate 10 to 20 times higher than vesicles reconstituted by the cholate-dialysis procedure. A small but reproducible stimulation of Na + transport by valinomycin in the presence of K + was observed.

Coupled transport of sodium and organic solutes.

Coupled transport of sodium and organic solutes.

Adenosine triphosphate-dependent retention of sodium ions by a sodium and potassium-activated adenosine triphosphatase preparation from erythrocyte membranes

An ATP-dependent retention of sodium ions by a preparation of human erythrocyte membrane fragments is reported. Several characteristics of this retention were correlated to the sodium and potassium-activated ATPase: nucleotide specificity, magnesium ion requirement, pH dependence curves, and species differences of man, rat, dog, and cat. The membrane preparation preferentially retained sodium ions as compared with potassium and rubidium ions. Low concentrations of potassium ions stimulated ATP-dependent sodium retention, and ouabain inhibited this activity by 60% in either the presence or the absence of K +.