Na,K-ATPase activity

ATP certainly fulfils the criteria for a NT. It is mostly synthesised by mitochondrial oxidative phosphorylation using glucose taken up by the nerve terminal. Much of that ATP is, of course, required to help maintain Na+/K+ ATPase activity and the resting membrane potential as well as a Ca +ATPase, protein kinases and the vesicular binding and release of various NTs. But that leaves some for release as a NT. This has been shown in many peripheral tissues and organs with sympathetic and parasympathetic innervation as well as in brain slices, synaptosomes and from in vivo studies with microdialysis and the cortical cup. There is also evidence that in sympathetically innervated tissue some extracellular ATP originates from the activated postsynaptic cell. While most of the released ATP comes from vesicles containing other NTs, some... 

The membrane-bound preparation from kidney is easily solubilized in non-ionic detergent and analytical ultracentrifugation shows that the preparation consists predominantly (80 85%) of soluble af units with 143000 [28]. The soluble a)S unit maintains full Na,K-ATPase activity, and can undergo the cation or nucleotide induced conformational transitions that are observed in the membrane-bound preparation. A cavity for occlusion of 2K or 3Na ions can be demonstrated within the structure of the soluble a)S unit [29], as an indication that the cation pathway is organized in a pore through the aji unit rather than in the interphase between subunits in an oligomer. 

Figure 4.8 Reduction of Na/K ATPase activity in isoiated guinea-pig hearts subjected to ischaemia/reperfusion and its prevention by various agents control non-ischaemic hearts (Nl) guinea-pig hearts subjected to global ischaemia for 2 h and subsequently reperfused for 1 h (IR). In other preparations, superoxide dismutase (SOD) 100 U/ml, catalase (CAT) 150 U/ml, dimethylsulphoxide (DMS) 50 mu, histidine (HIS) 10 mu, vitamin E (TOC)...

Na/K ATPase has been investigated recently. These studies were undertaken to investigate the direct modulatory effects of glutathione on Na/K ATPase activity in the absence of other factors present during ischaemia and reperfusion. 

The possible role of cellular glutathione status in the controlling sarcolemmal protein activity has been addressed by studying the effect of GSH, GSSG and several other thiol and disulphide compounds on Na/K ATPase activity using (1) an isolated bovine ventricular Na/K ATPase preparation (2) crude sarcolemmal preparations (biochemical studies) (c) Langendorff-perfiised isolated hearts (cytochemical studies) and (4) isolated ventricular myocytes (electrophysiologjcal studies). 

Although it is widely accepted that ischaemia/ reperfusion-induced oxidant stress is associated with a reduction of Na/K ATPase activity, it is difficult to determine which features of this process are responsible for this effect. A classical approach to this type of problem has been to determine the effect of the application of selected metabolites or agents on the activity of the enzyme of interest, an approach that has been exploited for the sarcolemmal Na/K ATPase and glutathione (Haddock et al., 1990). The application of GSH (O.l-l.OmM) induces a concentration-dependent increase in the activity of a bovine isolated ventricular Na/K ATPase preparation (determined by the ouabain-sensitive hydrolysis of ATP to release inorganic phosphate). In the presence of 1 mM GSH there was a 38% stimulation of activity compared to untreated control... 

If the cardiac redox state (reflected by fluctuations in the ratio of GSH and GSSG) is important in the regulation of the function of some enzymes, the manipulation of this ratio may result in parallel changes in enzyme activity. Thus, a reduction of Na/K ATPase activity associated with the application of 1 mM GSSG has been shown to be reversed and completely overcome, in a concentration-dependent manner, by the subsequent addition of GSH (O.l-l.OmM) (Haddock et al., 1991) (see Fig. 4.10). 

Figure 4.9 Effect of reduced glutathione (GSH) (0.25-1.0 ihm) and oxidized glutathione (GSSG) (0.25-1.0 mM) on ouabain-sensitive Na/K ATPase activity. An isolated Na/K ATPase preparation was prepared from fresh bovine ventricular tissue. Na/K ATPase activity was determined and quantified by the ouabain-sensitive hydrolysis of ATP to yield Inorganic phosphate. The rate of inorganic phosphate production was compared prior to and following the addition of either GSH or GSSG to the Incubation mixture. The data are presented as...

Additional studies have also demonstrated that the application of other disulphides, such as oxidized dithiothreitol and cystine, produce similar effects to GSSG over an identical concentration range (Haddock et al., 1991). Therefore the Na/K ATPase activity may be modified by disulphides derived from a variety of species. 

These data demonstrate that both GSH and GSSG have profound effects on Na/K ATPase activity and may act in concert to modify enzyme activity during oxidant stress. However, it should be recognized that the steric conformation of an isolated enzyme preparation in a chemically buffered solution may be considerably different to the native enzyme located in a dynamic lipid bilayer. For this reason, these investigations have been extended to include a variety of preparations in which the Na/K pump is in its native environment. 

The cytochemical quantification of ouabain-sensitive Na/K ATPase activity in 10 /im thick whole heart slices. 

Biochemical Analysis of Na/K ATPase Activity in Sarcolemmal Preparations... 

Whilst experimentally it is relatively easy to investigate the eflFect of the exogenous application of GSH and GSSG on cardiac Na/K ATPase activity, one further approach that has been exploited in many aspects of oxidant-induced cell injury has been the depletion of cellular glutathione levels. The hypothesized importance of GSH in the cell s antioxidant armoury would be expected to be reflected in an increased susceptibility to oxidant stress-... 

Figure 4.11 Effect of glutathione on the reduction of myocardial Na/K ATPase activity associated with Ischaemia and reperfuslon. Isolated rat hearts were perfused in the Langendorff mode with oxygenated Tyrode s solution for a control period of 10 min. This was immediately followed by a 60 min period of global, stop-flow ischaemia and 5 min subsequent reperfuslon. Glutathione (GSH) (1 mM) was added to the perfusion fluid 5 min prior to the onset of Ischaemia and throughout the reperfuslon period. The data are presented as means standard errors of the means (n = 6).

Haddock, P.S., Hearse, D.J. and Woodward, B. (1990). Effect of glutathione and other thiols on bovine heart Na/K ATPase activity. J. Mol. Cell. Cardiol. 22, S4. 

SM Schwartz, HE Bostwick, MS Medow. Estrogen modulates ileal basolateral membrane lipid dynamics and Na+-K+ ATPase activity. Am J Physiol 254 G687-G694, 1988. 

DF Cooperstein. (1987). Na+-K+-ATPase activity and transport process in toad corneal epithelium. Comp Biochem Physiol 87A 1119-1121. 

Gulyani, S. Mallick, B. N. (1993). Effect of rapid eye movement sleep deprivation on rat brain Na-K ATPase activity. J. Sleep Res. 2, 45-50. 

Kaur, S., Panchal, M., Faisal, M. et al. (2004). Long term blocking of GABA-A receptor in locus coeruleus by bilateral microinfusion of picrotoxin reduced rapid eye movement sleep and increased brain Na-K ATPase activity in freely moving normally behaving rats. Behav. Brain Res. 151, 185-90. 

The effects of Li+ upon hematopoiesis have been proposed to be due to two different systems modification of the activity of the membrane Na+/K+-ATPase, and the inhibition of adenylate cyclase. Monovalent cation flux, in particular Na+ transport, is known to influence the differentiation and proliferation of hematopoietic stem cells. For instance, ouabain, an effective inhibitor of the membrane Na+/K+-ATPase, blocks the proliferation of lymphocytes and has been shown to attenuate the Li+-induced proliferation of granulocyte precursors [208]. Conversely, Li+ can reverse the actions of amphotericin and monensin, which mediate Na+ transport and which inhibit CFU-GM, CFU-E, and CFU-MK colony formation in the absence of Li+ [209]. Therefore, the influence of Li+ upon normal physiological cation transport—for example, its influence upon Na+/K+-ATPase activity—may be partly responsible for the observed interference in hematopoiesis. 

Lemaire-Gony, S. and N. Mayer-Gostan. 1994. In vitro dose-response study of the effect of cadmium on eel (Anguilla anguilla) gill Na+/K+-ATPase activities. Ecotoxicol. Environ. Safety 28 43-52. 

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