Munekazu Shigekawa

Purification of cardiac (Na+,K+)-activated adenosine triphosphatase from rat.

A procedure is described for preparation of highly active (Na+,K+)-ATPase from rat heart which has a specific activity of 200-600 mumol Pi/mg/h. The procedure is simple and can be applied to small amounts of heart muscle (approximately 1 g). The ATPase activity was more than 90% sensitive to ouabain (at concentrations up to 1 mM). The ouabain sensitivity is biphasic with about 20% of the ATPase activity being inhibited at approximately 3 X 10(-7) M ouabain.

Regulatory Mechanism of NHE1 Isoform of Na+/H+ Exchanger in Cardiac and Other Tissues

Regulation of intracellular pH and cell volume is essential for the normal function of a cell. The Na+/H+ exchanger in the plasma membrane plays a major role in both functions by extruding cytoplasmic H+ in exchange for extracellular Na+. In cardiomyocytes, in which protons are continuously produced by high metabolic activity, elucidation of the regulatory mechanism of the Na+/H+ exchanger is particularly important, because intracellular pH is a key modulator of contractility and because the transporter plays a critical role in cardiac pathophysiology such as ischemia/reperfusion-associated cell injury. In these cells, the ubiquitous form (NHE1) of the transporter is predominantly expressed and its activity presumably is under the regulatory influence of a variety of extracellular and intracellular factors including many receptor agonists, osmotic stress, and cell ATP level, as in other cell types. Recent advances in the molecular mechanism of short-term regulation of NHE1 by these factors and its pathophysiological relevance are discussed in this chapter.

Mechanism of Inhibition of Na + -H + Exchanger (NHE1) by ATP Depletion: Implications for Myocardial Ischemia

Na+-H+ exchange activity is metabolic energy dependent and may be inhibited when cell ATP level is reduced during myocardial ischemia. We found that ATP depletion inhibits activity of the cardiac isofom of the Na+-H+ exchanger (NHE1) by decreasing its apparent affinity for cytoplasmic H+, but not its Vmax value. By using a set of deletion mutants of the regulatory cytoplasmic domain of NHE1, we identified a 26-amino-acid-containing segment required to confer sensitivity to ATP depletion. This segment is localized within the most amino-terminal subdomain of the cytoplasmic domain that is critically important for the maintenance of high pHi sensitivity of NHE1 under nomal physiological condltions, as well as for upregulation of pHi sensitivity induced by stimulation with growth factors.

Regulation of Plasma Membrane Ca-Pump ATPase of Vascular Smooth Muscle by cGMP

Relaxation of vascular smooth muscle can be induced by several agents, such as nitrovasodilators, natriuretic factor, and endothelium-derived relaxant factor (EDRF), which increase the cyclic guanosine monophosphate (cGMP) level within vascular smooth muscle cells (VSMCs; Fig. 1) [1, for review]. There is a good parallel between the cGMP accumulation and reduction of vascular tone [1,2]. cGMP-dependent protein kinase (G-PK) phosphorylates several proteins in VSMCs [3]. However, the function of these proteins and their relevance to vasodilation remain unclear. The Ca-pump ATPase in the plasma membrane of VSMCs presumably plays an important role in the relaxation of VSMCs through its A TP- and calmodulin-dependent Ca2+ extrusion. Therefore, we examined whether the plasma membrane Ca pump is a target for G-PK-dependent regulation.

REACTION MECHANISM OF ATP HYDROLYSIS BY SARCOPLASMIC RETICULUM WITH CaATP AS A SUBSTRATE

Publisher Summary This chapter discusses adenosine triphosphate (ATP) hydrolysis with CaATP as a substrate to obtain information about the role of divalent cation in ATP hydrolysis. The existence and properties of this type of ATP hydrolysis have not so far been established. The results indicated that the ATPase can turn over normally with CaATP as a substratebut at a very slow rate. Mg2+ was not required for the turnover of the ATPase per se. Sarcoplasmic reticulum (SR) vesicles were prepared from rabbit skeletal muscle. The amount of calcium bound to the ATPase was measured by a membrane filtration method. At appropriate intervals after the addition of EDTA to the reaction mixture, 0.1 ml of the reaction mixture was rapidly passed through a membrane filter (HA 0.45-Um Millipore filter) by suction at 2°C. Then, the membrane filter was washed three times with 2 ml of a solution containing imidazole/HCl (pH 7.0) and EDTA, the concentrations of which were the same as those in the reaction mixture.