Shigeo Wakabayashi

CYTOPLASMIC DOMAIN OF THE UBIQUITOUS NA+/H+ EXCHANGER NHE1 CAN CONFER CA2+RESPONSIVENESS TO THE APICAL ISOFORM NHE3

The Na+/H+ exchanger isoforms NHE1 and NHE3 are regulated differently by various stimuli. Calcium has been recognized as one of the major second messengers in such exchanger regulation. We previously proposed that Ca2+-induced activation of NHE1 occurs via displacement of its autoinhibitory domain from the H+ modifier site due to direct binding of Ca2+/calmodulin. To further validate this hypothesis, the functional role of the cytoplasmic domain was studied in both wild-type and chimeric exchangers, i.e. NHE1, NHE3, NHE1 with the cytoplasmic domain of NHE3(N1N3), and NHE3 with the cytoplasmic domain of NHE1(N3N1). After expression in exchanger-deficient fibroblasts (PS120), early response (<80 s) to external stimuli was assessed as 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+ uptake. Among stimuli tested (ionomycin, α-thrombin, phorbol ester, hyperosmotic stress, and platelet-derived growth factor) that are all known to activate NHE1, only ionomycin and thrombin induced a significant intracellular Ca2+ mobilization and early activation of 22Na+ uptake, implying that Ca2+ is a main regulator of NHE1 in the early phase of the agonist response. However, all the stimuli did not activate NHE3 or N1N3. In contrast, a significant stimulation of 22Na+ uptake in response to ionomycin and thrombin was observed in N3N1, accompanied by an alkaline shift of pHi sensitivity (∼0.2 pH units). Deletion of the cytoplasmic calmodulin-binding domain within N3N1 resulted in a constitutive alkaline shift of pHisensitivity and abolished the activation by ionomycin and thrombin. Together, these data reinforce our concept of Ca2+-induced activation of NHE1. Furthermore, they provide evidence for a functional interaction of the autoinhibitory domain of NHE1 with the H+-modifier site of a different isoform, NHE3.

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.

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.