The ubiquitously expressed Na(+)/H(+) exchanger isoform 1 (NHE1) functions as a major intracellular pH (pH(i)) regulatory mechanism in many cell types, and in some tissues its activity may contribute to ischemic injury. In the present study, cortical astrocyte cultures from wild-type (NHE1(+/+)) and NHE1-deficient (NHE1(-/-)) mice were used to investigate the role of NHE1 in pH(i) recovery and ischemic injury in astrocytes. In the absence of HCO(3)(-), the mean resting pH(i) levels were 6.86 +/- 0.03 in NHE1(+/+) astrocytes and 6.53 +/- 0.04 in NHE1(-/-) astrocytes. Removal of extracellular Na(+) or blocking of NHE1 activity by the potent NHE1 inhibitor HOE-642 significantly reduced the resting level of pH(i) in NHE1(+/+) astrocytes. NHE1(+/+) astrocytes exhibited a rapid pH(i) recovery (0.33 +/- 0.08 pH unit/min) after NH(4)Cl prepulse acid load. The pH(i) recovery in NHE1(+/+) astrocytes was reversibly inhibited by HOE-642 or removal of extracellular Na(+). In NHE1(-/-) astrocytes, the pH(i) recovery after acidification was impaired and not affected by either Na(+)-free conditions or HOE-642. Furthermore, 2 h of oxygen and glucose deprivation (OGD) led to an approximately 80% increase in pH(i) recovery rate in NHE1(+/+) astrocytes. OGD induced a 5-fold rise in intracellular [Na(+)] and 26% swelling in NHE1(+/+) astrocytes. HOE-642 or genetic ablation of NHE1 significantly reduced the Na(+) rise and swelling after OGD. These results suggest that NHE1 is the major pH(i) regulatory mechanism in cortical astrocytes and that ablation of NHE1 in astrocytes attenuates ischemia-induced disruption of ionic regulation and swelling.

Cultured human endothelial cells (EC) exposed to atherogenic low-density lipoprotein levels have increased reactive oxygen species (ROS) generation. The enzyme responsible for this ROS production elevation is unknown. We have examined for the presence of a functional leukocyte-type NADPH oxidase in EC to elucidate whether this enzyme could be the ROS source. The plasma membrane fraction of disrupted EC showed a reduced-minus-oxidized difference spectra with absorption peaks identical to those observed in the spectra of the leukocyte NADPH oxidase component, cytochrome b558. Western-blot analysis, using anti-gp91 -phox. anti -p22-phox. anti -p47-phox. and anti -p67-phox antibodies, demonstrated the protein expression of NADPH oxidase subunits in EC. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed the mRNA expression of gp91-phox, p22-phox, p47-phox, and p67-phox in EC. Sonicates from unstimulated EC produced no measurable superoxide; whereas, exogenously applied arachidonic acid activated superoxide generation in a manner that was dependent upon the presence of NADPH and both membrane and cytosolic fractions combined. Apocynin, a specific leukocyte NADPH oxidase inhibitor, was shown by Western-blot analysis of membrane and cytoplasmic fractions to inhibit the translocation of p47-phox to the membrane of stimulated EC. These findings support the presence of a functionally active leukocyte-type NADPH oxidase in EC. NADPH oxidase could be the major cellular ROS source in EC perturbation, which has been hypothesized to be a major contributing factor in the pathogenesis of atherosclerosis.

The outermost epidermal layer, the stratum corneum (SC), exhibits an acidic surface pH, whereas the pH at its base approaches neutrality. NHE1 is the only Na ؉ /H ؉ antiporter isoform in keratinocytes and epidermis, and has been shown to regulate intracellular pH. We now demonstrate a novel function for NHE1, as we find that it also controls acidification of extracellular "microdomains" in the SC that are essential for activation of pH-sensitive enzymes and the formation of the epidermal permeability barrier. NHE1 expression in epidermis is most pronounced in granular cell layers, and although the surface pH of NHE1 knockout mice is only slightly more alkaline than normal using conventional pH measurements, a more sensitive method, fluorescence lifetime imaging, demonstrates that the acidic intercellular domains at the surface and of the lower SC disappear in NHE1 ؊/؊ animals. Fluorescence lifetime imaging studies also reveal that SC acidification does not occur through a uniform gradient, but through the progressive accumulation of acidic microdomains. These findings not only visualize the spatial distribution of the SC pH gradient, but also demonstrate a role for NHE1 in the generation of acidic extracellular domains of the lower SC, thus providing the acidification of deep SC interstices necessary for lipid processing and barrier homeostasis.

An increasing body of evidence has demonstrated that NADPH oxidase plays a critical role in several early steps leading toward the development of atherosclerosis. These effects appear to be carried out by both the ability of O 3 2 to act as a small second messenger molecule, and potentially the oxidation of low density lipoprotein by O 3 2 . We describe a model for the initiation and development of atherosclerosis that suggests targeted inhibition of NADPH oxidase as a powerful site for prevention and treatment of this disease.z 2000 Federation of European Biochemical Societies.

The basolateral Na+-K+-2Cl− cotransporter (NKCC1) functions in the maintenance of cellular electrolyte and volume homeostasis. NKCC1-deficient ( Nkcc1 −/−) mice were used to examine its role in cardiac function and in the maintenance of blood pressure and vascular tone. Tail-cuff measurements demonstrated that awake Nkcc1 −/− mice had significantly lower systolic blood pressure than wild-type ( Nkcc1 +/+) mice (114.5 ± 2.2 and 131.8 ± 2.5 mmHg, respectively). Serum aldosterone levels were normal, indicating that extracellular fluid-volume homeostasis was not impaired. Studies using pressure transducers in the femoral artery and left ventricle showed that anesthetized Nkcc1 −/− mice have decreased mean arterial pressure and left ventricular pressure, whereas myocardial contraction parameters were not significantly different from those of Nkcc1 +/+ mice. When stimulated with phenylephrine, aortic smooth muscle from Nkcc1 +/+ and Nkcc1 −/−mice exhibited no significant differences in maximum contractility and only moderate dose-response shifts. In phasic portal vein smooth muscle from Nkcc1 −/− mice, however, a sharp reduction in mechanical force was noted. These results indicate that NKCC1 can be important for the maintenance of normal blood pressure and vascular tone.