Susceptibility of cloned K+ channels to reactive oxygen species.

Free radical-induced oxidant stress has been implicated in a number of physiological and pathophysiological states including ischemia and reperfusion-induced dysrhythmia in the heart, apoptosis of T lymphocytes, phagocytosis, and neurodegeneration. We have studied the effects of oxidant stress on the native K+ channel from T lymphocytes and on K+ channels cloned from cardiac, brain, and T-lymphocyte cells and expressed in Xenopus oocytes. The activity of three Shaker K+ channels (Kv1.3, Kv1.4, and Kv1.5), one Shaw channel (Kv3.4), and one inward rectifier K+ channel (IRK3) was drastically inhibited by photoactivation of rose bengal, a classical generator of reactive oxygen species. Other channel types (such as Shaker K+ channel Kv1.2, Shab channels Kv2.1 and Kv2.2, Shal channel Kv4.1, inward rectifiers IRK1 and ROMK1, and hIsK) were completely resistant to this treatment. On the other hand tert-butyl hydroperoxide, another generator of reactive oxygen species, removed the fast inactivation processes of Kv1.4 and Kv3.4 but did not alter other channels. Xanthine/xanthine oxidase system had no effect on all channels studied. Thus, we show that different types of K+ channels are differently modified by reactive oxygen species, an observation that might be of importance in disease states.

The Superoxide Synthases of Rose Cells1 : Comparison of Assays

In an effort to identify the enzymatic mechanism responsible for the synthesis of reactive oxygen species produced during the hypersensitive response, preparations of rose (Rosa damascena) cell plasma membranes, partially solubilized plasma membrane protein, and cytosol were assayed for the NADH- and NADPH-dependent synthesis of superoxide using assays for the reduction of cytochrome c (Cyt c), assays for the reduction of nitroblue tetrazolium, and assays for the chemiluminescence of N,N′-dimethyl-9,9′-biacridium dinitrate (lucigenin). Each assay ascribed the highest activity to a different preparation: the Cyt c assay to cytosol, the nitroblue tetrazolium assay to plasma membrane, and the lucigenin assay to the partially solubilized plasma membrane protein (with NADH). This suggests that no two assays measure the same set of enzymes and that none of the assays is suitable for comparisons of superoxide synthesis among different cell fractions. With the plasma membrane preparation, the presence of large amounts of superoxide-dismutase-insensitive Cyt c reductase confounded attempts to use Cyt c to measure superoxide synthesis. With the partially solubilized membrane protein, direct reduction of lucigenin probably contributed to the chemiluminescence. Superoxide synthesis detected with lucigenin should be confirmed by superoxide-dismutase-sensitive Cyt c reduction.

Comparative Biochemistry of the Oxidative Burst Produced by Rose and French Bean Cells Reveals Two Distinct Mechanisms1

Cultured cells of rose (Rosa damascena) treated with an elicitor derived from Phytophthora spp. and suspension-cultured cells of French bean (Phaseolus vulgaris) treated with an elicitor derived from the cell walls of Colletotrichum lindemuthianum both produced H2O2. It has been hypothesized that in rose cells H2O2 is produced by a plasma membrane NAD(P)H oxidase (superoxide synthase), whereas in bean cells H2O2 is derived directly from cell wall peroxidases following extracellular alkalinization and the appearance of a reductant. In the rose/Phytophthora spp. system treated with N,N-diethyldithiocarbamate, superoxide was detected by a N,N′-dimethyl-9,9′-biacridium dinitrate-dependent chemiluminescence; in contrast, in the bean/C. lindemuthianum system, no superoxide was detected, with or without N,N-diethyldithiocarbamate. When rose cells were washed free of medium (containing cell wall peroxidase) and then treated with Phytophthora spp. elicitor, they accumulated a higher maximum concentration of H2O2 than when treated without the washing procedure. In contrast, a washing treatment reduced the H2O2 accumulated by French bean cells treated with C. lindemuthianum elicitor. Rose cells produced reductant capable of stimulating horseradish (Armoracia lapathifolia) peroxidase to form H2O2 but did not have a peroxidase capable of forming H2O2 in the presence of reductant. Rose and French bean cells thus appear to be responding by different mechanisms to generate the oxidative burst.