The membrane potential of cytoplasts, derived from human neutrophils, was depolarized by the activation of the superoxide-generating NADPH-dependent oxidase. The extent of the depolarization was inhibited by diphenylene iodonium and was therefore due directly to the activity of the oxidase, which must be electrogenic. The extent of the depolarization was influenced by alteration of the delta pH across the cytoplast membrane, indicating that the outward translocation of H+ eventually compensates for superoxide generation. The depolarization of the potential is enhanced by Cd2+, a blocker of H+ currents, suggesting that the compensatory movement is via an H+ channel.

Imaging techniques, such as confocal microscopy and fluorescent activated cells scan are facilitating the study of responses at the single-cell level. Superoxide is reported to oxidise the nonfluorescent dihydrorhodamine 123 (DHR) to rhodamine 123. The generation of rhodamine 123 by human neutrophils, stimulated by the phorbol ester phorbol 12-myristate 13-acetate was inhibited slowly by diphenylene iodonium and rapidly by azide, but not by superoxide dismutase. In the absence of enzymes H,O, (but not 0; ) oxidised DHR slowly but the rate was greatly enhanced by peroxidases. The rhodamine 123 generated by phorbol-ester-stimulated neutrophils was observed to be located within the cell despite the fact that neutrophils failed to accumulate external rhodamine 123. This stimulated rise in cellular fluorescence was eliminated by excess extracellular catalase. It appears that H20,, released on the outside, crosses the plasma membrane where oxidation of DHR is catalysed by cellular peroxidases. Since in a mixed population DHR failed to distinguish between 0;-producing and non-producing HL60 cells it is not a suitable probe for single-cell observations. We conclude that DHR oxidation reports only the presence of H,O, and intracellular peroxidases, and not the generation of 0; by any one cell. Only peroxidase-containing cells fluoresce.Superoxide (0,) is a free radical formed by single electron donation to oxygen. Like all free radicals, it is highly reactive and will dismute rapidly to H,O,. 0; can be generated by a number of enzymes, e.g. xanthine oxidase, but the major source in the body is the NADPH oxidase of phagocytic white blood cells (neutrophils, macrophages, eosinophils). The 0; generated by the NADPH oxidase is widely studied as it is crucial to the role of these cells in the cellular immune response to bacterial infections [I -31. There are a number of assays for the detection of superoxide generation: (a) the superoxide-dismutase-inhibitable reduction of cytochrome c (the unpaired electron of 0 : is used to reduce cytochrome c ) ; (b) chemiluninescence (Ol, or a dismutation product, combines with luminol or lucigenin resulting in the liberation of light; (c) nitroblue tetrazolium reduction (superoxide or a product donate electrons to the water-soluble nitroblue tetrazolium resulting in the formation of the waterinsoluble formazan) ; (d) oxygen consumption using an oxygen electrode [4-61. There are also a number of fluorescent assays for the release of hydrogen peroxide. However these techniques monitor the generation of superoxide by populations of cells (-10'-10") and not the response of single cells.Dihydrofluroscein diacetate and dihydrorhodamine 123 (DHR) have recently become available commercially. Dihydrof-luroscein diacetate requires to be loaded into cells prior to the experiment and is trapped as a result of cleavage of the ester bonds by cellular estereases [7]. It is therefore only accessible to 0; or H,O, which is liberated in or diffuses into the cytosol of the cell and is not directly assess...

Women with endometriosis experience a range of problems for which they may or may not be adequately supported. This paper reports on one aspect of a study conducted at the Epworth Hospital, Melbourne, to identify the information needs of women facing laparoscopy for endometriosis. A number of focus groups were conducted that provided women with a forum for communicating their experiences of endometriosis and laparoscopy. The findings include the experiences of 61 women who described the lack of support, the struggles and the losses involved in living with endometriosis. By far the worst experience that these women described was the encounter with health professionals and the ways in which their symptoms were trivialised and dismissed. There is a great deal for nurses to learn about the experience of living with endometriosis if they are to support women with this chronic illness in their search for well-being.

The internal pH (pHi) of cytoplasts, derived from human neutrophils, falls 0.05 pH units upon activation of the superoxide-generating NADPH oxidase. The decrease in pHi is absent in diphenyleneiodonium-treated cytoplasts and therefore it is likely to arise directly from the activity of the oxidase. The addition of amiloride, to diminish the Na+/H+ exchanger, enhanced the extent of the internal acidification but not the initial rate. However the electroneutral Na+/H+ exchanger cannot be a contributor to H+ efflux to compensate for charge translocated by the oxidase. In the presence of Cd ions or valinomycin, phorbol-induced acidification of the cytosol was greatly increased, suggesting an inability to translocate the cytosolic H+ generated by an electrogenic oxidase. In the presence of both Cd and valinomycin the cytoplasts retained 0.8 H+ per O2-. generated. The rate of acidification of the external medium by stimulated cytoplasts is greatly reduced in the presence of Zn and valinomycin. Our results support the view that the plasma membrane of neutrophils contains Zn2+- or Cd2+-sensitive proton-conducting channels which maintain a stable membrane potential and pHi during the activity of the electrogenic NADPH oxidase.

1. Effects of arachidonic acid (AA) on proton and electron currents in human eosinophils were studied using the permeabilized-patch voltage-clamp technique, using an applied NH 4 + gradient to control pH i .2. Superoxide anion (O 2 _ ) release was assessed by cytochrome c reduction in human eosinophils. Significant O 2 _ release was stimulated by 5-10 µM AA.3. AA activated diphenylene iodinium (DPI)-inhibitable inward current reflecting electron efflux through NADPH oxidase. These electron currents (I e ) were elicited in human eosinophils at AA concentrations (3-10 µM) similar to those that induced O 2 _ release.4. The voltage-gated proton conductance (g H ) in eosinophils stimulated with AA was profoundly enhanced: H + current amplitude (I H ) increased 4.6 times, activation was 4 times faster, and the H + conductance-voltage (g H -V) relationship was shifted to substantially more negative voltages. The electrophysiological effects of AA resembled those reported for PMA, except that AA did not consistently slow r tail (deactivation of H + currents).5. The stimulation of both proton and electron currents by AA was reversible upon washout. Repeated exposure elicited repeated responses. The activation of H + currents by AA was dissociable from its activation of NADPH oxidase; H + currents were enhanced at low concentrations of AA that did not elicit detectable I e or when NADPH oxidase was inhibited by DPI.6. Most of the effects of AA on H + currents qualitatively resemble those reported in whole-cell studies, reflecting a more direct action than PMA. The results are compatible with AA being an immediate activator of both NADPH oxidase and proton channels in human eosinophils.