Reactive oxygen species are produced in the ovary. In luteal cells, peroxide abruptly inhibits LH-sensitive cAMP and progesterone production, and may serve a role as a mediator of luteolysis by such mechanisms. The objective of the present studies was to evaluate the acute actions of peroxide in rat granulosa cells. Peroxide at concentrations in the low micromolar range produced a marked and dose-dependent inhibition of FSH-sensitive cAMP accumulation and progesterone production, and depleted cell levels of ATP within 1 min. Longer treatment with peroxide (60 min) caused complete abrogation of the actions of FSH. Peroxide-induced depletion of ATP was prevented by 3-aminobenzamide, an inhibitor of DNA repair, but maintenance of cell levels of ATP did not prevent the anti-FSH effects of peroxide. Peroxide also abrogated cAMP accumulation and progesterone production in response to LH in granulosa cells. Unlike that seen with LH, inhibition of FSH-sensitive cyclic AMP accumulation by peroxide was partially reversed with isobutylmethyl xanthine, an inhibitor of cyclic AMP phosphodiesterase. Although peroxide inhibited cAMP accumulation in response to cholera toxin, it did not inhibit this same response to forskolin, which indicates that peroxide may interfere with G-protein-dependent activation of adenylate cyclase. Peroxide inhibited steroidogenesis in response to cholera toxin, forskolin, and 8-bromo-cAMP. The marked inhibitory actions of peroxide on gonadotropic hormone action and steroidogenesis in granulosa cells raise the possibility that peroxide may mediate events associated with loss of follicular function.

The flagellar motor of Escherichia coli (E. coli) is driven by a proton-motive force (PMF), hence it was of interest to determine whether the motor is symmetrical in the sense that it can be rotated by any polarity of PMF. For this purpose the cells had to be deenergized first. Conventional deenergization procedures caused irreversible loss of motility, presumably due to ATP-dependent degradative processes. However, E. coli cells deenergized by incubation with arsenate manifested a slow, reversible depletion of PMF. In this procedure there was a sufficiently long time window, during which a considerable proportion of the cells lost their motility and could be made to rotate again by an artificially-imposed PMF. The motors of these cells rotated in response to any PMF polarity, but positive and negative polarities rotated different sub-populations of cells and the direction was almost exclusively counterclockwise. The reason for the unidirectionality of the rotation was not the intervention of the chemotaxis system. A number of potential reasons are suggested. One is the arsenate effect on the motor function found previously [Margolin, Y., Barak, R. and Eisenbach, M. (1994) J. Bacteriol. 176, 5547-5549]. A possible interaction between arsenate and the motor is discussed.

The effect of arsenate on flagellar rotation in cytoplasm-free flagellated envelopes of Escherichia coli and Salmonella typhimurium was investigated. Flagellar rotation ceased as soon as the envelopes were exposed to arsenate. Inclusion of phosphate intracellularly (but not extracellularly) prevented the inhibition by arsenate.In a parallel experiment, the rotation was not affected by inclusion of an ATP trap (hexokinase and glucose) within the envelopes. It is concluded that arsenate affects the motor in a way other than reversible deenergization. This may be an irreversible damage to the cell or direct inhibition of the motor by arsenate. The latter possibility suggests that a process of phosphorylation or phosphate binding is involved in the motor function.An observation made a number of years ago was that cytoplasm-free envelopes of Escherichia coli or Salmonella typhimurium, tethered to glass by their flagella, can be made to rotate clockwise (CW) by inclusion of purified CheY in them (16). At that time this observation was intriguing, because CW rotation in intact bacteria was known to require intracellular ATP (1,3,10,17,20); however, these envelopes rotated CW in the apparent absence of ATP. (When envelopes are prepared, the internal content of the bacterial cell is diluted at the lysis step by more than 104-fold; therefore, their ATP levels are presumably negligible.) To verify that the CW-rotating envelopes indeed did not contain residual ATP, we treated them with arsenate, an effective ATP-depleting agent in intact bacteria (10,17). Surprisingly, arsenate immediately stopped not only the CW-rotating envelopes but also the counter-CW (CCW)-rotating, CheY-free envelopes. The purpose of this study is to discriminate between the potential reasons for the inhibition of flagellar rotation by arsenate. (It should be noted that even though the ATP requirement was later found to be for CheY phosphorylation [8,9,23] addition of the respiratory substrate D,L-lactic acid (2 mM) to the flow medium. Exogenous addition of arsenate (on top of the lactic acid) stopped the rotation of both CCW (Fig. 1) and CW (not shown) envelopes within 2 min. Removal of arsenate from the flow medium caused resumption of rotation, provided that the incubation time with arsenate was relatively short: about 67% of the envelopes resumed their rotation when the incubation time was 5 min or less, but only about 17% did so when the incubation time was 8 min or longer. The inhibitory effect of arsenate was much reduced when phosphate was included within the envelopes (Fig. 1), indicating (i) that arsenate acts in the envelopes as a competitive inhibitor of phosphate and (ii) that its site of action is intracellular. (It should be noted in this regard that E. coli has two primary Pi transport systems: the pst system which is ATP dependent, having a higher specificity for phosphate than for arsenate, and the pit system, which is a proton motive force [PMF]-dependent system having similar affinities for phosphate and arsenate [18]. The pit sys...

Galactose and other chemotactic attractants have been shown to trigger an apparent hyperpolarization in Escherichia coli (Eisenbach, M., 1982, Biochemistry, 21:6818-6825). The probe used to measure membrane potential in that study, tetraphenylphosphonium (TPP+), may respond also to surface-charge changes in the membrane. The distinction between true changes in membrane potential and changes in the surface charge of the membrane is crucial for the study of this phenomenon in bacterial chemotaxis. To distinguish between these parameters, we compared the response to galactose with different techniques: K+ distribution in the presence of valinomycin (measured with a K+-selective electrode), TPP+ distribution (measured with a TPP+-selective electrode) at different ionic strengths, absorbance changes of bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethineoxonol (oxonol V), and fluorescence changes of three probes with different mechanisms of response. All the techniques revealed stimulation by galactose of transient hyperpolarization, of comparable magnitude. This indicates the involvement of ion currents rather than alterations of local surface properties.

To examine whether or not sensory signaling in bacteria is by way of fluctuations in membrane potential, we studied the effect of clamping the potential on bacterial chemotaxis. The potential was clamped by valinomycin, a K+-specific ionophore, in the presence of K+. Despite the clamped potential, sensory signaling did occur: both Escherichia coli and Bacillus subtilis cells were still excitable and adaptable under these conditions. It is concluded that signaling in the excitation and adaptation steps of chemotaxis is not by way of fluctuations in the membrane potential.