Lipid rafts function in Ca2+ signaling responsible for activation of sperm motility and chemotaxis in the ascidian Ciona intestinalis.

Lipid rafts are specialized membrane microdomains that function as signaling platforms across plasma membranes of many animal and plant cells. Although there are several studies implicating the role of lipid rafts in capacitation of mammalian sperm, the function of these structures in sperm motility activation and chemotaxis remains unknown. In the ascidian Ciona intestinalis, egg-derived sperm activating- and attracting-factor (SAAF) induces both activation of sperm motility and sperm chemotaxis to the egg. Here we found that a lipid raft disrupter, methyl-ß-cyclodextrin (MCD), inhibited both SAAF-induced sperm motility activation and chemotaxis. MCD inhibited both SAAF-promoted synthesis of intracellular cyclic AMP and sperm motility induced by ionophore-mediated Ca(2+) entry, but not that induced by valinomycin-mediated hyperpolarization. Ca(2+)-imaging revealed that lipid raft disruption inhibited Ca(2+) influx upon activation of sperm motility. The Ca(2+)-activated adenylyl cyclase was clearly inhibited by MCD in isolated lipid rafts. The results suggest that sperm lipid rafts function in signaling upstream of cAMP synthesis, most likely in SAAF-induced Ca(2+) influx, and are required for Ca(2+)-dependent pathways underlying activation and chemotaxis in Ciona sperm.

Valinomycin induced energy-dependent mitochondrial swelling, cytochrome c release, cytosolic NADH/cytochrome c oxidation and apoptosis.

In valinomycin induced stimulation of mitochondrial energy dependent reversible swelling, supported by succinate oxidation, cytochrome c (cyto-c) and sulfite oxidase (Sox) [both present in the mitochondrial intermembrane space (MIS)] are released outside. This effect can be observed at a valinomycin concentration as low as 1 nM. The rate of cytosolic NADH/cyto-c electron transport pathway is also greatly stimulated. The test on the permeability of mitochondrial outer membrane to exogenous cyto-c rules out the possibility that the increased rate of exogenous NADH oxidation could be ascribed either to extensively damaged or broken mitochondria. Accumulation of potassium inside the mitochondria, mediated by the highly specific ionophore valinomycin, promotes an increase in the volume of matrix (evidenced by swelling) and the interaction points between the two mitochondrial membranes are expected to increase. The data reported and those previously published are consistent with the view that "respiratory contact sites" are involved in the transfer of reducing equivalents from cytosol to inside the mitochondria both in the absence and the presence of valinomycin. Magnesium ions prevent at least in part the valinomycin effects. Rather than to the dissipation of membrane potential, the pro-apoptotic property of valinomycin can be ascribed to both the release of cyto-c from mitochondria to cytosol and the increased rate of cytosolic NADH coupled with an increased availability of energy in the form of glycolytic ATP, useful for the correct execution of apoptotic program.

Interference of MI-D, a new mesoionic compound, on artificial and native membranes.

MI-D (4-phenyl-5-(4-nitrocinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride), a new mesoionic compound, decreased the rate of swelling induced by valinomycin-K+, as well as induced swelling in the presence of nigericin-K+. Shrinkage was also affected, suggesting interference with the inner mitochondrial membrane, which would affect both fluidity and elasticity. Fluorescence polarization of DPH and DPH-PA, probing the core and outer regions respectively, of the DMPC and native membranes, indicated that MI-D shifts the midpoint of phase transition to higher values and orders of the fluid phase. These alterations in membrane fluidity are thus related to MI-D effects on the energy-linked functions of mitochondria.

The effect of DDT on K+ transport in mouse liver mitochondria.

This study describes DDT-induced changes in membrane permeability of mitochondria and erythrocytes to K+ as monitored by a K+-selective electrode. DDT is a strong inhibitor of valinomycin-mediated K+ uptake and the corresponding H+ efflux and an inducer of K+ leakage out of mitochondria but not to any significant extent out of erythrocytes. The inhibition of K+ uptake and H+ efflux was a function of (a) preincubation time between mitochondria and DDT, (b) mitochondrial concentration, (c) the nature of the carrier solvent and (d) temperature. The kinetics of inhibition of K+ uptake showed that DDT is an uncompetitive inhibitor with respect to valinomycin and a competitive inhibitor with respect to K+. The efflux of endogenous K+ showed a sigmoid dependency on DDT concentration and was reduced to endogenous rates when the temperature was lowered below the gel-liquid crystalline phase transition of the lipids. It is suggested that the DDT-induced changes in membrane permeability are due to perturbation of the lipid phase and that its toxicity may be due in part to hyperpolarization of subcellular membranes.

Cation reversal of inhibition of growth by valinomycin in Streptococcus pyogenes and Clostridium sporogenes.

Study of the antimicrobial spectrum of valinomycin revealed that, in addition to the gram-positive bacteria reported in literature, Streptococcus pyogenes and Clostridium sporogenes are also susceptible to this antibiotic. The minimal inhibitory concentrations (MIC) of the antibiotic for S. pyogenes grown aerobically and anaerobically did not differ markedly, negating the hypothesis that oxidative phosphorylation is involved in the mechanism of action of this antibiotic. This conclusion is further strengthened by the inhibition of growth of C. sporogenes, an obligate anaerobe. In a medium with a low K(+) concentration, the MIC for S. pyogenes was 0.02 mug/ml, the lowest ever recorded for this antibiotic. The inhibition of growth of S. pyogenes and C. sporogenes was readily reversed by addition of K(+) to the medium, indicating a compensation for net efflux of K(+) from the cells when the transmembrane potential reached equilibrium. In contrast to these bacteria, Bacillus subtilis was less susceptible to the antibiotic when the potassium concentration of the medium was low. The addition of potassium in the presence of valinomycin increased the inhibition of growth, which appears to result from dissipation of metabolic energy as in the mitochondrial system.