Effect of coexposure to 50 Hz magnetic fields and an aneugen on human lymphocytes, determined by the cytokinesis block micronucleus assay.

Interference of 50 Hz extremely low frequency magnetic fields (ELF-MF) with the known aneugen vinblastine (VBL) on micronucleus formation was tested with the in vitro cytokinesis block micronucleus assay in human lymphocyte cultures. Isolated lymphocyte cultures were prepared from 18 individuals. Three groups of quadruplicate cultures from six unrelated individuals were exposed to 50 Hz ELF-MF of background (bkg), 80 and 800 microT, respectively, during the complete incubation period (72 h). Twenty-four hours after culture initiation, one replicate culture from each individual within each ELF-MF group was exposed to 0, 5, 10, or 15 ng/ml VBL. The isolated lymphocyte cultures were scored for the presence of micronuclei, the nuclear division index (NDI), and apoptosis. As expected, increased VBL concentration resulted in an increased micronucleus and apoptosis frequency and in a decreased NDI. In the presence of VBL, there was a systematic tendency for increased micronucleus and apoptosis frequency in the ELF-MF exposed groups compared to the bkg group. In the absence of VBL, we observed no statistically significant effect of ELF-MF on micronucleus induction or apoptosis frequency, but the NDI was significantly higher in the 800 microT group compared to the other groups, suggesting an effect of ELF-MF on cell proliferation. An interaction between ELF-MF and VBL on NDI was observed. This interaction reflected the drastic decrease in NDI due to coexposure to VBL.

Inositol 1,4,5-trisphosphate releases Ca2+ from vacuolar membrane vesicles of Saccharomyces cerevisiae.

Inositol 1,4,5-trisphosphate (IP3) induces a release of Ca2+ from vacuolar membrane vesicles of Saccharomyces cerevisiae. The amount released is dependent on IP3 concentration (concentration for half maximal effect, Km, apparent = 0.4 microM). Myo-inositol, and inositol 1,4-bisphosphate up to 50 microM have no effect on Ca2+ levels in the vesicles. The IP3-induced Ca2+ release is blocked by dantrolene and 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate-HCl (TMB-8), which are known to block Ca2+ release from Ca2+ stores in animal cells. IP3-induced release of Ca2+ also occurs when Ca2+ is accumulated by means of an artificial pH gradient, indicating that the effect of IP3 is not due to an effect on the vacuolar H(+)-ATPase. The IP3-induced Ca2+ release is not accompanied by a change in the pH gradient, which indicates that it is not due to a reversal of the Ca2+/nH+ antiport or to a decrease in delta pH by IP3. The present results suggest that IP3 may act as a second messenger in the mobilization of Ca2+ in yeast cells. As in plant cells, the vacuolar membrane of yeast seems to contain a Ca2+ channel, which can be opened by IP3. In this respect the vacuole could function as an IP3-regulated intracellular Ca2+ store, equivalent to the endoplasmic- and sarcoplasmic reticulum in animal cells, and play a role in Ca(2+)-dependent signal transduction in yeast cells.

A study of the mechanism by which inhibitors of the plasmamembrane ATPase enhance uptake of divalent cations in yeast.

The enhancement of divalent cation uptake in yeast provoked by the membrane ATPase inhibitors trifluoperazine, miconazole, compound 48/80, ethidium, DIO-9 and calmidazolium should be ascribed to an increase in cation permeability of the yeast rather than to hyperpolarisation of the yeast cell membrane. For trifluoperazine and miconazole it is unequivocally shown that the cells are hyperpolarized though for miconazole only transiently. Whether the other drugs also hyperpolarize the yeast cells is uncertain. The apparent hyperpolarisation caused by trifluoperazine and miconazole may be attributed to a specific increase in the K+ permeability of the yeast plasmamembrane evoked by these compounds.

All-or-none interactions of inhibitors of the plasma membrane ATPase with Saccharomyces cerevisiae.

The inhibitors of the yeast plasma membrane ATPase, Dio-9, miconazole, suloctidil, N,N'-dicyclohexycarbodiimide, triphenyltin and diethylstilbestrol provoke an all-or-none K+ loss from the cells. Some of the K+-depleted cells also show an increased permeability for the dye, Bromophenol blue, indicating that the barrier properties of these cells are drastically changed. Apart from this all-or-none process, a graded loss of K+ is also observed. These observations warn against the use of the inhibitors in studies aimed at evaluating the role of the ATPase in the energy transduction of membrane transport processes of yeasts.

Uptake and accumulation of Mn2+ and Sr2+ in Saccharomyces cerevisiae.

Initial uptake of Mn2+ and Sr2+ in the yeast Saccharomyces cerevisiae was studied in order to investigate the selectivity of the divalent cation uptake system and the possible involvement of the plasma-membrane ATPase in this uptake. The initial uptake rates of the two ions were not significantly different. This ruled out a direct role of the plasma-membrane ATPase, since this ATPase is specific for Mn2+ compared to Sr2+. After 1 h uptake, Mn2+ had accumulated 10-times more than Sr2+. Influx of Mn2+ and Sr2+ remained unchanged during that time, however. The differences in accumulation level found for Mn2+ and Sr2+ could be ascribed to a greater efflux of Sr2+ as compared with Mn2+. Probably this greater efflux of Sr2+ was only apparent, since differential extraction of the yeast cells revealed that Mn2+ is more compartmentalised than Sr2+, giving rise to a lower relative cytoplasmic Mn2+ concentration.

Factors affecting the inhibition of yeast plasma membrane ATPase by vanadate.

Inhibition of yeast plasma membrane ATPase by vanadate occurs only if either Mg2+ or MgATP2- is bound to the enzyme. The dissociation constant of the complex of vanadate and inhibitory sites is 0.14-0.20 microM in the presence of optimal concentrations of Mg2+ and of the order of 1 microM if the enzyme is saturated with MgATP2-. The dissociation constants of Mg2+ and MgATP2- for the sites involved are 0.4 and 0.62-0.73 mM, respectively, at pH 7. KCl does not increase the affinity of vanadate to the inhibitory sites as was found with (Na+ + K+)-ATPase. On the other hand, the effect of Mg2+ upon vanadate binding is similar to that upon (Na+ + K+)-ATPase, and the corresponding affinity constants of Mg2+ and vanadate for the two enzymes are of the same order of magnitude.

Cotransport of phosphate and sodium by yeast.

Phosphate uptake by yeast at pH 7.2 is mediated by two mechanisms, one of which has a Km of 30 micronM and is independent of sodium, and a sodium-dependent mechanism with a Km of 0.6 micronM, both Km values with respect to monovalent phosphate. The sodium-dependent mechanism has two sites with affinity for Na+, with affinity constants of 0.04 and 29 mM. Also lithium enhances phosphate uptake; the affinity constants for lithium are 0.3 and 36 mM. Other alkali ions do not stimulate phosphate uptake at pH 7.2. Ribidium has no effect on the stimulation of phosphate uptake by sodium. Phosphate and arsenate enhance sodium uptake at pH 7.2. The Km of this stimulation with regard to monovalent orthophosphate is about equal to that of the sodium-dependent phosphate uptake. The properties of the cation binding sites of the phosphate uptake mechanism and those of the phosphate-dependent cation transport mechanism have been compared. The existence of a separate sodium-phosphate cotransport system is proposed.