We investigated whether static electromagnetic fields (EMFs) at a flux density of 4.75 T, generated by an NMR apparatus (NMRF), could promote movements of Ca2+, cell proliferation, and the eventual production of proinflammatory cytokines in human peripheral blood mononuclear cells (PBMC) as well as in Jurkat cells, after exposure to the field for 1 h. The same study was also performed after activation of cells with 5 mg/ml phytohaemagglutinin (PHA). Our results clearly demonstrate that static NMRF exposure has neither proliferative, nor activating, nor proinflammatory effects on both normal and PHA activated PBMC. Moreover, the concentration of interleukin-1beta, interleukin-2, interleukin-6, interferon, and tumour necrosis factor alpha (TNFalpha) remained unvaried in exposed cells. Exposure of Jurkat cells statistically decreased the proliferation and the proliferation indexes, which 24 and 48 h after exposure were 0.7 +/- 0.29 and 0.87 +/- 0.12, respectively. Moreover, in Jurkat cells the [Ca2+]i was higher than in PBMC and was reduced significantly to about one half after exposure. This is consistent with the decrease of proliferation and with the low levels of IL-2 measured. On the whole, our data suggest that NMRF exposure failed to affect the physiologic behaviour of normal lymphomonocytes. Instead in Jurkat cells, by changing the properties of cell membranes, NMRF can influence Ca2+ transport processes, and hence Ca2+ homeostasis with improvement of proliferation.

To elucidate the mechanism of cell growth regulation by nitric oxide (NO) and the role played in it by Ca2+, we studied the relationship among intracellular Ca2+ concentration ([Ca2+]i), mitogen-activated protein kinases [extracellular signal-regulated protein kinase (ERK)] and proliferation in cell lines exposed to different levels of NO. Data showed that NO released by low [(z)-1-[2-aminiethyl]-N-[2-ammonioethyl]amino]diazen-1-ium-1,2diolate (DETA/NO) concentrations (10 microm) determined a gradual, moderate elevation in [Ca2+]i (46.8 +/- 7.2% over controls) which paralleled activation of ERK and potentiation of cell division. Functionally blocking Ca2+ or inhibiting calmodulin or MAP kinase kinase activities prevented ERK activation and antagonized the mitogenic effect of NO. Experimental conditions favouring Ca2+ entry into cells led to increased [Ca2+]i (189.5 +/- 4.8%), ERK activation and cell division. NO potentiated the Ca2+ elevation (358 +/- 16.8%) and ERK activation leading to expression of p21Cip1 and inhibition of cell proliferation. Furthermore, functionally blocking Ca2+ down-regulated ERK activation and reversed the antiproliferative effect of NO. Both the mitogenic and antimitogenic responses induced by NO were mimicked by a cGMP analogue whereas they were completely antagonized by selective cGMP inhibitors. These results demonstrate for the first time that regulation of cell proliferation by low NO levels is cGMP dependent and occurs via the Ca2+/calmodulin/MAP kinase kinase/ERK pathway. In this effect the amplitude of Ca2+ signalling determines the specificity of the proliferative response to NO possibly by modulating the strength of ERK activation. In contrast to the low level, the high levels (50-300 microm) of DETA/NO negatively regulated cell proliferation via a Ca2+-independent mechanism.

The relationship between nitric oxide (NO) and intracellular Ca2+ in hypoxic-ischemic brain damage is not known in detail. Here we used rat striatal slices perfused under low-oxygen and Ca2+-free conditions and cultured human astrocytoma cells incubated under similar conditions as models to study the dynamics of intracellular NO and Ca2+ in hypoxia-induced tissue damage. Exposure of rat striatal slices for 70 min to low oxygen tension elicited a delayed and sustained increase in the release of 45Ca2+. This was potentiated by the NO donors sodium nitroprusside (SNP) and spermine-NO and inhibited by N-omega-nitro-L-arginine methyl ester (L-NAME) or by the NO scavenger 2-phenyl-4,4,5,5 tetramethylimidazoline-1-oxyl-3-oxide (PTIO). A membrane-permeant form of heparin in combination with either ruthenium red (RR) or ryanodine (RY) also inhibited 45Ca2+ release. In human astrocytoma U-373 MG cells, hypoxia increased intracellular Ca2+ concentration ([Ca2+]i) by 67.2 +/- 13.1% compared to normoxic controls and this effect was inhibited by L-NAME, PTIO or heparin plus RR. In striatal tissue, hypoxia increased NO production and LDH release and both effects were antagonized by L-NAME. Although heparin plus RR or RY antagonized hypoxia-induced increase in LDH release they failed to counteract increased NO production. These data therefore indicate that NO contributes to hypoxic damage through increased intracellular Ca2+ mobilization from endoplasmic reticulum and suggest that the NO-Ca2+ signalling might be a potential therapeutic target in hypoxia-induced neuronal degeneration.

We investigated whether a combination of static electromagnetic field (EMF) at a flux density of 4.75 T together with pulsed EMF at a flux density of 0.7 mT generated by an NMR apparatus (NMRF), could promote movements of Ca(2+), cell proliferation, and the eventual production of proinflammatory cytokines in human lymphocytes as well as in Jurkat cells, after exposure to the field for 1 h. The same study was also performed after activation of cells with 5 micro g/ml phytohaemagglutinin (PHA) immediately before the exposure period. Our results clearly demonstrate that NMRF exposure increases the [Ca(2+)](i), without any proliferative, or activating, or proinflammatory effect on both normal and PHA stimulated lymphocytes. Accordingly, the levels of interferon gamma, tumor necrosis factor alpha, interleukin-1beta, interleukin-2, and interleukin-6 remained unvaried after exposure. Exposure of Jurkat cells statistically decreased the [Ca(2+)](i) and the proliferation. This is consistent with the low levels of IL-2 measured in supernatants of these cells after exposure. On the whole our data suggest that static and pulsed NMRF exposure contribute synergistically in the increase of the [Ca(2+)](i) without any activating or proinflammatory effect either in normal or in PHA challenged lymphocytes. In Jurkat cells, by changing the properties of cell membranes, NMRF exposure can influence Ca(2+) transport processes and hence Ca(2+) homeostasis, causing a marked decrease of proliferation.

The NO donor 3-Morpholinosydnonimine (SIN-1) releases NO in the presence of molecular oxygen. In this study, we evaluated the effect of SIN-1 on mitochondria of rat cortical synaptosomes. We demonstrated in vitro that the amount of ONOO(-) generated and H(2)O(2) formation directly correlated with SIN-1 concentration. The mean oxygen consumption by synaptosomal mitochondria was approximately 3.8 nmol of O(2) min(-1) mg(-1) protein, which decreased significantly in the presence of SIN-1 1 mM to 2.5 nmol O(2) min(-1) mg(-1). This decrease was not modified by catalase or Trolox, demonstrating that ONOO(-) was responsible for the effect. The same concentration of SIN-1 caused a significant decrease of ATP production by synaptosomal mitochondria and depolarized the mitochondrial membrane. Moreover, ROS production increased progressively and was completely inhibited by pre-incubation of synaptosomes with Trolox. Finally, phosphatidylserine was externalized and, at the same time, intrasynaptosomal lactate dehydrogenase decreased confirming both, the external membrane breakdown after the addition of SIN-1 and the damage to the synaptosomes.