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.

Experiments assessed whether long term exposure to 50 Hz pulsed electromagnetic fields with a peak magnetic field of 3 mT can alter the dynamics of intracellular calcium in human astrocytoma U-373 MG cells. Pretreatment of cells with 1.2 microM substance P significantly increased the [Ca(2+)](i). The same effect was also observed when [Ca(2+)](i) was evaluated in the presence of 20 mM caffeine. After exposure to electromagnetic fields the basal [Ca(2+)](i) levels increased significantly from 143 +/- 46 nM to 278 +/- 125 nM. The increase was also evident after caffeine addition, but in cells treated with substance P and substance P + caffeine we observed a [Ca(2+)](i) decrease after exposure. When we substituted calcium-free medium for normal medium immediately before the [Ca(2+)](i) measurements, the [Ca(2+)](i) was similar to that measured in the presence of Ca(2+). In this case, after EMFs exposure of cells treated with substance P, the [Ca(2+)](i), measured without and with addition of caffeine, declined from 824 +/- 425 to 38 +/- 13 nM and from 1369 +/- 700 to 11 +/- 4 nM, respectively, indicating that electromagnetic fields act either on intracellular Ca(2+) stores or on the plasma membrane. Moreover the electromagnetic fields that affected [Ca(2+)](i) did not cause cell proliferation or cell death and the proliferation indexes remained unchanged after exposure.

We evaluated the effects of 50 Hz pulsed electromagnetic fields (EMFs) with a peak magnetic field of 3 mT on human astrocytoma cells. Our results clearly demonstrate that, after the cells were exposed to EMFs for 24 h, the basal [Ca(2+)](i) levels increased significantly from 124+/-51 nM to 200+/-79 nM. Pretreatment of the cells with 1.2 microM substance P increased the [Ca(2+)](i) to 555+/-278 nM, while EMF exposure caused a significant drop in [Ca(2+)](i) to 327+/-146 nM. The overall effect of EMFs probably depends on the prevailing Ca(2+) conditions of the cells. After exposure, the proliferative responses of both normal and substance P-pretreated cells increased slightly from 1.03 to 1.07 and 1.04 to 1.06, respectively. U-373 MG cells spontaneously released about 10 pg/ml of interleukin-6 which was significantly increased after the addition of substance P. Moreover, immediately after EMF exposure and 24 h thereafter, the interleukin-6 levels were more elevated (about 40%) than in controls. On the whole, our data suggest that, by changing the properties of cell membranes, EMFs can influence Ca(2+) transport processes and hence Ca(2+) homeostasis. The increased levels of interleukin-6 after 24 h of EMF exposure may confirm the complex connection between Ca(2+) levels, substance P and the cytokine network.

Intracellular Ca2ϩ mobilization and release into mammal CSF plays a fundamental role in the etiogenesis of fever induced by the proinflammatory cytokine interleukin-1␤ (IL-1␤) and other pyrogens. The source and mechanism of IL-1␤-induced intracellular Ca 2ϩ mobilization was investigated using two experimental models. IL-1␤ (10 ng/ml) treatment of rat striatal slices preloaded with 45 Ca 2ϩ elicited a delayed (30 min) and sustained increase (125-150%) in spontaneous 45 Ca 2ϩ release that was potentiated by L-arginine (300 M) and counteracted by N--nitro-L-arginine methyl ester (L-NAME) (1 and 3 mM). The nitric oxide (NO) donors diethylamine/NO complex (sodium salt) (0.3 and 1 mM) and spermine/NO (0.1 and 0.3 mM) mimicked the effect of IL-1␤ on ] i ) (402 Ϯ 71.2% of baseline), which was abolished by 1 mM L-NAME. These data indicate that the NO/cGMP-signaling pathway is part of the intracellular mechanism transducing IL-1␤-evoked Ca 2ϩ mobilization in glial and striatal cells and that the ryanodine and the inositol-(1,4,5)-trisphosphate-sensitive Ca 2ϩ stores are involved. Key words: interleukin-1␤; nitric oxide; Ca 2ϩ release; human astrocytoma cells; rat striatum; cGMP; Ca 2ϩ stores; fever; neurotoxicityOur previous work on the mechanisms underlying the fever process showed that administration of interleukin-1␤ (IL-1␤) and other pyrogens into the lateral ventricle of rabbits was always accompanied by an increase in [Ca 2ϩ ] in the CSF. The antipyretic acetylsalicylic acid counteracted this effect and the increase in body temperature evoked by IL-1␤ (Palmi et al., 1992). The changes in brain [Ca 2ϩ ] were later shown to be strictly correlated with the temperature gain and with the increase in prostaglandin E 2 in CSF of these animals, whereas the antipyretic-anti-inflammatory agent dexamethasone antagonized both the fever and the increase in CSF [Ca 2ϩ ] induced by IL-1␤ (Palmi et al., 1994). The pyrogenic effect of IL-1␤ was also antagonized by lipocortin 5-(204 -212) peptide, a member of the annexin family that possesses the anti-inflammatory effects of glucocorticoids (Palmi et al., 1995) as well as by ventricular-cisternal perfusion with EGTA-enriched artificial CSF (Palmi et al., 1994).Together, these findings corroborated the involvement of Ca 2ϩ in thermoregulation (Myers and Veale, 1970;Palmi and Sgaragli, 1989), establishing the role of this ion in the intracellular signaling pathways that control the pyrogenic response to IL-1␤. Additional in vitro studies showed increased Ca 2ϩ efflux from rat striatum treated with IL-1␤ and antagonism of this effect by a specific IL-1 receptor antagonist protein. This explained the mechanism responsible for the increased Ca 2ϩ observed in CSF in vivo and also provided evidence that a specific receptor mediates Ca 2ϩ response (Palmi et al., 1996).The lag phase of the Ca 2ϩ response to IL-1␤ and the kinetic pattern of Ca 2ϩ release in these experiments were reminiscent of those of nitric oxide (NO) production by IL-1␤ in neurons (Bredt et al., 1991) and other c...

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.