The effects of the heavy-metal ions Cd2+ and Zn2+ on the homoeostasis of intracellular free Ca2+ in E367 neuroblastoma cells were examined using 19F-NMR spectroscopy with the fluorinated chelator probe 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N', N'-tetra-acetic acid (5F-BAPTA). First, the technique was used to quantify the uptake and intracellular free concentrations of the heavy metals after treatment of the cells with 20 microM CdCl2 or 100 microM ZnCl2. Secondly, metal-induced transients in intracellular free Ca2+ were recorded. Addition of 20 microM CdCl2, but not 100 microM ZnCl2, evoked a transient increase in Ca2+ from a resting level of 84 nM to approx. 190 nM within 15 min after addition of the metal. Zn2+ at 20 microM completely prevented the induction of a Ca2+ transient by Cd2+. Ca2+ was mobilized by Cd2+ from intracellular organelles, since depletion of these stores by thapsigargin abolished the effect of the toxic metal. Furthermore, 20 microM Cd2+ evoked a transient rise in cellular Ins(1,4,5)P3, reaching a maximum level within 5 min after addition of the metal. These results demonstrate that perturbation of the Ins(1,4,5)P3/Ca2+ messenger system is an early and discrete cellular effect of Cd2+.

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Substance P (SP), fibroblast growth factor (FGF) and epidermal growth factor (EGF) were recogruzed to stimulate proliferation of human skin fibroblasts in vitro. FGF and SP have competence factor activity, whereas EGF acts as a progression factor. Growth factor synergisms and antagonisms which affect fibroblast functions me part of a mul!ifaceted regulatoxy apparatus that involves the nervous system, the endocrine system and other modulators of cell function such as prostaglandins. The ability of eicosanoids to regulate proliferation of fibroblasts and the increased production of arachidonic acid metabolites in response to vatious growth factors, led us to mvesbgate the role of prostaglandins m cytokine-induced cell cycledependent human libroblast proliferation. We tested the effect of SP, EGF and FGF and their combinations on cell growth and the pattem of arachidonic acid metabolites release. A combination of SP and EGF syn@ticaUy stimulated fibroblast proliferation and prostaglandin E2 release, whereas addition of SP to FGF co-stimulated cultures did not affect cell growth. Inhibition of cyclooxygenase by acetylsalicylic acid augmented the growth response to all substances alone. In the v c e of acetysalicyhc acid, SP combined with FGF enhanced fibroblast proliferation, whereas a combination with EGF inhibited cellular growth with respect to growth induced by EGF alone. 'Ihe finindicate that eicosanoids may be important mediators of competence and progression hctor activities that may determine the effects of SP on fibroblast proliferation in a cytokine network. Wth respect to the question how the carcinogenic metalcadmium might stimulate cell proliferation and tumor growth, mechanisms of immediate early gene induction are most pertinent. Hence we have characterized the conditions of protooncogene induction by Cd2+ in PC12 cells at both the transcriptional and the translational level. Northem Blots showed that 5 pM Cd2+ caused a transient formation of c-fos mRNA, at which full induction is achieved after 30 min and the level declines within 2 h. Since Cd2+ may inhibit protein synthesis on our experimental conditions, we wished to make sure that the protooncogene induction really resulted in the formation of the protein product. Western Blots demonstrated the stimulation of c-Fos protein synthesis by low concentrations of Cd2+. A dose of 0.5 pM sufficed to induce the protein and with 1 pM a maximum induction was already achieved. The Fos protein appeared rapidly, with a maximum expression at I h. The protein level declined within 6-8 hours. Cd2+-stimulated expression of the Fos protooncoprotein was inhibited in the presence of the PKC-Inhibitor Bisindolylmaleimide, indicating a PKCdependent mechanism of heavy-metal induced prote oncogene expression. The nucleosome structure of eukaryotic chromatin is a barrier to transcription which must be unravelled to permit access of transcriptional activation proteins and RNA polymerases. The SWVSNF complex, an integral component of the RNA pol I1 holoenzyme, can disrupt nu...