INDUCTION OF METALLOTHIONEIN MESSENGER-RNA AND PROTEIN IN PRIMARY MURINE NEURON CULTURES

Metallothionein (MT)-I and -II are present in most if not all cells of the body and are readily inducible both in vivo and in vitro. MT-III is present only in brain, primarily in neurons, and is seemingly not i nducible. Because it is difficult to determine whether inducing agents reach neurons (in vivo) in sufficient concentrations, it was of inter est to directly test the inducibility of MT-III in vitro. A further ob jective was to examine the inducibility of MT-I and -II so that compar isons could be made to responses in glial cells. Cultures were establi shed from fetal (Days 14-17) CF-1 mice. Cells were treated after they achieved a mature phenotype (around 6 days) with various concentration s of the following MT inducers: dexamethasone (Dex), cadmium (Cd), zin c (Zn), or inorganic mercury (Hg). MT protein was quantified by the Cd -hemoglobin assay at 24, 48, 72, 96, and 120 hr. All inducers produced significant increases in MT protein between 24 and 96 hr. MT protein increased in a dose-dependent manner, and maximum (up to 6-fold over c ontrols) increases were seen at 3, 30, 100, and 1000 mu M for Cd, Hg, Zn, and Dex, respectively. The expression of MT-I, -II, and -III mRNA was examined by dot-blot analysis 6 hr following the addition of induc ers at concentrations known to maximally stimulate the induction of MT protein. Zinc and Cd produced 2.5 to 3.5-fold increases in MT-I and - II mRNA, whereas Dex and Hg produced 1.5- to 2.5-fold increases. Howev er, all inducers assayed decreased MT-III mRNA about 30-60%. The prese nt results indicate that MT-I and -II are inducible in neurons as they are in astrocytes, but the basal level and induced level of MT protei n is about one-third in neurons as in astrocytes. Unlike MTI and -II, MT-III is apparently not inducible under the present experimental cond itions. (C) 1996 Academic Press, Inc.