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The effects of chronic pharmacological modulation of L‐type Ca2+ channel activity on the cell surface expression of Na+ channels were examined in GH3 cells. Prolonged inhibition (4–5 days) of L‐channels with nimodipine caused a 50–60 % decrease in the peak amplitude of whole‐cell Na+ currents recorded with the patch‐clamp technique. On the contrary, prolonged exposure to the L‐channel agonist Bay K 8644 induced an ≈2.5‐fold increase in peak Na+ current. In both cases, there were only minor changes in cell capacitance and no significant changes in Na+ channel gating properties. Measurements of the specific binding of radiolabelled saxitoxin to intact cells showed that nimodipine treatment reduced the number of cell surface Na+ channels, whereas treatment with Bay K 8664 produced the opposite effect. The dual regulation of Na+ channel abundance explained the mentioned changes in Na+ current amplitude. Plasma membrane Na+ channels had a half‐life of ≈17 h both in control cells and in cells treated with Bay K 8644, as estimated from the rate of decay of peak Na+ current after inhibition of protein synthesis with cycloheximide. Actinomycin D, an inhibitor of gene transcription, and also cycloheximide, occluded the stimulatory effect of Bay K 8644 on Na+ current density when measured over a 24 h period. These findings indicate that the entry of Ca2+ through L‐type channels influences in a positive way the number of functional Na+ channels in GH3 cells, and suggest that Ca2+ influx stimulates either Na+ channel gene expression or the expression of a regulatory protein that promotes translocation of pre‐assembled Na+ channels into the plasma membrane.

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In clonal pituitary GH 3 cells, spontaneous action potentials drive the opening of Ca v 1 (L-type) channels, leading to Ca 2+ transients that are coupled to prolactin gene transcription. Nerve growth factor (NGF) has been shown to stimulate prolactin synthesis by GH 3 cells, but the underlying mechanisms are unknown. Here we studied whether NGF influences prolactin gene expression and Ca 2+ currents. By using RT-PCR, NGF (50 ng ml −1 ) was found to augment prolactin mRNA levels by ∼80% when applied to GH 3 cells for 3 days. A parallel change in the prolactin content was detected by Western blotting. Both NGF-induced responses were mimicked by an agonist (Bay K 8644) and prevented by a blocker (nimodipine) of L-type channels. In whole-cell patch-clamp experiments, NGF enhanced the L-type Ca 2+ current by ∼2-fold within 60 min. This effect reversed quickly upon growth factor withdrawal, but was maintained for days in the continued presence of NGF. In addition, chronic treatment (≥ 24 h) with NGF amplified the T-type current, which flows through Ca v 3 channels and is thought to support pacemaking activity. Thus, NGF probably increases the amount of Ca 2+ that enters per action potential and may also induce a late increase in spike frequency. MC192, a specific antibody for the p75 neurotrophin receptor, but not tyrosine kinase inhibitors (K252a and lavendustin A), blocked the effects of NGF on Ca 2+ currents. Overall, the results indicate that NGF activates the p75 receptor to cause a prolonged increase in Ca 2+ influx through L-type channels, which in turn up-regulates the prolactin mRNA.

It has been reported that the main function of tau protein is to stabilize microtubules and promote the movement of organelles through the axon in neurons. In Alzheimer's disease, tau protein is the major constituent of the paired helical filament, and it undergoes post-translational modifications including hyperphosphorylation and truncation. Whether other functions of tau protein are involved in Alzheimer's disease is less clear. We used SH-SY5Y human neuroblastoma cells as an in vitro model to further study the functions of tau protein. We detected phosphorylated tau protein as small dense dots in the cell nucleus, which strongly colocalize with intranuclear speckle structures that were also labelled with an antibody to SC35, a protein involved in nuclear RNA splicing. We have shown further that tau protein, phosphorylated at the sites recognized by pT231, TG-3, and AD2 antibodies, is closely associated with cell division. Different functions may be characteristic of phosphorylation at specific sites. Our findings suggest that the presence of tau protein is involved in separation of sister chromatids in anaphase, and that tau protein also participates in maintaining the integrity of the DNA (pT231, prophase) and chromosomes during cell division (TG-3).

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