Transient oxidative stress in SH-SY5Y human neuroblastoma cells results in caspase dependent and independent cell death and tau proteolysis.

The effects of an oxidative insult on cell survival and tau metabolism were investigated in human neuroblastoma SH-SY5Y cells. In this treatment paradigm cells were exposed to the membrane permeant oxidant tert-butylhydroperoxide (tBHP) for 40 min, returned to fresh media and cell survival/death was monitored during the post-treatment period. Cell viability decreased significantly by 6 hr after tBHP exposure, and by 24 hr lactate dehydrogenase (LDH) release was 40.1 +/- 8.8% in tBHP treated cells compared to 8.1 +/- 4.7% in control cells. This oxidative stress paradigm also resulted in significant activation of caspase-3 by 2 hr post-treatment and nuclear apoptotic morphology. Furthermore, tBHP treatment also resulted in delayed tau proteolysis that was first evident 2 hr post-treatment. Treatment of the cells with the general caspase inhibitor Boc-Asp(OMe)-Fluoromethylketone (BAF) completely inhibited caspase-3 activation in response to tBHP, and delayed, but did not prevent cell death. BAF treatment also decreased tau proteolysis. In vitro, recombinant tau was readily proteolyzed by active recombinant caspase-3 into a stable breakdown product. Further tau in the cell lysates was cleaved by active recombinant caspase-3 at a rate, and to an extent similar to that observed for the well-established caspase-3 substrate poly(ADP-ribose)polymerase (PARP). These results suggest that oxidative stress-induced cell death occurs through both caspase-dependent and-independent pathways, and that tau is likely an in situ substrate of caspase-3.

Effect of streptomycin on wall-stress-induced arrhythmias in the working rat heart.

OBJECTIVE: To assess whether streptomycin, an inhibitor of mechano-sensitive cation channels, has an effect on arrhythmias-induced by an increase of ventricular wall stress in the rat heart. METHODS: The isolated working rat heart preparation was used. Arrhythmias were induced by increasing the afterload (i.e., aortic pressure) against which the left ventricle (LV) pumped for 20 s. This led to an increase of LV pressure, stretch of the LV and an increase in LV wall stress. The number of ventricular premature beats induced by each afterload step was compared in the absence and presence of streptomycin, a compound known to block mechano-sensitive cation channels in the heart. RESULTS: Perfusion with 200 microM streptomycin caused a significant reduction in wall-stress-induced arrhythmias. The effect of streptomycin on arrhythmias reached steady-state within 10 min of application. In the presence of streptomycin, arrhythmias elicited by a 40 mmHg afterload increase were reduced to 38% of control. Arrhythmias induced by an 80 mmHg afterload increase were reduced to 61% of control. Complex arrhythmias (ventricular tachycardia) induced by an afterload increase were also reduced in the presence of 200 microM streptomycin. There was no change in inotropic state with streptomycin, as assessed either by cardiac output or by maximum developed LV pressure. Streptomycin 50 microM (a typical therapeutic plasma concentration in patients) had no effect on wall-stress-induced arrhythmias. CONCLUSIONS: The results were inconsistent with streptomycin acting by modulating inositol phosphate production, or altering the level of intracellular calcium or inotropic state. The anti-arrhythmic effect of streptomycin appears more consistent with inhibition of mechano-sensitive cation channels, suggesting that these ion channels might be involved in causing wall-stress-induced arrhythmias.

Structure and myofiber-specific expression of the rat muscle regulatory gene MRF4.

We have cloned an 11.3-kb rat genomic DNA fragment encompassing the muscle regulatory factor 4 (MRF4) protein-coding sequence, 8.5 kb of 5'-flanking sequence, and 1.0 kb of 3'-flanking sequence. In order to study MRF4 gene expression, the rat myogenic cell line, L6J1-C, which expresses the endogenous MRF4 gene only in differentiated myofibers, was transfected stably with the full-length genomic clone and various 5' deletions. RNase protection assays demonstrated that MRF4 genes containing as little as 430 bp of 5'-flanking sequence exhibited an increase in expression as the cells differentiated into myofibers, indicating that elements responsible for fiber-specific expression are contained within this cloned DNA fragment. Similar up-regulation was observed with genes containing 1.5 kb of 5'-flanking sequence. Interestingly, MRF4 genes containing 5.0 kb and 8.5 kb of 5'-flanking sequence were up-regulated to even higher levels, suggesting that additional myofiber-specific regulatory elements located between 1.5 and 5.0 kb upstream from the coding region play a role in regulating the expression of this muscle-specific gene.