Effects of endotoxic shock on neuronal NOS and calcium transients in rat cardiac myocytes.

OBJECTIVE

The effects of endotoxic shock on transcriptional and translational pattern of nitric oxide synthase isoforms (NOSs) and cytoplasmic calcium were investigated.

METHODS

Male SD rats injected with lipopolysaccharides or saline were sacrificed after 6 and 20 h. Cardiac myocytes were enzimatically isolated from the excised hearts and evaluated for: (1) expression of constitutive (e and n) and inducible (i) NOSs by RT-PCR; (2) NOSs protein levels by Western blot, enzymatic activities by a radioimmunometric assay and nitric oxide metabolites by spectrophotometry; (3) calcium transients by Indo-1 fluorescence.

RESULTS

Increase in iNOS mRNA, and decrease in e and nNOS mRNAs were observed in cardiac myocytes isolated 6h after LPS injection with recovery to basal levels at 20 h. Significant down-regulation of e and nNOS protein levels (p < 0.01) and calcium-dependent activity (p < 0.05) were detected at 20 h. Serum TNF-alpha increased after 6 and 20 h (p < 0.05), whereas NO metabolites rose only after 20 h (p < 0.0001). The diastolic calcium increased 6 h from LPS injection (p < 0.0001) and remained significantly higher after 20 h. Calcium transients amplitude was not affected by LPS injection.

CONCLUSIONS

Endotoxic shock stimulates iNOS and down-regulates expression of nNOS in purified cardiac myocytes, but endogenous NO production does not likely affect calcium transients.