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 20h. 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 6 h after LPS injection with recovery to basal levels at 20h. Significant down-regulation of e and nNOS protein levels (p < 0.01) and calcium-dependent activity (p < 0.05) were detected at 20h. Serum TNF-alpha increased after 6 and 20h (p < 0.05), whereas NO metabolites rose only after 20h (p < 0.0001). The diastolic calcium increased 6h 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. (c) 2004 Elsevier Ltd. All rights reserved.