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.
Comini
Laura
L
Fondazione Salvatore Maugeri IRCCS, Center of Cardiovascular Pathophysiology, Via Pinidolo 23, 25064 Gussago, Brescia, Italy. lcomini@fsm.it
Boraso
Antonella
A
Bachetti
Tiziana
T
Bernocchi
Palmira
P
Pasini
Evasio
E
Bastianon
Daniela
D
Curello
Salvatore
S
Terracciano
Cesare Maria
CM
Ceconi
Claudio
C
Ferrari
Roberto
R
D000818:Animals; D002118:Calcium / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D032383:Myocytes, Cardiac / Q000201:enzymology / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012772:Shock, Septic / Q000201:enzymology / Q000378:metabolism*
D007527:Isoenzymes; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496322:Nos1 protein, rat; C496318:Nos2 protein, rat; C496316:Nos3 protein, rat; D002118:Calcium