HYPOXIA INDUCED iNOS EXPRESSION IN RATS IS
AGE DEPENDENT A S Warner*, K M Saint, M A Arstall, J D
Horowitz The Queen Elizabeth Hospital, University of
Adelaide, SA Inducible nitric oxide synthase (iNOS)
expression has been reported in cardiac myocytes in myocardial infarction and
in hibernating myocardium but the mechanisms of induction remain unclear.
Previous studies show the expression of iNOS in cardiac myocytes in response to
hypoxia but others have yielded conflicting results. We investigated the effects
of hypoxia (1% oxygen) and anoxia (0% oxygen) on primary cultures of rat
cardiac myocytes. Methods: Primary cultures of rat
cardiac myocytes were grown from Day 1-3 Sprague-Dawley neonates, Day 7 neonates and from adults. Interleukin
1b
(IL-1b)-induced
iNOS expression was used as a positive control. Neonatal cultures were exposed
to hypoxia or anoxia for 48 hours while adult cultures were exposed for 6
hours. Total RNA was extracted using Trizol LS reagent (Life Technologies) and
analysed by RT PCR and by Northern Blotting using digoxigenin labeled RNA
probes (Roche). 18S was used as a comparison for loading differences. Results: Myocytes grown in
normoxic conditions showed no iNOS expression in Day 1-3 or Day 7 cultures.
iNOS expression was seen in all the IL-1b treated positive
controls. In Day 1-3 cultures, neither hypoxia nor anoxia resulted in any iNOS
mRNA detectable on RT PCR or Northern blotting. In contrast, iNOS expression
was seen in Day 7 neonatal rat cardiac myocyte cultures in response to both
hypoxia and anoxia on RT PCR and Northern analysis. In adult cells, hypoxia and
anoxia resulted in increased iNOS expression compared to normoxic controls. Conclusion: Hypoxia does induce iNOS expression in cardiac myocytes from
Day 7 neonatal and older rats, but not in Day 1-3 neonatal rats. Furthermore,
as Day 1-3 neonatal rat cardiac myocytes are able to express iNOS in response
to IL-1b,
it suggests that the mechanism of iNOS induction in response to hypoxia may be
cytokine independent.
