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In all cell types, the maintenance of normal cell volume is an essential homeostatic function. Relatively little is known about the induction of apoptosis by hyperosmotic stress and its molecular mechanism in terminally differentiated cardiac myocytes. We compared the apoptotic response of cultured neonatal rat cardiomyoctes to hyperosmotic stress by sorbitol (SOR) with those induced by doxorubicin (Doxo) or angiotensin II (Ang II). We also examined the apoptotic-signaling pathway stimulated by the hyperosmotic stress. Apoptosis was assessed by the observation of: (1) cell viability, (2) DNA fragmentation detected by the TUNEL method and by agarose gel electrophoresis, and (3) poly(ADP-ribose)polymerase (PARP) degradation, and Bcl-XS and Bcl-XL levels by Western blot analysis. Exposure of cardiomyocytes to 0.3 M SOR for 24 h resulted in decreased cell viability and increased generation of oligosomal DNA fragments (2.5-fold of controls). At this time, 83 +/- 5% of SOR-treated myocytes were TUNEL-positive (vs 23.7 +/- 6.8% in controls; P<0.01). PARP levels also decreased by approximately 42% when cardiac myocytes were exposed to SOR. Hyperosmotic stress induced a more rapid and stronger apoptotic response in cardiomyocytes than Doxo or Ang II. In addition, SOR increased 3.2-fold Bcl-XS proapoptotic protein without changes in Bcl-XL antiapoptotic protein levels and in the p53-transactivating activity. Taken together, these results strongly suggest that hyperosmotic stress triggers cardiac myocyte apoptosis in a p53-independent manner, being earlier and stronger than apoptosis induced by Doxo and Ang II.

Abstract-Angiotensin I is a substrate for both ACE and for neutral endopeptidase 24.11 (NEP). We hypothesized that high ACE expression is related to low NEP activity. Accordingly, circulating and tissue NEP and ACE activities were measured by fluorometry in homozygous rats (F 0 and F 2 ) for the Lewis microsatellite allele (LL, low ACE) and for the Brown Norway microsatellite allele (BB, high ACE). Plasma, lung, and aortic ACE activities in F 0 and F 2 were higher in BB rats than in LL rats (PϽ0.01), whereas left ventricular ACE activity was similar in both genotypes. In contrast, NEP activity in the LL group was higher in the serum, aorta, and lungs in F 0 and F 2 homozygous (PϽ0.05 Key Words: angiotensin-converting enzyme Ⅲ angiotensin Ⅲ polymorphism Ⅲ neutral endopeptidase A ngiotensin I-converting enzyme plays an important role in the regulation of the renin angiotensin system (RAS) by hydrolyzing angiotensin (Ang) I to Ang II and degrading bradykinin to bradykinin-(1-7) and Ang-(1-7) to Ang-(1-5), both inactive peptides. In this way, ACE activity influences circulating and tissue levels of Ang II, contributes to regulate the vasculature tone, and may have some effects on cardiac and vascular mass and structure. 1 Neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) is a transmembrane zinc metalloendopeptidase, present at the surface of several tissues. 2 NEP also participates in the RAS, cleaving Ang I and Ang II. 3,4 The action of NEP on Ang I generates Ang-(1-7), an heptapeptide with vasodilator activity. The physiological effects of Ang-(1-7) are opposite those of Ang II and would favor a blood pressure-lowering effect under conditions of high Ang II activity. 3,4 In humans, the ACE gene insertion/deletion (I/D) polymorphism is due to the presence (insertion) or absence (deletion) of a 287-bp sequence in the intron 16 on the chromosome 17q23 5 and determines plasma ACE levels by 50%. The D allele is associated with increased ACE and possibly increased Ang II levels. 6,7 Recent evidence suggests that the DD genotype-with higher plasma ACE activity-is associated with higher risk of hypertension in men. 8 A similar polymorphism has been observed in normotensive rats, whereas homozygous rats for the Lou and Lewis microsatellite alleles have lower ACE activity than that of homozygous rats for the Brown Norway microsatellite allele (BB). 9 It remains unknown whether NEP activity-because of its interaction with Ang I and Ang II as well as with bradykinin-is modulated by ACE expression. Because higher ACE activity will decrease Ang I and bradykinin levels and increase Ang II levels, we hypothesized that an inverse relationship between ACE expression (genetically determined) and NEP activity might exist. To test this hypothesis, NEP activity was determined in serum and in different tissues from male Lewis homozygous (LL, genetically with low ACE activity) and BB rats (genetically with high ACE activity) in F 0 and in homozygous F 2. These rats were also characterized by their ACE genotype and circ...

Patients with acute myocardial infarction had increased levels of oxidative stress associated with a reduction in enzymatic antioxidant reserve. Administration of GIK solution did not improve these abnormalities among patients undergoing primary angioplasty.

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