Isolated rat neonatal cardiac myocytes were subjected to immersion in hypoxic (PO2 < 2 mm Hg),
glucose-free
Tyrode's solution for 5 h followed by concomitant reoxygenation and staining with the membrane-impermeant fluorophore,
propidium iodide, in normoxic (PO2 > 150 mm Hg),
serum-free culture media for 15 min in order to assess sarcolemmal damage indicative of myocyte viability due to
hypoxia/reoxygenation injury. Prior to hypoxic exposure, cells were pretreated for 90 min with the
angiotensin-converting enzyme inhibitor cyclopenta[b]
pyrrole-2-
carboxylic acid, 1-[2-[(1-carboxy-3-phenylpropyl)amino]-l-oxopropyl]octahydro-[2S-[1[R* (R*)]2 alpha, 3a beta, 6a beta]] (
ramiprilat), concomitantly with
ramiprilat and H-D-
Arg-Arg-Pro-
Hyp-Gly-Thi-Ser-D-
Tic-Oic-Arg-
OH (
bradykinin B2 receptor antagonist
HOE 140), the bioactive
peptide Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (
bradykinin) or concomitantly with
bradykinin and
HOE 140.
Hypoxia/reoxygenation injury to untreated control cardiac myocytes was characterized by a significant loss of sarcolemmal integrity measured at 75 +/- 4% of total cell fluorescence (mean +/- S.E., n = 42 cultures). Compared to
propidium iodide staining of the above untreated control myocytes, those pretreated with 30 or 100 microM
ramiprilat showed a significant reduction of
propidium iodide staining to 45 +/- 9% and 40 +/- 8% (n = 9, P < 0.05) of untreated controls, respectively. Pretreatment with the protective concentrations of
ramiprilat concomitant with 10 microM
HOE 140 abolished the significant reduction in
propidium iodide staining observed with
ramiprilat alone. Similarly, pretreatment with 10 or 100 nM
bradykinin significantly reduced
propidium iodide staining to 35 +/- 5% and 60 +/- 10% (n = 6, P < 0.05) of the untreated hypoxic controls, respectively. In addition, concomitant pretreatment with protective concentrations of
bradykinin and 10 microM
HOE 140 also abolished the significant reduction in
propidium iodide staining observed with
bradykinin alone. The results indicate that the
angiotensin-converting enzyme inhibitor ramiprilat has a protective effect on isolated cardiac myocytes exposed to
hypoxia/reoxygenation and that this effect is most likely related to a local action of
bradykinin on the cardiac myocyte via the activation of the
kinin B2 receptor.