Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 103, Issue 12, Pages 4759-4764Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0506589103
Keywords
mitochondria; L-arginine pools; spatial confinement
Categories
Funding
- NCI NIH HHS [CA 00405] Funding Source: Medline
- NHLBI NIH HHS [R01 HL065455, HL 065455] Funding Source: Medline
- NIA NIH HHS [R01 AG025017, R01 AG 021523, R01 AG021523, AG 025017] Funding Source: Medline
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Cardiac myocytes contain two constitutive NO synthase (NOS) isoforms with distinct spatial locations, which allows for isoform-specific regulation. One regulatory mechanism for NOS is substrate (L-arginine) bioavailability. We tested the hypothesis that arginase (Arg), which metabolizes L-arginine, constrains NOS activity in the cardiac myocyte in an isoform-specific manner. Arg activity was detected in both rat heart homogenates and isolated myocytes. Although both Arg I and II mRNA and protein were present in whole heart, Arg 11 alone was found in isolated myocytes. Arg inhibition with S-(2-boronoethyl)-L-cysteine (BEC) augmented Ca2+-dependent NOS activity and NO production in myocytes, which did not depend on extracellular L-arginine. Arg II coimmunoprecipited with NOS1 but not NOS3. isolation of myocyte mitochondrial fractions in combination with immuno-electron microscopy demonstrates that Arg II is confined primarily to the mitochondria. Because NOS1 positively modulates myocardial contractility, we determined whether Arg inhibition would increase basal myocardial contractility. Consistent with our hypothesis, Arg inhibition increased basal contractility in isolated myocytes by a NOS-dependent mechanism. Both the Arg inhibitors N-hydroxynor-L-arginine and BEC dose-dependently increased basal contractility in rat myocytes, which was inhibited by both nonspecific and NOS1-specific NOS inhibitors N-G-nitro-L-arginine methyl ester and S-methyl-L-thiocitrulline, respectively. Also, BEC increased contractility in isolated myocytes from WT and NOS3 but not NOS1 knockout mice. We conclude that mitochondrial Arg 11 negatively regulates NOS1 activity, most likely by limiting substrate availability in its microdomain. These findings have implications for therapy in pathophysiologic states such as aging and heart failure in which myocardial NO signaling is disrupted.
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