High-glucose-induced regulation of intracellular ANG II synthesis and nuclear redistribution in cardiac myocytes

The prevailing paradigm is that cardiac ANG II is synthesized in the extracellular space from components of the circulating and/or local renin-angiotensin system. The recent discovery of intracrine effects of ANG II led us to determine whether ANG II is synthesized intracellularly in neonatal rat ventricular myocytes (NRVM). NRVM, incubated in serum-free medium, were exposed to isoproterenol or high glucose in the absence or presence of candesartan, which was used to prevent angiotensin type 1 (AT(1)) receptor-mediated internalization of ANG II. ANG II was measured in cell lysates and the culture medium, which represented intra- and extracellularly synthesized ANG II, respectively. Isoproterenol increased ANG II concentration in cell lysates and medium of NRVM in the absence or presence of candesartan. High glucose markedly increased ANG II synthesis only in cell lysates in the absence and presence of candesartan. Western analysis showed increased intracellular levels of angiotensinogen, renin, and chymase in high-glucose-exposed cells. Confocal immunofluorocytometry confirmed the presence of ANG II in the cytoplasm and nucleus of high-glucose-exposed NRVM and along the actin filaments in isoproterenol-exposed cells. ANG II synthesis was dependent on renin and chymase in high-glucose-exposed cells and on renin and angiotensin-converting enzyme in isoproterenol-exposed cells. In summary, the site of ANG II synthesis, intracellular localization, and the synthetic pathway in NRVM are stimulus dependent. Significantly, NRVM synthesized and retained ANG II intracellularly, which redistributed to the nucleus under high-glucose conditions, suggesting a role for an intracrine mechanism in diabetic conditions.