Pulmonary Arteries Show Differences in the Effects of Angiotensin II Stimulation of Mitochondrial Superoxide on Regulation of Heme Biosynthesis and Soluble Guanylate Cyclase Expression

Abstract

It is well established that Angiotensin II (AngII) increases mitochondrial superoxide levels through the AT1 receptor. Ferrochelatase (FECH), the mitochondrial heme‐generating enzyme, has an iron‐sulfur cluster required for its stability and activity, which could potentially be disrupted by superoxide. We have observed that 24 hr organoid culture of bovine coronary arteries (BCAs) with AngII caused a depletion of mitochondrial superoxide dismutase‐2 (SOD‐2), FECH and soluble guanylate cyclase (sGC), as well as an increase in deficiency in heme (detected by depletion of cytochrome oxidase subunit‐4 (COx4), but not catalase). The heme precursor δ‐aminolevulinic acid (ALA) prevents these effects of AngII through a mechanism that appears to involve promoting protoporphyrin IX (PpIX) accumulation and its stimulation of sGC/cGMP‐activation of protein kinase G (PKG). Thus, in this study we investigated the effects of AngII on 24 hr organoid cultured endothelium‐denuded bovine pulmonary arteries (BPAs). AngII increased mitochondrial (MitoSox/HPLC) and extra‐mitochondrial/cytosolic superoxide (DHE/HPLC) and protoporphyrin IX (PpIX) levels (detected by its surface fluorescence), and it depleted SOD‐2 (western blot) and Cox4 (western blot) protein expression. However AngII did not deplete protein expression of FECH, sGC and catalase (western blot). ALA prevented AngII‐elicited increases in mitochondrial superoxide, and depletion of SOD‐2 and COx4. Furthermore, co‐organoid culture of BPAs with AngII in presence of cGMP‐independent activation of PKG (DHEA (dehydroepiandrosterone)) and cGMP‐mediated activation of PKG (8‐bromo‐cGMP) reverses the increases in mitochondrial and extra‐mitochondrial superoxide induced by AngII in BPA. The increase in PpIX and depletion of heme, but not FECH, suggests that increased mitochondrial superoxide in AngII‐treated BPA is impairing the availability of iron for heme generation from PpIX. Since agents which bind the heme site of sGC appear to protect it from depletion when the sGC heme is oxidized, the observed AngII‐elicited increase in PpIX in BPA may prevent the depletion of sGC. In contrast, AngII did not increase PpIX in BCA, and the depletion of both FECH and heme appears to be a more dominant factor in AngII‐treated BCA. While increased PKG stimulation by PpIX generation from ALA may function to attenuate AngII‐induced increases in mitochondrial superoxide and depletion of heme in both BPA and BCA, and the increase in PpIX observed in BPA treated with AngII may function to prevent the depletion of sGC, and perhaps FECH.Support or Funding InformationSupported by NIH grant: R01HL115124