Myristoylated Peptide Inhibitor Research Articles

Protein Kinase A-mediated Phosphorylation Modulates Cytochrome c Oxidase Function and Augments Hypoxia and Myocardial Ischemia-related Injury

We have investigated the effects of hypoxia and myocardial ischemia/reperfusion on the structure and function of cytochrome c oxidase (CcO). Hypoxia (0.1% O(2) for 10 h) and cAMP-mediated inhibition of CcO activity were accompanied by hyperphosphorylation of subunits I, IVi1, and Vb and markedly increased reactive O(2) species production by the enzyme complex in an in vitro system that uses reduced cytochrome c as an electron donor. Both subunit phosphorylation and enzyme activity were effectively reversed by 50 nm H89 or 50 nm myristoylated peptide inhibitor (MPI), specific inhibitors of protein kinase A, but not by inhibitors of protein kinase C. In rabbit hearts subjected to global and focal ischemia, CcO activity was inhibited in a time-dependent manner and was accompanied by hyperphosphorylation as in hypoxia. Additionally, CcO activity and subunit phosphorylation in the ischemic heart were nearly completely reversed by H89 or MPI added to the perfusion medium. Hyperphosphorylation of subunits I, IVi1, and Vb was accompanied by reduced subunit contents of the immunoprecipitated CcO complex. Most interestingly, both H89 and MPI added to the perfusion medium dramatically reduced the ischemia/reperfusion injury to the myocardial tissue. Our results pointed to an exciting possibility of using CcO activity modulators for controlling myocardial injury associated with ischemia and oxidative stress conditions.

Mesangial cell filamentous actin disassembly and hypocontractility in high glucose are mediated by PKC-ζ

In high glucose (HG), mesangial cells (MCs) lose their contractile response to endothelin-1 (ET-1) coincidently with filamentous (F)-actin disassembly. We postulated that these MC phenotypic changes are mediated by altered protein kinase C (PKC) isozyme activity, myosin light chain (MLC(20)) phosphorylation, or Ca(2+) signaling. MCs were growth arrested for 24 h in 0.5% fetal bovine serum (FBS)-DMEM in 5.6 (normal glucose; NG) or 30 mM glucose (high glucose; HG). In HG, the planar area was reduced [2,608 +/- 135 vs. 3,952 +/- 225 (SE) microm(2) in NG, P < 0.01, n = 31] with no contractile response to 0.1 microM ET-1. Mannitol did not affect cell size or ET-1 response. Confocal imaging of fluo 3- loaded cells revealed that the peak intensity of ET-1-induced Ca(2+) signaling was not altered in HG vs. NG. Immunoblotting of phosphorylated MLC(20) showed that HG increased mono- and decreased unphosphorylated MLC(20) (42 +/- 16 and 49 +/- 15 vs. 13 +/- 3 and 80 +/- 4% of total in NG, P < 0.05, n = 3), but the peak phosphorylation responses to ET-1 were identical in NG and HG. ET-1 stimulated translocation of PKC-delta and -epsilon from cytosolic to membrane and particulate fractions identically in NG and HG but did not cause PKC-zeta translocation. In HG, membrane accumulation of PKC-zeta was observed. Membrane PKC-zeta activity measured by immunoprecipitation and (32)P phosphorylation of PKC-epsilon pseudosubstrate peptide was 190 +/- 18% of NG (P < 0.01, n = 4), which was completely inhibited by pretreatment with a myristoylated peptide inhibitor (ZI). In HG, pretreatment with ZI for 24 h restored normal MC size and contractile and F-actin disassembly responses to ET-1. In conclusion, in HG, decreased MC size is due to decreased F-actin assembly, and loss of contractile response to ET-1 occurs in the presence of normal Ca(2+) signaling and normal MLC(20) phosphorylation. In HG, altered F-actin and contractile functions in MCs are mediated by PKC-zeta.