Limits...
Activation of protease calpain by oxidized and glycated LDL increases the degradation of endothelial nitric oxide synthase.

Dong Y, Wu Y, Wu M, Wang S, Zhang J, Xie Z, Xu J, Song P, Wilson K, Zhao Z, Lyons T, Zou MH - J. Cell. Mol. Med. (2008)

Bottom Line: Neither eNOS reduction nor any of these other effects were observed in cells exposed to native LDL.Reduction of intracellular Ca(2+) levels abolished eNOS reduction by HOG-LDL, as did pharmacological or genetic through calcium channel blockers or calcium chelator BAPTA or inhibition of NAD(P)H oxidase (with apocynin) or inhibition of calpain (calpain 1-specific siRNA).Consistent with these results, HOG-LDL impaired acetylcholine-induced endothelium-dependent vasorelaxation of isolated mouse aortas, and pharmacological inhibition of calpain prevented this effect.

View Article: PubMed Central - PubMed

Affiliation: Harold Hamm Oklahoma Diabetes Center, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.

ABSTRACT
Oxidation and glycation of low-density lipoprotein (LDL) promote vascular injury in diabetes; however, the mechanisms underlying this effect remain poorly defined. The present study was conducted to determine the effects of 'heavily oxidized' glycated LDL (HOG-LDL) on endothelial nitric oxide synthase (eNOS) function. Exposure of bovine aortic endothelial cells with HOG-LDL reduced eNOS protein levels in a concentration- and time-dependent manner, without altering eNOS mRNA levels. Reduced eNOS protein levels were accompanied by an increase in intracellular Ca(2+), augmented production of reactive oxygen species (ROS) and induction of Ca(2+)-dependent calpain activity. Neither eNOS reduction nor any of these other effects were observed in cells exposed to native LDL. Reduction of intracellular Ca(2+) levels abolished eNOS reduction by HOG-LDL, as did pharmacological or genetic through calcium channel blockers or calcium chelator BAPTA or inhibition of NAD(P)H oxidase (with apocynin) or inhibition of calpain (calpain 1-specific siRNA). Consistent with these results, HOG-LDL impaired acetylcholine-induced endothelium-dependent vasorelaxation of isolated mouse aortas, and pharmacological inhibition of calpain prevented this effect. HOG-LDL may impair endothelial function by inducing calpain-mediated eNOS degradation in a ROS- and Ca(2+)-dependent manner.

Show MeSH

Related in: MedlinePlus

Pharmacological or genetic inhibition of calpain prevents reduction of eNOS by HOG-LDL. Western blot analysis of (A) total eNOS as well as (B) dimeric and monomeric eNOS in BAECs exposed to HOG-LDL in the presence or absence of the indicated concentration of the calpain inhibitor III, MDL28170. n= 3, *P < 0.01 versus control, #P < 0.01 versus HOG-LDL-treated groups. (C) Effect of the other calpain inhibitors, calpeptin (20 μM), ALLM (20 μM), ALLN (20 μM) or E-64 (15 μM), on total eNOS levels in HOG-LDL-exposed BAECs. *P < 0.01 versus untreated controls or n-LDL, #P < 0.01 versus HOG-LDL. (D) Western blot analysis of eNOS, and calpain 1 in HOG-LDL-stimulated HUVECs transfected with calpain 1 siRNA or scrambled siRNA for 48 hrs. *P < 0.01 versus control, #P < 0.01 versus HOG-LDL. The blot is a representative of four blots obtained from four separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2821526&req=5

fig03: Pharmacological or genetic inhibition of calpain prevents reduction of eNOS by HOG-LDL. Western blot analysis of (A) total eNOS as well as (B) dimeric and monomeric eNOS in BAECs exposed to HOG-LDL in the presence or absence of the indicated concentration of the calpain inhibitor III, MDL28170. n= 3, *P < 0.01 versus control, #P < 0.01 versus HOG-LDL-treated groups. (C) Effect of the other calpain inhibitors, calpeptin (20 μM), ALLM (20 μM), ALLN (20 μM) or E-64 (15 μM), on total eNOS levels in HOG-LDL-exposed BAECs. *P < 0.01 versus untreated controls or n-LDL, #P < 0.01 versus HOG-LDL. (D) Western blot analysis of eNOS, and calpain 1 in HOG-LDL-stimulated HUVECs transfected with calpain 1 siRNA or scrambled siRNA for 48 hrs. *P < 0.01 versus control, #P < 0.01 versus HOG-LDL. The blot is a representative of four blots obtained from four separate experiments.

Mentions: As HOG-LDL increased calpain activity and eNOS export to the cytoplasm, we determined if selective pharmacologic or genetic inhibition of calpains attenuated the reduction in eNOS elicited by HOG-LDL in BAECs. Calpain inhibitors alone did not alter the levels of total, dimeric or monomeric eNOS (data not shown). However, treatment of cells with calpain inhibitor III (MDL28170), calpeptin, ALLM, ALLN or E64 prior to HOG-LDL exposure prevented reduction of total, dimeric and monomeric eNOS (Fig. 3A–C). To exclude off-target effects of calpain inhibitors, we tested the effect of genetic calpain inhibition on eNOS reduction by HOG-LDL. As the siRNA against bovine calpain was not available, we performed these experiments on HUVECs, which, like BAECs, express both eNOS and calpain. Transfection of calpain-specific siRNA, but not control siRNA, reduced calpain protein levels by 60% in HUVECs (Fig. 3D). Calpain 1-specific siRNA partially prevented eNOS reduction by HOG-LDL, whereas control siRNA had no effect (Fig. 3D).


Activation of protease calpain by oxidized and glycated LDL increases the degradation of endothelial nitric oxide synthase.

Dong Y, Wu Y, Wu M, Wang S, Zhang J, Xie Z, Xu J, Song P, Wilson K, Zhao Z, Lyons T, Zou MH - J. Cell. Mol. Med. (2008)

Pharmacological or genetic inhibition of calpain prevents reduction of eNOS by HOG-LDL. Western blot analysis of (A) total eNOS as well as (B) dimeric and monomeric eNOS in BAECs exposed to HOG-LDL in the presence or absence of the indicated concentration of the calpain inhibitor III, MDL28170. n= 3, *P < 0.01 versus control, #P < 0.01 versus HOG-LDL-treated groups. (C) Effect of the other calpain inhibitors, calpeptin (20 μM), ALLM (20 μM), ALLN (20 μM) or E-64 (15 μM), on total eNOS levels in HOG-LDL-exposed BAECs. *P < 0.01 versus untreated controls or n-LDL, #P < 0.01 versus HOG-LDL. (D) Western blot analysis of eNOS, and calpain 1 in HOG-LDL-stimulated HUVECs transfected with calpain 1 siRNA or scrambled siRNA for 48 hrs. *P < 0.01 versus control, #P < 0.01 versus HOG-LDL. The blot is a representative of four blots obtained from four separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2821526&req=5

fig03: Pharmacological or genetic inhibition of calpain prevents reduction of eNOS by HOG-LDL. Western blot analysis of (A) total eNOS as well as (B) dimeric and monomeric eNOS in BAECs exposed to HOG-LDL in the presence or absence of the indicated concentration of the calpain inhibitor III, MDL28170. n= 3, *P < 0.01 versus control, #P < 0.01 versus HOG-LDL-treated groups. (C) Effect of the other calpain inhibitors, calpeptin (20 μM), ALLM (20 μM), ALLN (20 μM) or E-64 (15 μM), on total eNOS levels in HOG-LDL-exposed BAECs. *P < 0.01 versus untreated controls or n-LDL, #P < 0.01 versus HOG-LDL. (D) Western blot analysis of eNOS, and calpain 1 in HOG-LDL-stimulated HUVECs transfected with calpain 1 siRNA or scrambled siRNA for 48 hrs. *P < 0.01 versus control, #P < 0.01 versus HOG-LDL. The blot is a representative of four blots obtained from four separate experiments.
Mentions: As HOG-LDL increased calpain activity and eNOS export to the cytoplasm, we determined if selective pharmacologic or genetic inhibition of calpains attenuated the reduction in eNOS elicited by HOG-LDL in BAECs. Calpain inhibitors alone did not alter the levels of total, dimeric or monomeric eNOS (data not shown). However, treatment of cells with calpain inhibitor III (MDL28170), calpeptin, ALLM, ALLN or E64 prior to HOG-LDL exposure prevented reduction of total, dimeric and monomeric eNOS (Fig. 3A–C). To exclude off-target effects of calpain inhibitors, we tested the effect of genetic calpain inhibition on eNOS reduction by HOG-LDL. As the siRNA against bovine calpain was not available, we performed these experiments on HUVECs, which, like BAECs, express both eNOS and calpain. Transfection of calpain-specific siRNA, but not control siRNA, reduced calpain protein levels by 60% in HUVECs (Fig. 3D). Calpain 1-specific siRNA partially prevented eNOS reduction by HOG-LDL, whereas control siRNA had no effect (Fig. 3D).

Bottom Line: Neither eNOS reduction nor any of these other effects were observed in cells exposed to native LDL.Reduction of intracellular Ca(2+) levels abolished eNOS reduction by HOG-LDL, as did pharmacological or genetic through calcium channel blockers or calcium chelator BAPTA or inhibition of NAD(P)H oxidase (with apocynin) or inhibition of calpain (calpain 1-specific siRNA).Consistent with these results, HOG-LDL impaired acetylcholine-induced endothelium-dependent vasorelaxation of isolated mouse aortas, and pharmacological inhibition of calpain prevented this effect.

View Article: PubMed Central - PubMed

Affiliation: Harold Hamm Oklahoma Diabetes Center, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.

ABSTRACT
Oxidation and glycation of low-density lipoprotein (LDL) promote vascular injury in diabetes; however, the mechanisms underlying this effect remain poorly defined. The present study was conducted to determine the effects of 'heavily oxidized' glycated LDL (HOG-LDL) on endothelial nitric oxide synthase (eNOS) function. Exposure of bovine aortic endothelial cells with HOG-LDL reduced eNOS protein levels in a concentration- and time-dependent manner, without altering eNOS mRNA levels. Reduced eNOS protein levels were accompanied by an increase in intracellular Ca(2+), augmented production of reactive oxygen species (ROS) and induction of Ca(2+)-dependent calpain activity. Neither eNOS reduction nor any of these other effects were observed in cells exposed to native LDL. Reduction of intracellular Ca(2+) levels abolished eNOS reduction by HOG-LDL, as did pharmacological or genetic through calcium channel blockers or calcium chelator BAPTA or inhibition of NAD(P)H oxidase (with apocynin) or inhibition of calpain (calpain 1-specific siRNA). Consistent with these results, HOG-LDL impaired acetylcholine-induced endothelium-dependent vasorelaxation of isolated mouse aortas, and pharmacological inhibition of calpain prevented this effect. HOG-LDL may impair endothelial function by inducing calpain-mediated eNOS degradation in a ROS- and Ca(2+)-dependent manner.

Show MeSH
Related in: MedlinePlus