Abstract
Heme oxygenase-1 knockout, Hmox1(−/−), mice exhibit exacerbated vascular lesions after ischemia-reperfusion and mechanical injury. Surprisingly, we found no studies that reported contractile responses and sensitivity to vasorelaxants in Hmox1(−/−) mice. The contractile responses [superior mesenteric arteries (SMA), from female Hmox1(−/−) mice] exhibited increased sensitivity to phenylephrine (p < 0.001). Cumulative addition of acetylcholine relaxed SMA, with the residual contraction remaining 2 times higher in Hmox1(−/−) mice (p < 0.001). Sodium nitroprusside (SNP, an NO donor) and 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole [YC-1; acts directly on soluble guanylate cyclase (sGC)] led to further relaxation, yet the residual contraction remained 2 to 3 times higher in Hmox1(−/−) than Hmox1(+/+) mice (p < 0.001). Branches from Hmox1(−/−) mesenteric and renal arteries also showed reduced relaxation (p < 0.025). Relaxation of SMA was measured to 4-({(4-carboxybutyl) [2-(5-fluoro-2-{[4′-(trifluoromethyl) biphenyl-4-yl] methoxy}phenyl)ethyl]amino}benzoic acid (BAY 60-2770), which is a more effective activator of oxidized/heme-free sGC; and to 5-cyclopropyl-2-{1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl}-pyrimidin-4-ylamine (BAY 41-2272), a more effective stimulator of reduced sGC. Hmox1(−/−) arteries were 15 times more sensitive to BAY 60-2770 (p < 0.025) than were Hmox1(+/+) arteries. Pretreatment with 1H-[1,2,4]oxadiazolo[3,4-a]quinoxalin-1-one (ODQ), an oxidizer of sGC, predictably shifted the BAY 60-2770 response of Hmox1(+/+) to the left (p < 0.01) and BAY 41-2272 response to the right (p < 0.01). ODQ had little effect on the responses of Hmox1(−/−) arteries, indicating that much of sGC was oxidized/heme-free. Western analyses of sGC in SMA indicated that both α1and β1 subunit levels were reduced to <50% of Hmox1(+/+) level (p < 0.025). These findings support the hypothesis that the antioxidant function of Hmox1 plays a significant role in the maintenance of sGC in a reduced state, which is resistant to degradation and is sensitive to NO. This function may be especially important in reducing vascular damage during ischemia-reperfusion injury.
Footnotes
This work was supported in part by the National Institutes of Health National Heart, Lung, and Blood Institute [Grants R01-HL59976 (to W.D.), R01-HL82816 (to R.J.K.)] and the National Institutes of Health National Institute on Alcohol Abuse and Alcoholism [Grant R01-AA14945 (to R.J.K.)].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.169755.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- Hmox1
- heme oxygenase-1
- sGC
- soluble guanylate cyclase
- GK
- protein kinase G
- ZnPPIX
- zinc protoporphyrin-IX
- bp
- base pair(s)
- SMA
- superior mesenteric artery(ies)
- RA
- renal artery branch
- FA
- femoral artery
- PS
- physiological solution
- PE
- phenylephrine
- SNP
- sodium nitroprusside
- ACh
- acetylcholine
- YC-1
- 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole
- BAY 41-2272
- 5-cyclopropyl-2-{1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl}-pyrimidin-4-ylamine
- 5-HT
- 5-hydroxytryptamine
- Ado
- adenosine
- ODQ
- 1H-[1,2,4]oxadiazolo[3,4-a]quinoxalin-1-one
- BAY 60-2770
- 4-({(4-carboxybutyl) [2-(5-fluoro-2-{[4′-(trifluoromethyl)biphenyl-4-yl]methoxy}phenyl)ethyl]amino}benzoic acid
- eNOS
- endothelial nitric-oxide synthase
- BAY 58–2667
- 4-[(4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino)methyl[benzoic] acid.
- Received April 27, 2010.
- Accepted July 2, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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