Septic impairment of capillary blood flow requires nicotinamide adenine dinucleotide phosphate oxidase but not nitric oxide synthase and is rapidly reversed by ascorbate through an endothelial nitric oxide synthase-dependent mechanism.
OBJECTIVE
To determine the roles of nitric oxide synthase (NOS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the impairment of capillary blood flow in sepsis and in the reversal of this impairment by ascorbate.


METHODS
Prospective, controlled laboratory study.


METHODS
Animal laboratory in research institute.


METHODS
Adult male wild type (WT), neuronal nitric oxide synthase (nNOS)-/-, inducible NOS (iNOS)-/-, endothelial NOS (eNOS)-/-, and gp91phox-/- mice.


METHODS
Sepsis was induced by feces injection into peritoneum (FIP). A bolus of ascorbate or NADPH oxidase inhibitor apocynin was injected intravenously at 6 hrs post-FIP. Alternatively, NOS cofactor (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) or nitric oxide donor S-nitroso-N-acetylpenicillamine was superfused on the surface of the extensor digitorum longus muscle.


RESULTS
Capillary blood flow impairment and NOS activity in the extensor digitorum longus muscle were measured by intravital microscopy and by enzymatic assay, respectively. Sepsis at 6 hrs impaired flow in WT mice. Apocynin, and knockout of gp91phox but not of any NOS isoforms, rescued this impairment. Constitutive NOS activity was unaffected by sepsis, but it was abolished by nNOS knockout (iNOS activity was negligible in all mice). Ascorbate rapidly (10 mins) rescued impaired flow in WT, nNOS-/-, iNOS-/- but not eNOS-/- mice. Ascorbate also improved survival of WT mice after FIP. BH4 and SNAP rescued flow in WT mice, while BH4 failed to rescue it in eNOS-/- mice.


CONCLUSIONS
Capillary blood flow impairment in septic skeletal muscle requires NADPH oxidase but not NOS, and it is rapidly reversed by ascorbate and BH4 through an eNOS-dependent mechanism.
Tyml
Karel
K
Lawson Health Research Institute, The Centre for Critical Illness Research, Department of Medical Biophysics, University of Western Ontario, London, Canada. ktyml@lhsc.on.ca
Li
Fuyan
F
Wilson
John X
JX
D000098:Acetophenones / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001205:Ascorbic Acid / Q000494:pharmacology; D002196:Capillaries / Q000187:drug effects*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008833:Microcirculation; D018482:Muscle, Skeletal / Q000098:blood supply* / Q000378:metabolism; D009249:NADP / Q000494:pharmacology; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000378:metabolism / Q000502:physiology*; D018805:Sepsis / Q000378:metabolism*
D000098:Acetophenones; D000975:Antioxidants; D009249:NADP; C056165:acetovanillone; D019001:Nitric Oxide Synthase; D019255:NADPH Oxidases; D001205:Ascorbic Acid