Immunofluorescence microscopy to assess enzymes controlling nitric oxide availability and microvascular blood flow in muscle.
OBJECTIVE
The net production of NO by the muscle microvascular endothelium is a key regulator of muscle microvascular blood flow. Here, we describe the development of a method to quantify the protein content and phosphorylation of endothelial NO synthase (eNOS content and eNOS ser(1177) phosphorylation) and NAD(P)H oxidase expression.


METHODS
Human muscle cryosections were stained using antibodies targeting eNOS, p-eNOS ser(1177) and NOX2 in combination with markers of the endothelium and the sarcolemma. Quantitation was achieved by analyzing fluorescence intensity within the area stained positive for the microvascular endothelium. Analysis was performed in duplicate and repeated five times to investigate CV. In addition, eight healthy males (age 21 ± 1 year, BMI 24.4 ± 1.0 kg/m(2)) completed one hour of cycling exercise at ~65%VO(2max) . Muscle biopsies were taken from the m. vastus lateralis before and immediately after exercise and analyzed using the new methods.


RESULTS
The CV of all methods was between 6.5 and 9.5%. Acute exercise increased eNOS serine(1177) phosphorylation (fold change 1.29 ± 0.05, p < 0.05).


CONCLUSIONS
These novel methodologies will allow direct investigations of the molecular mechanisms underpinning the microvascular responses to insulin and exercise, the impairments that occur in sedentary, obese and elderly individuals and the effect of lifestyle interventions.
Exercise Metabolism Research Group, School of Sport and Exercise Sciences, University of Birmingham, Edgbaston, Birmingham, UK.
Matthew
M
Cocks
Sam O
SO
Shepherd
Christopher S
CS
Shaw
Juul
J
Achten
Matthew L
ML
Costa
Anton J M
AJ
Wagenmakers
D000328:Adult; D000818:Animals; D001783:Blood Flow Velocity / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006801:Humans; D008297:Male; D008833:Microcirculation / Q000502:physiology*; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D019255:NADPH Oxidases / Q000096:biosynthesis*; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000096:biosynthesis*; D010766:Phosphorylation / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar
D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D019255:NADPH Oxidases