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Immunofluorescence microscopy to assess enzymes controlling nitric oxide availability and microvascular blood flow in muscle

journal contribution
posted on 2012-10-01, 00:00 authored by M Cocks, S O Shepherd, Chris ShawChris Shaw, J Achten, M L Costa, A J M Wagenmakers
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.

History

Journal

Microcirculation

Volume

19

Issue

7

Pagination

642 - 651

Publisher

Wiley

Location

Chichester, Eng.

ISSN

1073-9688

eISSN

1549-8719

Language

eng

Publication classification

C Journal article; C1.1 Refereed article in a scholarly journal

Copyright notice

2012, Wiley