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

Cocks, Matthew, Shepherd, Sam O., Shaw, Christopher S., Achten, Juul, Costa, Matthew L. and Wagenmakers, Anton J.M. 2012, Immunofluorescence microscopy to assess enzymes controlling nitric oxide availability and microvascular blood flow in muscle, Microcirculation, vol. 19, no. 7, pp. 642-651, doi: 10.1111/j.1549-8719.2012.00199.x.

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Title Immunofluorescence microscopy to assess enzymes controlling nitric oxide availability and microvascular blood flow in muscle
Author(s) Cocks, Matthew
Shepherd, Sam O.
Shaw, Christopher S.ORCID iD for Shaw, Christopher S. orcid.org/0000-0003-1499-0220
Achten, Juul
Costa, Matthew L.
Wagenmakers, Anton J.M.
Journal name Microcirculation
Volume number 19
Issue number 7
Start page 642
End page 651
Total pages 10
Publisher Wiley
Place of publication Chichester, Eng.
Publication date 2012-10
ISSN 1073-9688
1549-8719
Keyword(s) eNOS
NAD(P)H oxidase
muscle microvascular endothelial function
immunofluorescence microscopy
Adult
Animals
Blood Flow Velocity
Endothelium, Vascular
Gene Expression Regulation, Enzymologic
Humans
Male
Microcirculation
Microscopy, Fluorescence
Middle Aged
Nitric Oxide
Nitric Oxide Synthase Type III
Phosphorylation
Rats
Rats, Wistar
Summary 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 ser1177 phosphorylation) and NAD(P)H oxidase expression.

METHODS: Human muscle cryosections were stained using antibodies targeting eNOS, p-eNOS ser1177 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/m2) completed one hour of cycling exercise at ~65%VO2max. 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 serine1177 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.
Language eng
DOI 10.1111/j.1549-8719.2012.00199.x
Field of Research 111699 Medical Physiology not elsewhere classified
Socio Economic Objective 970111 Expanding Knowledge in the Medical and Health Sciences
HERDC Research category C1.1 Refereed article in a scholarly journal
ERA Research output type C Journal article
Copyright notice ©2012, Wiley
Persistent URL http://hdl.handle.net/10536/DRO/DU:30089570

Document type: Journal Article
Collections: Faculty of Health
School of Exercise and Nutrition Sciences
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