Nitric oxide synthase in critically ischaemic muscle and alterations in isoform expression during revascularization surgery.
BACKGROUND
Dysfunction of the nitric oxide pathway is implicated in peripheral arterial disease. Nitric oxide synthase (NOS) isoforms and NOS activity were studied in muscle from patients with critical leg ischaemia (CLI). Alterations in NOS during revascularization surgery were also assessed.


METHODS
Muscle biopsies were taken from patients with CLI undergoing amputation and also from patients undergoing femorodistal bypass at the start of surgery, after arterial clamping and following reperfusion. The presence of NOS within muscle sections was confirmed using reduced nicotinamide adenine dinucleotide phosphate diaphorase histochemistry. NOS isoform distribution was studied by immunohistochemistry. NOS mRNA and protein levels were measured using real-time reverse transcriptase-polymerase chain reaction and western blotting. NOS activity was assessed with the citrulline assay.


RESULTS
All three NOS isoforms were found in muscle, associated with muscle fibres and microvessels. NOS I and III protein expression was increased in CLI (P = 0.041). During revascularization, further ischaemia and reperfusion led to a rise in NOS III protein levels (P = 0.008). NOS activity was unchanged.


CONCLUSIONS
Alterations in NOS I and III occurred in muscle from patients with CLI and further changes occurred during bypass surgery.
Tsui
J C S
JC
Department of Surgery, Royal Free and University College Medical School, London, UK. jcstsui@hotmail.com
Baker
D M
DM
Shaw
S G
SG
Shi-Wen
X
X
Dashwood
M R
MR
D000368:Aged; D000369:Aged, 80 and over; D001706:Biopsy; D015153:Blotting, Western; D001026:Coronary Artery Bypass; D003324:Coronary Artery Disease / Q000201:enzymology*; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D007511:Ischemia / Q000201:enzymology*; D007527:Isoenzymes / Q000378:metabolism; D007866:Leg / Q000098:blood supply*; D008297:Male; D008875:Middle Aged; D018482:Muscle, Skeletal / Q000098:blood supply* / Q000201:enzymology / Q000473:pathology; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D007527:Isoenzymes; D019001:Nitric Oxide Synthase