Experimental Physiology (1995), 80, 237-247
Pr-inted in Great Br-itain
THE EFFECT OF NG-NITRO-L-ARGININE METHYL
ESTER UPON HINDLIMB BLOOD FLOW RESPONSES
TO MUSCLE CONTRACTION IN THE
S. M. POUCHER
Cardiovascular and Metabolism Department, ZENECA Pharmaceuticals, Mereside, Alderley Park, Macclesfield,
Cheshire SKJO 4TG, UK
(MANUSCRIPT RECEIVED 8 JULY 1994, ACCEPTED 4 OCTOBER 1994)
The aim of the present experiment was to investigate the relative contribution of nitric oxide
produced in endothelial cells to functional and reactive hyperaemia in the hindlimb of
anaesthetized cats. Cats (2-5-3.4 kg) were anaesthetized with alphadalone-alphalaxone, and
breathed spontaneously following tracheotomy. Left hindlimb blood flow was measured with a
flow probe and hyperaemia responses were monitored following 10 s occlusion of the left
external iliac artery and during 20 min stimulation of the sciatic and femoral nerves at 3 Hz. This
was repeated following nitric oxide synthase inhibition with NG-nitro-L-arginine methyl ester
(L-NAME, 100 mg kg-', i.v.). Following L-NAME administration, baseline hindlimb blood flow
and arterial blood pressure were restored by infusion of sodium nitroprusside (range, 0-3-2-25 ,ug
kg-l min-', i.v.). Following arterial occlusion, L-NAME reduced the peak reactive hyperaemia
(6-5 ± 0-8 vs. 4.5 + 1.0 ml min-' kg-', P <0.05) and blood flow repayment (9-9 + 2-3
6-1 + 2-6 ml, P < 0-05) responses. In contrast, the total functional hyperaemia response during
hindlimb contraction was not altered (264-7 + 68-2 vs. 264-4 + 62-8 ml kg-', n.s.). The results of
the study suggest that the production of nitric oxide from endothelial cells does not contribute to
functional hyperaemia in contracting skeletal muscle, but plays a large role in reactive
hyperaemia. The results imply that flow-dependent dilatation of feed arteries is mediated by NO
in reactive hyperaemia.
The concept of flow-dependent vasodilatation of feed arteries was first proposed by Hilton
(1959) and is believed to be an essential response in the promotion and maintenance of
microcirculatory flow during hyperaemia (Segal, 1992). Endothelium-derived relaxing
factor (EDRF) has been demonstrated to be responsible for flow-induced iliac artery
dilatation in anaesthetized dogs (Snow, McAuliffe, Moors & Brownlie, 1994) and in hypoxic
vasodilatation in isolated guinea-pig hearts (Park, Rubin, Gross & Levi, 1992). If the flow-
dependent vasodilatation is an important component of the total vascular response in a
physiological setting, for example, either following a period of arterial occlusion or during
skeletal muscle contraction, then inhibition of the effects of EDRF would be expected to
reduce the observed blood flow responses.
The aim of the present experiment was to investigate the relative contribution of EDRF to
the metabolic stimulation of functional and reactive hyperaemia in the hindlimb of
anaesthetized cats. An understanding of the regulation of skeletal muscle blood flow may
lead to the development of drugs for treatment of ischaemic disease. In the present study
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S. M. POUCHER
nitric oxide synthase was inhibited using NG-nitro-L-arginine methyl ester (L-NAME). One
of the problems seen when using inhibitors of the enzyme, nitric oxide synthase, is the effect
upon resting organ blood flow and systemic blood pressure, which, if not accounted for, can
give misleading results. Therefore, in the current series ofexperiments the pressor and vaso-
constrictor effects ofL-NAME were controlled by infusion of sodium nitroprusside.
Aprevious communication of some of the results has been made (Poucher, 1993).
Cats (2.5-3-4 kg, n =7) were anaesthetized with alphadalone-alphalaxone (0-98 :038 w/v, Saffan,
10-5 + 0.3 mg kg-', I.v.). A tracheotomy was performed and the animals breathed spontaneously.
Anaesthesia was maintained throughout the whole of the experiment by the continuous infusion of
alphadalone-alphalaxone (0.3 + 0-05 mg kg-' min-', i.v.). Arterial blood samples were withdrawn
periodically, from the brachial artery, throughout each experiment and the pH, PO andPco measured
(Coming 280 blood gas analyser, Medfield, MA, USA). Metabolic acidosis was corrected by intra-
venous injection of 10 M NaHCO3. Rectal temperature was recorded and maintained at 38-4 + 0-09 °C
bymeans of athermostatically controlled heating blanket (Harvard Instruments, Edenbridge, UK).
Systemic arterial bloodpressure was recorded through a catheter in the right common carotid artery
and hindlimb perfusion pressure was recorded at the bifurcation of the abdominal aorta through a
catheter inserted in the left femoral artery. All pressures were measured using strain gauge manometers
(PDCR 75, Druck Ltd, Barendeecht, Netherlands) attached to DC bridge amplifiers (Lectromed, MT8P,
St Peter, Jersey). Strain gauge manometers were calibrated with a column of mercury at the start of each
experiment. Pulse rate was derived electronically from the systemic arterial blood pressure.
The aortic bifurcation wasexposed through a mid-line abdominal incision. Hindlimb blood flow was
measured using a wrap-round electromagnetic flow probe (Carolina Medical Instruments, King, NC,
USA; 5 mm circumference) placed on the left external iliac artery. Zero blood flow was determined by
mechanical occlusion of a snare placed on the artery distal to the flow probe. The snare was also used
for the 10 s occlusion of blood flow for the reactive hyperaemia responses. The flow probe was
calibrated in situ at the end of the experiment with the animal's own blood before the cats were killed
with an overdose of anaesthetic.
The left sciatic and femoral nerves were exposed, crushed and stimulating electrodes placed around
them distal to the site of crush. Contraction of the left hindlimb was mediated by stimulation of the
sciatic and femoral nerves at 3 Hz (1OV, 0-1 ms pulse duration) for 20 min. Combined tension
produced by the tibialis anterior (TA) and extensor digitorum longus (EDL) was measured using an
isometric strain gauge (Grass FTC 10, Quincy, MA, USA). Blood pressure, pulse rate, blood flow and
muscle tension were recorded on an eight-channel pen recorder (Graftech Linearcorder, Mk 8 WR3500,
EDRFrelease, indicated by the blood flow response to acetylcholine chloride (10/ugkg-') administered
into the left extemal iliac artery, was inhibited by administration of L-NAME (100 mg kg-1, i.v.). The
effectupon systemic haemodynamics at rest was restored by the infusion of sodium nitroprusside (SNP;
mean 1-36 jug kg-' min -l, range 0.3-2-25 jug kg-' mnin-, i.v.).
Following stabilization of the preparation and, if required, correction of the animal's acid-base
balance, the left iliac artery was occluded for 10 s. The peak blood flow, area under the curve (AUC)
above baseline hindlimb blood flow of the reactive hyperaemia response, and the time (t50) to 50 %
recovery of blood flow from the peak response were measured. Upon recovery, the sciatic and femoral
nerves were stimulated for 20 min and the functional hyperaemia response measured. Blood flow was
also measured for 20 min following cessation of stimulation. The hyperaemia response during muscle
contraction and recovery were measured as vascular conductance (calculated as mean hindlimb blood
flow/mean hindlimb perfusion pressure). The area under the curve for blood flow and conductance
responses was calculated for contraction and recovery periods. An additional reactive hyperaemia
responsewas measuredfollowing iliac artery occlusion.
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NITRIC OXIDE AND FUNCTIONAL HYPERAEMIA
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