Forearm reactive hyperaemia is not mediated by nitric oxide in healthy volunteers

Abstract

Aims

To determine the role of nitric oxide (NO) in forearm reactive hyperaemia in healthy human subjects.

Methods

Ten healthy subjects aged 19–34 years underwent brachial artery cannulation. Forearm circulatory arrest was achieved by means of an upper arm cuff inflated to 200 mmHg for 5 min. The blood flow responses during reactive hyperaemia were measured using venous occlusion plethysmography following a 10 min intra-arterial influsion of 8 μmol min⁻¹N-monomethyl l-arginine (l-NMMA) and following matching placebo administered in random order. Results were analysed by repeated measures anova and t-tests.

Results

l-NMMA resulted in a significant reduction of basal forearm blood flow indicating inhibition of basal NO release (P = 0.005). There was no significant difference between the blood flow responses during reactive hyperaemia following l-NMMA and placebo (P = 0.97).

Conclusions

Nitric oxide production does not make a significant contribution to the vasodilatation associated with reactive hyperaemia in the human forearm.

Introduction

Reactive hyperaemia is the rapid increase in blood flow which follows transient arterial occlusion. Studies in animals have indicated that NO in particular is involved in the reactive hyperaemic response [1, 2]. Some authors have measured reactive hyperaemia in resistance vessels and the microvasculature as a marker for endothelial function in human subjects in vivo [3, 4]. Few studies have investigated the role of endothelium-derived NO in reactive hyperaemia in humans and the results of these studies are conflicting [5–7].

The aim of this study was to determine whether endothelium-derived NO is involved in reactive hyperaemia in the forearm of healthy human subjects.

Methods

Ten healthy, nonsmoking volunteers (seven male, three female) mean age 25 years (range 19–34) were recruited. Each subject gave written informed consent for the study which had the approval of the ethics committee of the Queen’s University of Belfast. Subjects underwent medical history and measurement of blood pressure and serum cholesterol concentration. Using 1% lignocaine for local anaesthesia, a 27 gauge needle was inserted into the brachial artery. After a period of at least 30 min, baseline forearm blood flow (FBF) was measured by venous occlusion plethysmography. An indium-gallium strain gauge coupled to an electronically calibrated plethysmograph (Medasonics model SPG16, Mountain View, CA) was connected to a chart recorder Medasonics model R12B). During measurements of FBF, the hand was excluded from the circulation by a wrist cuff inflated to 200 mmHg. FBF was measured in the experimental arm to determine the effects of l-NMMA on reactive hyperaemia and in the control arm to exclude systemic effects of the drug. An upper arm cuff was inflated to 40 mmHg for 5 s and released for 5 s between measurements.

The NO synthase inhibitor, N-mono-methyl-l-arginine (l-NMMA) was infused at 8 μmo1 min⁻¹ for 10 min. This dose was selected to ensure adequate suppression of stimulated NO production [8]. FBF was measured on completion of the infusion. Forearm circulatory arrest was achieved by means of an upper arm cuff inflated to 200 mmHg for 5 min. FBF was measured immediately and continuously on deflation of the cuff for a 2 min period. A wash-out period (at least 30 min) was allowed until FBF returned to baseline. Placebo was then infused for 10 min and the hyperaemic response repeated. The order of administration of l-NMMA or placebo was randomised. Results are expressed as FBF (ml. 100 ml⁻¹.min⁻¹) and analysed by Student’s t-tests to assess drug effects on basal flow. FBF was expressed as change from baseline and the hyperaemic response following ll-NMMA and placebo compared using repeated measures analysis of variance and summary measures.

Results

l-NMMA reduced basal blood flow by 39% in the experimental arm (Figure 1; P = 0.005). There was no difference in FBF in the control arm before and following l-NMMA (3.9±0.4 vs 4.1±0.5 (mean±s.e.mean)). There was no significant difference in reactive hyperaemia following l-NMMA or placebo (Figure 2; P = 0.97), although peak response appeared slightly greater following l-NMMA. The mean difference between treatments was 1.42 ml 100 ml⁻¹ min⁻¹ (95% CI 0.59, 2.25). The area under the curve was similar with placebo and l-NMMA (775.8±90.0 vs 918.0±195.7 arbitrary units; P = 0.52).

Figure 1.

Forearm blood flow at baseline and following placebo and l-NMMA (BL1 baseline prior to placebo; BL2 baseline prior to l-NMMA; mean and 95% CI).

Figure 2.

Forearm reactive hyperaemia to 5 min circulatory occlusion following placebo (○) and l-NMMA (•); (mean and 95% CI).

Discussion

We have demonstrated that reactive hyperaemia in the human forearm is not influenced by l-NMMA. This finding suggests that NO is not involved in the hyperaemic response to ischaemia in the forearm of healthy human volunteers.

The dose of l-NMMA infused was sufficient to inhibit NO production as there was a significant reduction in resting FBF due to reduced basal NO production. Vallance et al. have demonstrated inhibition of basal and stimulated release of NO with lower doses of l-NMMA than the dose used in this study [8]. It is unlikely that the effects of l-NMMA had worn off during the 5 min ischaemic period as only slight reduction of effects have been demonstrated 15 min following infusion [8]. There was no effect of l-NMMA on FBF in the control arm indicating that the dose used did not cause systemic effects.

If NO is involved in reactive hyperaemia, we should have demonstrated at least an attenuation of the response following infusion of l-NMMA. Our results show slightly greater amplitude of hyperaemia following l-NMMA, although this could be explained by the preconstricted baseline.

Studies that have investigated the role of NO in the forearm resistance vasculature have yielded conflicting results. Meredith et al. demonstrated a 16% reduction in peak hyperaemic blood flow and a 23% reduction in total hyperaemia following l-NMMA [5]. Tagawa et al. demonstrated no effect of l-NMMA on peak reactive hyperaemia but a 20–30% reduction in total hyperaemic blood flow [6]. These findings could be partly explained by the effect of l-NMMA on baseline FBF. Engelke et al. demonstrated little or no effect of l-NMMA on peak or total hyperaemic blood flow when the effect of l-NMMA on baseline FBF was accounted for [7]. In all studies, l-NMMA had a similar effect on baseline FBF.

Information in animal studies is more consistent, but the results should not be extrapolated to human subjects. Reactive hyperaemia following coronary artery occlusion in dogs was substantially decreased by removal of the endothelium and inhibition of NO synthase [2]. NO has been found to contribute to the duration but not the peak amplitude of reactive hyperaemia [9].

Reactive hyperaemia is distinct from flow-mediated dilatation which has been used to assess endothelial function noninvasively in peripheral conduit arteries, using high resolution ultrasound [10, 11]. In this technique, reactive hyperaemia is utilized as a tool to mediate the increase in blood flow with measurements made proximal to the ischaemic vascular bed. This dilatation has been demonstrated to be inhibited by l-NMMA and is therefore likely to be endothelium dependent. The regulation of vascular tone may differ in peripheral conduit arteries and arterioles as l-NMMA reduces blood flow in small resistance vessels but does not alter the diameter of larger arteries [12]. Furthermore, the mechanisms involved may depend on the duration of ischaemia.

It is likely that reactive hyperaemia has a multifactorial aetiology. Early investigators suggested increased levels of histamine [13] although Landowne & Thompson failed to demonstrate a reduction in reactive hyperaemia measured in the human lower limb following oral or intravenous antihistamines [14]. Factors which have been demonstrated to contribute to reactive hyperaemia include prostaglandins, adenosine and ATP-sensitive potassium channels [15, 16].

Although we have demonstrated that NO does not contribute to postischaemic dilatation in the human forearm circulation, other endothelium-dependent mediators such as prostaglandins may be involved.