Enfoque de la investigación 86

In 6 subjects, hand warming was used to stimulate sustained increases in blood flow in the radial artery. Immersion of the hand in cold water resulted in decreased radial artery blood flow compared to that normally seen in subjects at room temperature. With hand warming to between 42 °C and 45 radial artery blood flow increased from 0.016 ± 0.001 m to 0.113 ± 0.012 m during infusion of saline (mean 8.9 ± 1.5 fold increase) (Figure 3.4.4). This resulted in a mean radial artery dilatation of 11.7 ± 1.8%. Infusion of L-NMMA (4 pmol/min) had no significant effect on radial artery blood flow or dilatation during hand warming (Figure 3.4.4). In 3 of these subjects, a further 10 minute infusion of L-NMMA at 16 pmol/min (to compensate for the dilutional effect of increased forearm blood flow) had no additional effect on dilatation associated with hand warming than that seen during L-NMMA 4 pmol/min (radial dilatation was 13.3 ± 2.5% before and 13.1 ± 1.3% in the presence of L- NMMA).

Aspirin (1200 mg), had no significant effect on radial artery dilatation following hand warming (6.9 ± 1.4% before vs. 7.4 ± 1.0% after, P = NS) as seen with FMD following reactive hyperaemia.

To determine whether dilatation following hand warming was mediated by increased blood flow or through other mechanisms, the direct effects of hand warming on radial artery dilatation were assessed in 5 subjects. When radial artery blood flow was maintained at baseline levels, by inflating a wrist cuff to between diastolic and systolic blood pressure, the dilatation during hand warming was significantly attenuated compared to when hyperaemia was unopposed (2.7 ± 1.1% vs 8.1 ± 1.6% respectively, P = 0.01).

0.31 saline noradrenaline L-NMMA 0.2- E h-

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Saline Noradrenaline L-NMMA

Figure 3.4.3 Effect o f L-NMMA and noradrenaline on reactive hyperaemia and flow-mediated dilatation (FMD). (a) Neither noradrenaline or L-NM MA had any significant effect on the reactive hyperaemic flow envelope, (b) In contrast L- NM M A but not noradrenaline significantly inhibited FMD compared to saline.

0.15 0.10 E I - > 0.05 0.00 10 0 5 15 Time (minutes) 15 10 5 0 5 0 5 10 15 Time (minutes)

Figure 3.4.4 Effect of L-NMMA on hyperaemia (velocity-time integral (VTl)) in response to hand warming and flow-mediated dilatation (FMD). Hand warming caused a gradual but sustained increase in blood flow (upper panel) which was associated with a large and sustained dilatation of the radial artery (lower panel). The hyperaemic response was not significantly different during infusion of L- NMMA I from that seen with saline Q .

3.4.4 Discussion

This study demonstrates that under normal physiological conditions, NO synthesis is important in mediating conduit artery dilatation in response to intense and transient flow increases, but that under basal conditions or when the flow increase is maintained, alternative mechanisms o f vasodilatation predominate. Understanding the mechanisms of these heterogeneous responses of the endothelium to flow has implications for the design and interpretation of endothelial function tests and the treatment of early atherosclerosis.

Under normal physiological conditions both conduit and resistance arteries maintain a constant state of dilatation, the degree of which is determined by the relative balance between vasodilator and constrictor influences. In the present study, local infusion of the NO synthase inhibitor L-NMMA, noradrenaline and oral aspirin were used to probe the mechanisms of regulation of conduit artery tone under different conditions of blood flow. Reduced blood flow, during cuff inflation, resulted in significant radial artery constriction, confirming the presence o f tonic flow-mediated dilatation under normal resting conditions. If NO is important in maintaining basal conduit artery dilatation, then during infusion of L-NMMA, constriction of the artery should occur under resting conditions and not be enhanced by a further reduction in flow. In this study, L-NMMA had no effect on radial artery diameter under resting conditions and there was a trend to increased radial artery constriction during periods of reduced flow indicating minimal basal NO mediated dilatation in conduit arteries.

Previous reports have demonstrated a role for NO in conduit artery dilatation in response to a brief episode of reactive hyperaemia (Joannides et al, 1995; Lieberman

et al, 1996). This finding was confirmed in the present study in which FMD following reactive hyperaemia was almost completely abolished during infusion of L-NMMA, an effect that was not explained by any change in the flow stimulus. In contrast, L-NMMA did not significantly alter the dilator response to a sustained flow stimulus caused by local hand warming. Radial artery dilatation during hand warming was negligible, if radial artery blood flow was maintained at basal values excluding the possibility that the dilatation was a non-specific response to the stimulus used (i.e. hand warming).

These findings have physiological, pathophysiological and therapeutic implications. These findings suggest that a physiological role o f the NO pathway is to provide a mechanism to limit the degree to which shear stress is elevated in response to rapid changes in blood flow (Vallance et al, 1989) and imply that there is adaptation of the response of the NO pathway. Whether this occurs because of reduced NO production or desensitisation to the effects of NO is unclear, but understanding how the pathway is switched off might have implications for understanding how activity of the NO pathway is reduced in cardiovascular disease.

The mechanism(s) of dilatation in response to sustained flow is at present unclear, but might involve endothelial or non-endothelial dependent pathways. Inhibition of cyclooxygenase with aspirin had no effect on basal radial artery diameter or dilatation in response to either flow stimulus, implying that prostanoids are not important determinants of tone in this vessel under these conditions (Joannides et al,

1995). One possibility is that neuronal components are involved, although in animal experiments, FMD was preserved following surgical or pharmacological denervation (Lie et al, 1970; Hilton, 1959; Fleisch, 1935). This is an area for investigation in the future. An alternative explanation for these findings is that other vasodilator mechanisms are able to compensate for reduced NO synthesis, but that their rate of response to intense and rapidly changing stimuli is limited compared to the NO pathway. Further, experiments with blockade of multiple pathways will be needed to test this hypothesis.

In summary, these data indicate that whilst NO is an important regulator o f conduit artery tone in response to brief increases in blood flow, the maintenance of arterial dilatation under basal conditions or during sustained hyperaemia is mediated by NO- independent mechanisms. These data have important scientific implications for the interpretation of tests of endothelial function and the development of therapeutic strategies to replace or enhance endogenous NO activity. Further studies to elucidate the mechanism of this phenomenon will help to clarify its role and that of the NO pathway in physiological and pathophysiological conditions.