Jeremy Bellien

Michele Iacob

Helene Eltchaninoff

Ryad Bourkaib

Christian Thuillez

Dr Robinson Joannides
email: robinson.joannides@chu-rouen.fr

artery flow-mediated dilatation during NOS inhibition in humans

Keywords: pdfauthor=Jeremy Bellien, Michele Iacob, Helene Eltchaninoff, Ryad Bourkaib, Christian Thuillez, Robinson Joannides, pdfkeywords=Medicine, pdfcreator=HAL, pdfproducer=PDFLaTeX, pdfsubject= pharmacology, angiotensin antagonist, endothelium, nitric oxide, prostacyclin, endothelium-derived hyperpolarizing factor

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Endothelium plays a crucial role in conduit artery flow-mediated dilatation (FMD) and participates in the control of arterial tone and trophicity by releasing vasoactive factors mainly nitric oxide (NO) [START_REF] Joannides | Nitric oxide is responsible for flowdependent dilatation of human peripheral conduit arteries in vivo[END_REF][START_REF] Mullen | Heterogenous nature of flowmediated dilatation in human conduit arteries in vivo. Relevance to endothelial dysfunction in hypercholesterolemia[END_REF]. A decrease in arterial NO availability is associated with abnormal vasomotion, smooth muscle cell proliferation and migration, monocyte adhesion and procoagulant state that characterize endothelial dysfunction and contribute to the pathophysiology of cardiovascular diseases [START_REF] Mombouli | Endothelial dysfunction: from physiology to therapy[END_REF][START_REF] Quyyumi | Prognostic value of endothelial function[END_REF]. Increasing reported data demonstrate that the local activation of the renin-angiotensin system participates in the development of endothelial dysfunction through the stimulation of angiotensin II type 1 (AT 1 ) receptor subtype and the inactivation of NO [START_REF] Fleming | Signaling by the angiotensin-converting enzyme[END_REF][START_REF] Nickenig | The AT(1)-type angiotensin receptor in oxidative stress and atherogenesis. Part I: Oxidative stress and atherogenesis[END_REF][START_REF] Mollnau | Effects of angiotensin II infusion on the expression and function of NAD(P)H oxidase and components of nitric oxide/cGMP signaling[END_REF].

AT 1 receptor antagonist are specific agents that block the effects of angiotensin II mediated through AT 1 receptors thus preserving the potential beneficial activity of the angiotensin II type 2 (AT 2 ) receptors on vascular tone and blood pressure [START_REF] Nickenig | The AT(1)-type angiotensin receptor in oxidative stress and atherogenesis. Part I: Oxidative stress and atherogenesis[END_REF][START_REF] You | High blood pressure reduction reverses angiotensin II type 2 receptor-mediated vasoconstriction into vasodilation in spontaneously hypertensive rats[END_REF]. In humans, despite some conflicting results [START_REF] Anderson | Comparative study of ACE-inhibition, angiotensin II antagonism, and calcium channel blockade on flowmediated vasodilatation in patient with coronary disease (BANFF study)[END_REF][START_REF] Ghiadoni | Different effect of antihypertensive drugs on conduit artery endothelial function[END_REF], the administration of AT 1 receptor antagonists enhances the FMD of conduit arteries in cardiovascular diseases associated with endothelial dysfunction [START_REF] Giannattasio | Radial artery flow-mediated dilatation in heart failure patients. Effects of pharmacological and nonpharmacological treatment[END_REF][START_REF] Hornig | AT1-receptor antagonism improves endothelial function in coronary artery disease by a bradykinin/B2receptor-dependent mechanism[END_REF][START_REF] Koh | Comparison of effects of losartan, irbesartan, and candesartan on flow-mediated brachial artery dilation and on inflammatory and thrombolytic markers in patients with systemic hypertension[END_REF][START_REF] Prasad | Acute and chronic angiotensin-1 receptor antagonism reverses endothelial dysfunction in atherosclerosis[END_REF][START_REF] Wassmann | Angiotensin II type 1 receptor antagonism improves hypercholesterolemia-associated endothelial dysfunction[END_REF]. However, the action mechanisms of these agents on endothelial function, to our knowledge, have not been fully investigated in humans. In patients with coronary artery disease, the beneficial effect of AT 1 receptor blockade on the endothelial function of peripheral conduit arteries is mainly related to the increase in both NO and bradykinin availability [START_REF] Hornig | AT1-receptor antagonism improves endothelial function in coronary artery disease by a bradykinin/B2receptor-dependent mechanism[END_REF][START_REF] Prasad | Acute and chronic angiotensin-1 receptor antagonism reverses endothelial dysfunction in atherosclerosis[END_REF]. In addition, it has been demonstrated in animal models with hypertension or renal failure that chronic treatment with antagonists can also enhance the endothelium-dependent dilatation of isolated mesenteric arteries during combined inhibition of NO and prostacyclin (PGI 2 ) synthesis [START_REF] Goto | Renin-angiotensin blockade improves endothelial dysfunction in hypertension[END_REF][START_REF] Koobi | AT1 receptor blockade improves vasorelaxation in experimental renal failure[END_REF]. Under these experimental conditions, this beneficial effect of AT 1 receptor blockade is due to the release of an endothelium-dependent hyperpolarizing factor (EDHF) [START_REF] Goto | Renin-angiotensin blockade improves endothelial dysfunction in hypertension[END_REF][START_REF] Koobi | AT1 receptor blockade improves vasorelaxation in experimental renal failure[END_REF] that has not yet been identified but could be related to cytochrome P450 (CYP)derived products [START_REF] Fleming | Cytochrome P450 epoxygenase as EDHF synthase(s)[END_REF]. In humans, whether AT 1 receptor blockade can prevent or not the decrease in conduit artery FMD during NO deficiency by an endothelial alternative pathway remains unknown.

In this context, the main objective of this work was to evaluate in humans the effect of the acute administration of an AT 1 receptor antagonist on the FMD of peripheral conduit arteries before and during NO-synthase inhibition. Moreover, in an exploratory approach, we tested whether the effect of AT 1 receptor antagonist on FMD during NO deficiency remains constantly during the inhibition of PGI 2 and CYP-related product synthesis.

METHODS

Subjects

A total of 18 healthy male volunteers were included in this study. None had evidence of hypertension (blood pressure>140/90 mmHg), hyperlipidemia (total serum cholesterol>200 mg/dl), or clinical evidence of cardiovascular disease and diabetes on the basis of previous

Instrumentation

Measurements were performed at 08:00 in the morning, one hour after a fat free breakfast without tea or coffee, while subjects were supine in a quiet air-conditioned room, maintained at a constant temperature (22-24°C). A 27-gauge needle was inserted under local anesthesia (lidocaine 1%) into the brachial artery of the non-dominant arm to allow the local infusion of physiological saline and pharmacological agents at constant rate (1 ml/min). Systemic blood pressure and heart rate were measured by means of a brachial cuff oscillometric device (Dinamap, 8103, Critikon, Johnson & Johnson Santé, France). Radial internal diameter, blood flow velocity and digital blood pressure were continuously measured using a high-precision echotracking device (Nius 02, Asulab, Switzerland; 10 MHz) coupled to a Doppler device (Doptek, Deltex, UK; 8 MHz) and a finger photoplethysmograph (Finapres 2300, Ohmeda, CO) as previously described [START_REF] Joannides | Nitric oxide is responsible for flowdependent dilatation of human peripheral conduit arteries in vivo[END_REF][START_REF] Bellien | Role of nitric oxide in sustained peripheral conduit artery FMD[END_REF][START_REF] Bellien | Evidence for a basal release of a cytochrome-related endothelium-derived hyperpolarizing factor in the radial artery in humans[END_REF][START_REF] Joannides | Role of basal and stimulated release of nitric oxide in the regulation of radial artery caliber in humans[END_REF][START_REF] Joannides | Evaluation of the determinants of flow-mediated radial artery vasodilatation in humans[END_REF][START_REF] Tardy | Non invasive estimate of the mechanical properties of peripheral arteries from ultrasonic and photoplethysmographic measurements[END_REF] This device allows the measurement of radial artery internal diameter with a resolution on the diameter evolution of <1 µm [START_REF] Tardy | Non invasive estimate of the mechanical properties of peripheral arteries from ultrasonic and photoplethysmographic measurements[END_REF]. Radial artery flow was based on the measurement of velocity and diameter. In absence of change in mean blood pressure, the variations of radial artery flow observed with the pharmacological agents reflects the changes in radial vascular resistance which is usually calculated as the ratio of mean blood pressure to radial artery flow [START_REF] Benjamin | Measuring forearm blood flow and interpreting the responses to drugs and mediators[END_REF].

Study protocols

Randomized double-blind placebo-controlled cross-over study: Effect of AT 1 receptor blockade on radial artery FMD before and during NO-synthase inhibition

Twelve healthy subjects (23±1 years) were explored on 2 separate days with a two-week washout period between each day. Subjects were allowed to rest 30 minutes after instrumentation. Then heart rate and blood pressure were recorded and telmisartan (80 mg per os) or placebo were administered. After 2 hours, blood pressure, heart rate, radial artery blood flow and diameter were recorded for 5 minutes. Then, an arterial occlusion cuff placed on the wrist was inflated for 10 minutes at 180 mm Hg and was deflated to allow reactive hyperemia with the continuous measurements of all parameters. Based on preliminary results, we used a 10-min occlusion protocol to induce a maximal increase in radial artery diameter [START_REF] Joannides | Evaluation of the determinants of flow-mediated radial artery vasodilatation in humans[END_REF] with a significant participation of NO [START_REF] Bellien | Role of nitric oxide in sustained peripheral conduit artery FMD[END_REF]. After radial artery blood flow and diameter had returned to baseline, the subjects received sodium nitroprusside (SNP) at the incremental doses of 1.31, 2.62, and 5.24 µg.mg.min -1 .L -1 (5, 10 and 20 nmol.min -1 .L -1 ), 3 min for each dose to assess the endothelium-independent dilatation [START_REF] Joannides | Role of basal and stimulated release of nitric oxide in the regulation of radial artery caliber in humans[END_REF]. Once radial artery diameter had returned to baseline, subjects received the NO-synthase inhibitor N G -monomethyl-L-arginine (L-NMMA, Clinalfa, Switzerland) at the dose of 1.5 mg.min -1 .L -1 (8 µmol.min -1 .L -1 ) for 7 min. This dose of L-NMMA induced a prolonged inhibition of NO-synthase of at least 40 min and blocks the forearm and radial artery vasodilatation to acetylcholine administrated at doses producing maximal regional effect without systemic hemodynamic change [START_REF] Joannides | Role of basal and stimulated release of nitric oxide in the regulation of radial artery caliber in humans[END_REF]. Five min after L-NMMA infusion, all parameters were again measured at baseline and the same occlusionischemia and SNP infusion protocol was repeated. Baseline and peak measurements represented means of values obtained during 15 and 3 cardiac cycles respectively. The continuous measurement of radial artery parameters allows to precisely estimate the maximal values of diameter and flow occurring after cuff deflation. Thus, radial artery FMD was calculated as the percent change of radial artery diameter in response to post-ischemic hyperemia with the peak diameter after cuff deflation and the basal diameter before cuff inflation (figure 1): FMD (%) = (peak diameter -basal diameter)x 100 / basal diameter.

Moreover, because the modifications in the hyperemic stimulus influence the magnitude of radial artery FMD, we also measured the parameters characteristic of this stimulus: the peak flow representing the maximal value of flow after cuff deflation and the area under the curve of flow (AUC of flow) representing the duration of the increase in flow (figure 1) [START_REF] Joannides | Evaluation of the determinants of flow-mediated radial artery vasodilatation in humans[END_REF].

The echotracking measures were performed by the same observer (MI) and were read with the assistance of 2 other investigators (JB, RJ) all blinded to the subjects and treatments.

Six healthy subjects (23±2 years) participated in a complementary open study. Radial artery

FMD was evaluated in response to four reactive hyperemias (wrist occlusion: 10 min) performed successively. First, the radial artery FMD was evaluated under control conditions prior to telmisartan administration. Then, each subject received a single oral dose of telmisartan (80 mg) followed, 90 min later, by a single oral dose of aspirin (500 mg) to block vascular cyclooxygenase activity 30 min after administration and during the next 6 hours [START_REF] Bellien | Evidence for a basal release of a cytochrome-related endothelium-derived hyperpolarizing factor in the radial artery in humans[END_REF][START_REF] Heavey | Aspirin causes shortlived inhibition of bradykinin-stimulated protacyclin production in man[END_REF]. Thirty minutes after aspirin, a second reactive hyperemia was performed. After an additional 30 min resting period and the return to baseline measurements, subjects received L-NMMA at 1.5 mg.min -1 .L -1 (8 µmol/min/L) for 7 min followed by the third reactive hyperemia to evaluate a possible interaction between PGI 2 and NO during FMD. Finally, after obtaining baseline measurements again, a last reactive hyperemia was performed after the coinfusion during 7 min of L-NMMA at the same dose as previously with fluconazole, a potent inhibitor of endothelial cytochrome P450 (CYP) 2C9 located in human arteries in the endothelium and identified as EDHF synthase [START_REF] Fleming | Cytochrome P450 epoxygenase as EDHF synthase(s)[END_REF]. In fact, ex-vivo experiments have demonstrated that CYP 2C9 produces the 11,12-epoxyeicosatrienoic acid that induces the hyperpolarization and relaxation of vascular smooth muscle cells [START_REF] Archer | Endothelium-derived hyperpolarizing factor in human internal mammary artery is 11,12-epoxyeicosatrienoic acid and causes relaxation by activating smooth muscle BK(Ca) channels[END_REF]. In addition, we have recently demonstrated using fluconazole that a CYP-derived EDHF is involved with NO in the control of the basal radial artery diameter [START_REF] Bellien | Evidence for a basal release of a cytochrome-related endothelium-derived hyperpolarizing factor in the radial artery in humans[END_REF]. Fluconazole was infused at the dose of 0.37 mg.min -1 .L -1 (1.2 µmol.min -1 .L -1 ) to obtain a calculated local concentration, according to a radial artery flow at baseline and during reactive hyperemia (10 to 100 ml/min), at least two times higher that the in vitro Ki of CYP 2C9 and with a weaker activity on other CYP enzymes [START_REF] Bellien | Evidence for a basal release of a cytochrome-related endothelium-derived hyperpolarizing factor in the radial artery in humans[END_REF][START_REF] Venkatakrishnan | Effects of the antifungal agents on oxidative drug metabolism: clinical relevance[END_REF].

Statistical analysis

Results are expressed as mean ±SEM. In the randomized double-blind study, the effect of treatments and L-NMMA on measured parameters were analyzed by ANOVA with treatment (placebo and telmisartan), periods (control and L-NMMA) and subjects as factors and mean blood pressure as a covariate. In addition, the analysis of FMD was performed using the variation of radial artery flow during hyperemia as an additional covariate. Analysis was performed, only when ANOVA was considered significant, using a modified paired t-test adjusted for multiple comparisons. In the exploratory study, the effect of the inhibitors on radial artery FMD and peak flow were analyzed by ANOVA with inhibitors and subjects as

RESULTS

Randomized double-blind study

Systemic hemodynamical parameters (table 1)

Two hours after treatment administration, a significant decrease in mean blood pressure (P<0.05) and heart rate (P<0.001) was observed in both treatment groups. However, this decrease was not significantly different between treatment groups. The local administration of L-NMMA did not modify the systemic hemodynamical parameters in either groups (table 2).

Radial artery parameters at baseline

In control period, compared with placebo, telmisartan did not modify the basal radial artery flow and diameter (table 2). L-NMMA did not modify the radial artery diameter but decreased radial artery flow similarly in either treatment groups (both P<0.05) suggesting, in absence of change in mean blood pressure, a similar increase in local radial vascular resistance.

Post-ischemic hyperemia

Deflation of the occluding cuff was associated, in all cases, with an increase in radial artery flow (figure 1A, P<0.001). Concerning the hyperemic stimulus, in control period, telmisartan did not modify either the peak or the AUC of flow and L-NMMA did not modify these parameters in both treatment groups.

Radial artery flow-mediated dilatation

Deflation of the occluding cuff was associated, in all cases, with an increase in radial artery diameter (figure 1B, P<0.001). In control period, the radial FMD was not modified by telmisartan (9.9±0.7%) as compared with placebo (10.9±0.6%). L-NMMA reduced the radial FMD in the placebo group (P<0.05) but not in the telmisartan group. Thus, the radial FMD after L-NMMA was enhanced by telmisartan (12.6±1.2%) as compared with placebo (9.3±0.8%, P<0.05).

Radial artery endothelium-independent dilatation

SNP induced a dose-dependent increase in radial artery blood flow (figure 2A, P<0.05) and diameter (figure 2B, P<0.001) in the two treatment groups that was not modified by telmisartan. L-NMMA enhanced the increase in radial artery diameter at each dose of SNP to the same extent in both treatment groups (both P<0.05) without affecting the increase in radial artery blood flow.

Complementary study (table 3)

None of the inhibitors administrated affected the systemic hemodynamical parameters.

Compared with control conditions before telmisartan and inhibitors administration, the basal radial artery flow was not modified in telmisartan-treated subjects by aspirin but was reduced to the same extent by aspirin+L-NMMA and aspirin+L-NMMA+fluconazole (both P<0.05).

The basal radial artery diameter, compared with control conditions before telmisartan, was not modified in telmisartan-treated subjects by aspirin or aspirin+L-NMMA but in fact was reduced by aspirin+L-NMMA+fluconazole (P<0.05). Deflation of the occluding cuff was associated, in all cases, with an increase in radial artery flow and diameter (both P<0.001).

The radial artery FMD, compared with control conditions before telmisartan, was not modified in telmisartan-treated subjects by aspirin or aspirin+L-NMMA but was decreased by aspirin+L-NMMA+fluconazole (P<0.05) with no modification in peak flow.

DISCUSSION

The main result of the present study performed with healthy subjects was that the acute angiotensin II type 1 receptor blockade by telmisartan maintains the peripheral conduit artery FMD during local NO-synthase inhibition which suggests the development of an endothelial compensatory mechanism to prevent the effects of a decrease in NO availability. This effect of AT 1 receptor blockade on the endothelium-dependent dilatation of conduit arteries appears to be independent of PGI 2 availability and could in fact be related to an increase in the release of an EDHF. This double-bind study was designed to explore, in healthy subjects, the effect of AT 1 receptor blockade on the FMD of the radial artery, a model of peripheral conduit artery [START_REF] Joannides | Nitric oxide is responsible for flowdependent dilatation of human peripheral conduit arteries in vivo[END_REF][START_REF] Mullen | Heterogenous nature of flowmediated dilatation in human conduit arteries in vivo. Relevance to endothelial dysfunction in hypercholesterolemia[END_REF][START_REF] Hornig | AT1-receptor antagonism improves endothelial function in coronary artery disease by a bradykinin/B2receptor-dependent mechanism[END_REF][START_REF] Bellien | Role of nitric oxide in sustained peripheral conduit artery FMD[END_REF][START_REF] Bellien | Evidence for a basal release of a cytochrome-related endothelium-derived hyperpolarizing factor in the radial artery in humans[END_REF][START_REF] Joannides | Role of basal and stimulated release of nitric oxide in the regulation of radial artery caliber in humans[END_REF][START_REF] Joannides | Evaluation of the determinants of flow-mediated radial artery vasodilatation in humans[END_REF][START_REF] Hornig | Vitamin C improves endothelial function of conduit arteries in patients with chronic heart failure[END_REF], before and during induced NO deficiency. In the placebo treated group, the local NO-synthase inhibition obtained using L-NMMA decreased the basal radial artery flow without modifying radial artery diameter confirming, in absence of change in mean blood pressure, the role of NO in the control of the basal vascular resistance in humans [START_REF] Joannides | Nitric oxide is responsible for flowdependent dilatation of human peripheral conduit arteries in vivo[END_REF][START_REF] Hornig | AT1-receptor antagonism improves endothelial function in coronary artery disease by a bradykinin/B2receptor-dependent mechanism[END_REF][START_REF] Bellien | Role of nitric oxide in sustained peripheral conduit artery FMD[END_REF][START_REF] Bellien | Evidence for a basal release of a cytochrome-related endothelium-derived hyperpolarizing factor in the radial artery in humans[END_REF][START_REF] Joannides | Role of basal and stimulated release of nitric oxide in the regulation of radial artery caliber in humans[END_REF][START_REF] Hornig | Vitamin C improves endothelial function of conduit arteries in patients with chronic heart failure[END_REF]. In addition, L-NMMA enhanced the radial artery dilatation to SNP demonstrating an hypersensitivity of the smooth muscle cells to exogenous NO which has been previously observed both in vitro [START_REF] Moncada | Development and mechanism of a specific supersensitivity to nitrovasodilators after inhibition of vascular nitric oxide synthesis in vivo[END_REF] and in vivo [START_REF] Bellien | Role of nitric oxide in sustained peripheral conduit artery FMD[END_REF][START_REF] Bellien | Evidence for a basal release of a cytochrome-related endothelium-derived hyperpolarizing factor in the radial artery in humans[END_REF][START_REF] Joannides | Role of basal and stimulated release of nitric oxide in the regulation of radial artery caliber in humans[END_REF]. In fact, removal of the endogenous NOmediated vasodilator tone in conduit arteries has been shown to lead to a specific hypersensitivity to exogenous NO provided by nitrovasodilators [START_REF] Moncada | Development and mechanism of a specific supersensitivity to nitrovasodilators after inhibition of vascular nitric oxide synthesis in vivo[END_REF]. Thus, this result demonstrates, under experimental conditions, an effective inhibition of NO release by L-NMMA in the radial artery wall. In contrast, the increase in radial artery blood flow in response to SNP infusion was not affected by L-NMMA. As previously stressed [START_REF] Joannides | Role of basal and stimulated release of nitric oxide in the regulation of radial artery caliber in humans[END_REF], this could be related to the weak vasodilator effect observed with SNP infusion in resistance arteries. Furthermore, L-NMMA decreased the radial artery FMD in the placebo group demonstrating the role of NO in the endothelium-dependent dilatation of peripheral conduit arteries in response to a sustained increase in flow induced by a prolonged period of ischemia of 10 min [START_REF] Bellien | Role of nitric oxide in sustained peripheral conduit artery FMD[END_REF][START_REF] Joannides | Role of basal and stimulated release of nitric oxide in the regulation of radial artery caliber in humans[END_REF]. However, this decrease in the magnitude of FMD was only 15% when compared to experiments that have previously demonstrated a major role of NO in this response using shorter periods of ischemia of 3 to 8 min [START_REF] Joannides | Nitric oxide is responsible for flowdependent dilatation of human peripheral conduit arteries in vivo[END_REF][START_REF] Mullen | Heterogenous nature of flowmediated dilatation in human conduit arteries in vivo. Relevance to endothelial dysfunction in hypercholesterolemia[END_REF][START_REF] Hornig | Vitamin C improves endothelial function of conduit arteries in patients with chronic heart failure[END_REF]. This reduced effect could result from the dose of L-NMMA that could have been insufficient to fully inhibit the endothelial NO-synthase. Moreover, as previously underlined [START_REF] Mullen | Heterogenous nature of flowmediated dilatation in human conduit arteries in vivo. Relevance to endothelial dysfunction in hypercholesterolemia[END_REF], this moderate decrease in FMD after L-NMMA could also be explained by the decrease in the role of NO in peripheral conduit artery FMD during sustained hyperemic stimulation suggesting the involvement of other endothelial vasodilator factors under these experimental conditions.

Clinical Science Immediate Publication. Published on 24 Nov 2006 as manuscript CS20060236

Furthermore, in accordance with previous results [START_REF] Joannides | Nitric oxide is responsible for flowdependent dilatation of human peripheral conduit arteries in vivo[END_REF][START_REF] Mullen | Heterogenous nature of flowmediated dilatation in human conduit arteries in vivo. Relevance to endothelial dysfunction in hypercholesterolemia[END_REF][START_REF] Venkatakrishnan | Effects of the antifungal agents on oxidative drug metabolism: clinical relevance[END_REF][START_REF] Costa | Intravascular source of adenosine during forearm ischemia in humans. Implications for reactive hyperemia[END_REF][START_REF] Tagawa | Role of nitric oxide in reactive hyperemia in human forearm vessels[END_REF], L-NMMA did not modify the radial artery peak flow during hyperemia supporting the absence of contribution of NO in the maximal arteriolar vasodilatation in response to forearm ischemia. In addition, L-NMMA did not further decrease the duration of hyperemia in the present study in contrast to previous results obtained with brief duration of ischemia [START_REF] Joannides | Nitric oxide is responsible for flowdependent dilatation of human peripheral conduit arteries in vivo[END_REF][START_REF] Mullen | Heterogenous nature of flowmediated dilatation in human conduit arteries in vivo. Relevance to endothelial dysfunction in hypercholesterolemia[END_REF][START_REF] Hornig | Vitamin C improves endothelial function of conduit arteries in patients with chronic heart failure[END_REF]. A similar negative result was also reported after a duration of ischemia of 15 minutes [START_REF] Mullen | Heterogenous nature of flowmediated dilatation in human conduit arteries in vivo. Relevance to endothelial dysfunction in hypercholesterolemia[END_REF] and could be related to the decrease in the relative contribution of NO compared with the direct effect of adenosine in the late phase of flow increase during sustained hyperemia [START_REF] Costa | Intravascular source of adenosine during forearm ischemia in humans. Implications for reactive hyperemia[END_REF].

As regards the acute blockade of the AT 1 receptor, this was due to telmisartan, a high affinity antagonist, administered orally at a single dose of 80 mg which represents the maximal therapeutic dose [START_REF] Stangier | Inhibitory effect of telmisartan on the blood pressure response to angiotensin II challenge[END_REF][START_REF] Timmermans | Pharmacological properties of angiotensin II receptor antagonists[END_REF]. Systemic hemodynamical and vascular parameters were obtained two hours after treatment intake corresponding to the time of the maximal inhibitory effect of the drug and during the first two hours of its maximal activity [START_REF] Stangier | Inhibitory effect of telmisartan on the blood pressure response to angiotensin II challenge[END_REF]. In fact, a single administration of this dose of telmisartan has been shown to prevent in healthy volunteers 90% of the pressure increase in response to exogenous angiotensin II demonstrating under similar conditions than ours an effective and near maximal degree of AT 1 receptor blockade [START_REF] Stangier | Inhibitory effect of telmisartan on the blood pressure response to angiotensin II challenge[END_REF]. As expected from our population, the acute AT 1 receptor blockade caused a mild but non significant decrease in blood pressure [START_REF] Stangier | Inhibitory effect of telmisartan on the blood pressure response to angiotensin II challenge[END_REF]. In fact, this absence of decrease in blood pressure, which was also observed by Stangier and coworkers [START_REF] Stangier | Inhibitory effect of telmisartan on the blood pressure response to angiotensin II challenge[END_REF], is not an indicator of a non significant AT 1 receptor blockade but confirms the absence or the low involvement of the renin-angiotensin system in the short-term regulation of blood pressure in sodium-replete healthy subjects [START_REF] Newby | Endogenous angiotensin II contributes to basal peripheral vascular tone in sodium deplete but not sodium replete man[END_REF][START_REF] Baan | Effects of sodium depletion on the role of AT1-and alpha-adrenergic receptors in the regulation of forearm vascular tone in humans[END_REF]. However, as a negative relationship was demonstrated between blood pressure and the magnitude of conduit artery FMD, we used blood pressure as a covariate into the statistical analysis to avoid any passive effect of this parameter on the responses observed [START_REF] Iiyama | Impaired endothelial function with essential hypertension assessed by ultrasonography[END_REF]. During the control period, telmisartan did not significantly modify the radial artery flow at baseline nor the hyperemic response confirming that the reninangiotensin system, under physiological conditions, was not involved in the acute control of basal and post-ischemic peripheral vascular tone [START_REF] Prasad | Acute and chronic angiotensin-1 receptor antagonism reverses endothelial dysfunction in atherosclerosis[END_REF][START_REF] Hornig | Vitamin C improves endothelial function of conduit arteries in patients with chronic heart failure[END_REF][START_REF] Newby | Endogenous angiotensin II contributes to basal peripheral vascular tone in sodium deplete but not sodium replete man[END_REF][START_REF] Baan | Effects of sodium depletion on the role of AT1-and alpha-adrenergic receptors in the regulation of forearm vascular tone in humans[END_REF]. Moreover, telmisartan did not modify the magnitude of radial artery FMD as previously reported in healthy subjects using repeated administration of losartan and the angiotensin converting enzyme inhibitor quinapril [START_REF] Wilmink | Effect of angiotensin-converting enzyme inhibition and angiotensin II type 1 receptor antagonism on postprandial endothelial function[END_REF]. In contrast, during endothelial NO-synthase inhibition, the acute AT 1 receptor blockade prevented the decrease in the radial artery FMD observed in the placebo-treated group. This effect was not related to a modification in the smooth muscle cells sensitivity after AT 1 receptor antagonist administration because the endothelium-independent dilatation assessed Clinical Science Immediate Publication. Published on 24 Nov 2006 as manuscript CS20060236 by SNP was comparable in both treatment groups before and after L-NMMA. In addition, it appears unlikely that telmisartan could have increased the release of NO during FMD by enhancing endothelial NO-synthase activity to a level which makes this enzyme insensitive to the dose of L-NMMA administered. In fact, the persistence of the hypersensitivity to SNP observed in the telmisartan group after L-NMMA demonstrated the effective decrease of NO availability in the arterial wall. Moreover, the magnitude of this hypersensitivity was similar as that after placebo which demonstrated that, even if not maximal, a stable level of NOsynthase inhibition was achieved in both treatment groups. This last point shows that maintaining FMD during NO-synthase inhibition following AT 1 receptor blockade was not related to the increase in NO availability. Furthermore, this effect was not related to an increase in the hyperemic stimulus because telmisartan did not significantly affect the peak flow nor the duration of the increase in flow. Therefore, this result suggests the development of an endothelial compensatory mechanism after AT 1 receptor antagonists administration, probably the increase in the release by the endothelium, in response to flow, of an other vasodilator factor to compensate for the loss of NO synthesis. It is unlikely that telmisartan could have increased the release of PGI 2 under our conditions as we observed, in the complementary study, that the cyclooxygenase inhibitor aspirin administered alone or associated with L-NMMA no longer affected the radial artery FMD in the healthy subjects treated with the AT 1 receptor antagonist when compared to control. This result is consistent with previous reported data showing that a compensatory increase in PGI 2 release was only observed after chronic NO-synthase inhibition in animals [START_REF] Puybasset | Coronary and systemic hemodynamic effects of sustained inhibition of nitric oxide synthesis in conscious dogs -evidence for cross talk between nitric oxide and cyclooxygenase in coronary vessels[END_REF] and that the inhibition of cyclooxygenase did not modify radial artery FMD in response to sustained and non sustained hyperemic stimulations even in presence of L-NMMA [START_REF] Joannides | Nitric oxide is responsible for flowdependent dilatation of human peripheral conduit arteries in vivo[END_REF][START_REF] Mullen | Heterogenous nature of flowmediated dilatation in human conduit arteries in vivo. Relevance to endothelial dysfunction in hypercholesterolemia[END_REF].

Since the combined inhibition of NO and PGI 2 synthesis did not affect radial artery FMD in the telmisartan-treated volunteers, this beneficial effect of AT 1 receptor blockade on the conduit artery endothelial vasomotor function during induced NO deficiency could be explained by the increase in the release of an EDHF. This hypothesis is consistent with previous experiments performed in aged and hypertensive animals and in experimental renal failure demonstrating that AT 1 receptor antagonists administration can enhance the EDHFmediated dilatation of mesenteric arteries in response to acetylcholine during NO-synthase and cyclooxygenase inhibition [START_REF] Goto | Renin-angiotensin blockade improves endothelial dysfunction in hypertension[END_REF][START_REF] Koobi | AT1 receptor blockade improves vasorelaxation in experimental renal failure[END_REF][START_REF] Kansui | Angiotensin II receptor antagonist improves age-related endothelial dysfunction[END_REF]. Although the EDHF involved in this effect has not been identified, we coinfused fluconazole with L-NMMA to evaluate the role of CYP epoxygenases in radial artery FMD during AT 1 receptor blockade. In fact, previous ex-vivo studies have shown that FMD is mediated by CYP-related metabolites during NO deficiency in human coronary and mice mesenteric arterioles [START_REF] Huang | Epoxyeicostrienoic acids are released to mediate shear stress-dependent hyperpolarization of arteriolar smooth muscle[END_REF][START_REF] Miura | Flow-induced dilatation of human coronary arterioles. Important role of Ca 2+ -activated K + channels[END_REF]. In addition, an interaction between NO and an endothelial CYP-dependent vasodilator pathway participates in the regulation of exercise-induced skeletal muscle blood flow in humans in vivo [START_REF] Hillig | Cytochrome P450 2C9 plays an important role in the regulation of exercise-induced skeletal muscle blood flow and oxygen uptake in humans[END_REF]. Under these conditions, the basal radial artery diameter was reduced by the combination of aspirin, L-NMMA and fluconazole according to our previous study performed in healthy subjects without treatment that showed the role of a CYP-related EDHF in the regulation of radial artery diameter at baseline [START_REF] Bellien | Evidence for a basal release of a cytochrome-related endothelium-derived hyperpolarizing factor in the radial artery in humans[END_REF]. During reactive hyperemia, with combined blockade of both NO and PGI 2 synthesis, the infusion of fluconazole was associated with a lesser increase in diameter with no modification in peak flow compared to the control experiments. This demonstrates for the first time that a CYP-dependent vasodilator pathway is involved under these experimental conditions in human peripheral conduit artery FMD. Although the reduction in the magnitude of radial artery FMD via the addition of fluconazole in subjects treated with telmisartan was similar with that observed after L-NMMA in the placebo group, additional dedicated experiments are required to confirm whether the administration of AT 1 receptor antagonists increases the influence of a CYP-dependent vasodilator pathway to maintain conduit artery FMD during the loss of NO synthesis in humans.

Furthermore, it would be interesting to evaluate if the endothelial alternative mechanism involved in the beneficial effect of AT 1 receptor blockade could compensate a greater magnitude of decrease in NO-dependent FMD similar to those provided by L-NMMA during shorter occlusion periods. In addition, whether the development of this NO-and PGI 2independent mechanism following the administration of AT 1 receptor antagonists is effective in pathological states characterized by a decrease in NO availability warrants further investigation. This effect could be particularly important to maintain an adequate vascular conductance and cardiovascular coupling and to limit the progression of cardiovascular diseases in particular atherosclerosis.

Conclusion

These results demonstrate that acute AT 1 receptor blockade opposes the decrease in conduit artery flow-mediated dilatation during induced NO deficiency in humans suggesting the development of an endothelial compensatory mechanism. Although this mechanism is independent of PGI 2 , it may be related to the release of an EDHF.

  medical history and complete medical examination, normal electrocardiogram and recent routine laboratory tests. No participants were smokers or taking medication at the time of the study. The forearm volume was measured in each subject by using the water displacement method based on the Archimedes' principle and drug infusion rates were normalized to 1 liter tissue forearm by alteration of the drug concentration in the solvent while the pump speed of infusion was maintained constant. The research was carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association and was approved by the Consultative Committee for the Protection of Persons Engaged in Biomedical Research of Haute-Normandie. All participants gave informed written consent after full explanation of the purpose, nature and risk of all procedures used.

Clinical

  Science Immediate Publication. Published on 24 Nov 2006 as manuscript CS20060236 Exploratory study: Role of PGI 2 and EDHF on radial artery FMD during AT 1 receptor blockade.

Figure 1 .

 1 Figure 1. Typical recording of the mean values of radial artery diameter (A) and blood flow (B) at baseline before cuff occlusion, during hand ischemia and after deflation of the cuff during hyperemia. Radial artery flow-mediated dilatation (FMD) was calculated as the percent change of radial artery diameter during hyperemia. In addition, peak flow and the area under the curve of flow (AUC of flow) were determined to characterize the hyperemic stimulus.

Figure 2 .

 2 Figure 2. Bar graphs show the values of radial artery peak flow (A) and the area under the curve (AUC) of flow (B) during reactive hyperemia in the placebo (open bars) and telmisartan groups (solid bars) before (control) and after infusion of L-NMMA. All results are mean ±SEM of 12 subjects.

Figure 3 .

 3 Figure 3. Bar graphs show the percent change from baseline of radial artery diameter in response to reactive hyperemia in the placebo (open bars) and telmisartan groups (solid bars) before (control) and after infusion of L-NMMA. All results are mean ±SEM of 12 subjects. *: P<0.05.

Figure 4 .

 4 Figure 4. Line graphs show radial artery blood flow (A) and diameter (B) measured at baseline and during the incremental dose of sodium nitroprusside in the placebo group before ( ) and after ( ) infusion of L-NMMA and in the telmisartan group before ( ) and after ( ) infusion of L-NMMA. All results are mean ±SEM of 12 subjects. P: dose effect and *: P<0.05 vs before L-NMMA.

  Figure 1

  Figure 4

Clinical Science Immediate Publication. Published on 24 Nov 2006 as manuscript CS20060236

Clinical Science Immediate Publication. Published on 24 Nov 2006 as manuscript CS20060236

Clinical Science Immediate Publication. Published on 24 Nov 2006 as manuscript CS20060236 factors

  and basal values of flow and diameter as a cofactor. A value of P<0.05 was considered statistically significant.

	Clinical

Science Immediate Publication. Published on 24 Nov 2006 as manuscript CS20060236

Clinical Science Immediate Publication. Published on 24 Nov 2006 as manuscript CS20060236

Clinical Science Immediate Publication. Published on 24 Nov 2006 as manuscript CS20060236

Clinical Science Immediate Publication. Published on 24 Nov 2006 as manuscript CS20060236

Clinical Science Immediate Publication. Published on 24 Nov 2006 as manuscript CS20060236

Table 1 .

 1 Randomized double-blind study: systemic hemodynamical parameters measured at baseline before and 2 hours after placebo and telmisartan administration Values are mean ±SEM of 12 subjects. SBP, DBP and MBP indicate respectively, systolic, diastolic and mean blood pressure, HR, heart rate.

	ANOVA (P)

Science Immediate Publication. Published on 24 Nov 2006 as manuscript CS20060236

Table 2 .

 2 Randomized double-blind study: systemic hemodynamical and radial artery parameters measured at baseline before and during NO-synthase inhibition in the placebo-and telmisartan-treated

	groups
						ANOVA (P)
	Parameters	Treatment	Control	L-NMMA	Period Treatment	Period x Treatment
	Mean blood pressure (mmHg)	Placebo Telmisartan	84 ± 2 83 ± 2	86 ± 2 85 ± 2	0.35	0.40	0.99
	Heart rate (bpm)	Placebo Telmisartan	60 ± 2 64 ± 3	60 ± 2 65 ± 3	0.30	0.91	0.90
	Radial artery flow (ml/min)	Placebo Telmisartan	13.7 ± 1.7 9.7 ± 0.8* 15.4 ± 2.0 12.3 ± 1.6*	0.03	0.90	0.70
	Radial artery diameter (mm)	Placebo Telmisartan 2.54 ± 0.06 2.53 ± 0.08 2.56 ± 0.06 2.56 ± 0.05	0.13	0.58	0.51
	Values are mean ±SEM of 12 subjects.

*: P<0.05 vs control.

Clinical Science

Immediate Publication. Published on 24 Nov 2006 as manuscript CS20060236

ACKNOWLEDGMENTS

This study was partly supported by a grant from the laboratory GlaxoSmithKline and was carried out with the help of the CIC-INSERM-Rouen. The authors thank Richard Medeiros (Medical Editor, Rouen University Hospital) for his valuable advice in editing the manuscript. Clinical Science Immediate Publication. Published on 24 Nov 2006 as manuscript CS20060236


Jan T Groothuis

Nynke Van Dijk

Walter Ter Woerds

Wouter Wieling

Maria Te

MD Maria Te Hopman
email: m.hopman@fysiol.umcn.nl

Leg crossing with muscle tensing, a physical counter-manoeuvre to prevent syncope, enhances leg blood flow

Keywords: orthostatic intolerance, physical counter-manoeuvres, leg blood flow, leg vascular conductance, head-up tilt Clinical Science Immediate

In patients with orthostatic intolerance the mechanisms to maintain blood pressure fail. A physical counter-manoeuvre to postpone or even prevent orthostatic intolerance in these patients is leg crossing combined with muscle tensing. Although the central haemodynamic effects of physical countermanoeuvres are well documented, not much is known about the peripheral haemodynamic events.

Therefore, the purpose of the present study was to examine the peripheral haemodynamic effects of leg crossing combined with muscle tensing during 70° head-up tilt (HUT). Thirteen healthy subjects were monitored for 10 minutes in the supine position followed by 10 minutes in 70° HUT and, finally, 2 minutes of leg crossing with muscle tensing in 70° HUT. Mean arterial blood pressure (MAP), heart rate, stroke volume, cardiac output and total peripheral resistance were measured continuously by Portapres. Leg blood flow was measured using Doppler ultrasound. Leg vascular conductance was calculated as leg blood flow divided by MAP. Results show a significant increase in MAP (13 mmHg), stroke volume (27%) and cardiac output (18%), a significant decrease in heart rate (-5 bpm) and no change in total peripheral resistance during the physical counter-manoeuvre when compared to baseline 70° HUT. A significant increase in leg blood flow (325 ml/min) and leg vascular conductance (2.9 AU) were seen during the physical counter-manoeuvre when compared to baseline 70° HUT. The present study indicates that the physical counter-manoeuvre of leg crossing combined with muscle tensing clearly enhances leg blood flow and at the same time elevates mean arterial blood pressure.

Introduction

Orthostatic stresses are common daily events in humans. In the upright position a gravitational displacement of blood from the thorax to the venous vascular beds of the legs, buttock and abdomen occurs. During orthostasis approximately 600 -700 ml of blood is transferred to the regions below the diaphragm [START_REF] Rowell | Reflex control during orthostasis[END_REF][START_REF] Smith | General responses to orthostatic stress[END_REF], which is known as 'venous pooling'. This results in a reduced venous return to the heart and a fall in central venous pressure with a consequent decrease in cardiac filling, stroke volume and cardiac output [START_REF] Smit | Pathophysiological basis of orthostatic hypotension in autonomic failure[END_REF]. The cardiovascular system in healthy humans is exquisitely adapted to maintain sufficient cardiac output in the upright posture. Neural pathways of the autonomic nervous system mediate rapid short-term adjustments to orthostatic stress, by increasing total peripheral resistance and heart rate. Concurrently, an increase in muscle tone in the legs prevents excessive pooling and promotes the venous return when upright [START_REF] Smit | Pathophysiological basis of orthostatic hypotension in autonomic failure[END_REF]. In patients with orthostatic intolerance these regulatory mechanisms fail to maintain blood pressure in the upright posture [START_REF] Rowell | Reflex control during orthostasis[END_REF][START_REF] Jardine | Autonomic control of vasovagal syncope[END_REF][START_REF] Krediet | Management of Vasovagal Syncope: Controlling or Aborting Faints by Leg Crossing and Muscle Tensing[END_REF][START_REF] Van Lieshout | Muscle Tensing During Standing : Effects on Cerebral Tissue Oxygenation and Cerebral Artery Blood Velocity[END_REF]. Patients with autonomic failure, who suffer from severe orthostatic hypotension due to failure of sympathetic neural vasomotor control [START_REF] Smit | Pathophysiological basis of orthostatic hypotension in autonomic failure[END_REF], can promote venous return and raise cardiac output by applying physical counter-manoeuvres that raise muscle skeletal tone, e.g. leg crossing and muscle tensing [START_REF] Smit | Pathophysiological basis of orthostatic hypotension in autonomic failure[END_REF][START_REF] Bouvette | Role of physical countermaneuvers in the management of orthostatic hypotension: efficacy and biofeedback augmentation[END_REF][START_REF] Harkel | Effects of leg muscle pumping and tensing on orthostatic arterial pressure: a study in normal subjects and patients with autonomic failure[END_REF][START_REF] Van Lieshout | Physical manoeuvres for combating orthostatic dizziness in autonomic failure[END_REF][START_REF] Van Lieshout | Syncope, cerebral perfusion, and oxygenation[END_REF].

Also patients with vasovagal syncope have successfully used these physical counter-manoeuvres to counteract their orthostatic intolerance [START_REF] Krediet | Management of Vasovagal Syncope: Controlling or Aborting Faints by Leg Crossing and Muscle Tensing[END_REF][START_REF] Van Lieshout | Muscle Tensing During Standing : Effects on Cerebral Tissue Oxygenation and Cerebral Artery Blood Velocity[END_REF][START_REF] Wieling | Nonpharmacological treatment of reflex syncope[END_REF]. Leg crossing and muscle tensing, lead to an increase in mean arterial blood pressure and postpone or even prevent syncope [START_REF] Krediet | Management of Vasovagal Syncope: Controlling or Aborting Faints by Leg Crossing and Muscle Tensing[END_REF][START_REF] Harkel | Effects of leg muscle pumping and tensing on orthostatic arterial pressure: a study in normal subjects and patients with autonomic failure[END_REF][START_REF] Wieling | Nonpharmacological treatment of reflex syncope[END_REF][12][13]].

It has recently been shown that the blood pressure raising effect of physical counter-manoeuvres in patients with orthostatic intolerance is solely due to an increase in cardiac output [12,13]. However, leg vascular resistance itself could be affected in several ways during performance of the physical counter-manoeuvre. On the one hand, it has been suggested that leg vascular resistance would increase as a consequence of compression of the large arteries in the legs by the squeezing manoeuvre [START_REF] Van Lieshout | Muscle Tensing During Standing : Effects on Cerebral Tissue Oxygenation and Cerebral Artery Blood Velocity[END_REF][START_REF] Wieling | Nonpharmacological treatment of reflex syncope[END_REF]14]. Some questions may be raised by this latter suggestion since a very high pressure is needed to compress the large arteries and compression will only occur at the sight of squeezing, i.e. a small part of the thigh and not the entire leg. On the other hand vasodilatation is known to occur during muscle

Methods

Subjects

Seventeen healthy, normotensive subjects, nine males and eight females, volunteered to participate in this study. The subjects' medical history did not reveal any episodes of vasovagal syncope, cardiovascular diseases or any other morbidity. None of the subjects used any medication and all subjects were non-smokers. All subjects gave written informed consent. The study has been carried out in accordance with the Declaration of Helsinki (2000) and was approved by the medical ethical committee of our institution.

Experimental procedures and protocol

Subjects refrained from caffeine, alcohol and nicotine for at least 12 hours and did not eat for 2 hours prior to testing. The experiment took place in the morning, in a quiet, temperature-controlled room (between 22°C and 24°C). Before the experiment the subjects received instructions on how to perform the physical counter-manoeuvre. They practised leg crossing combined with muscle tensing standing on the tilt table. This physical counter-manoeuvre consisted of crossing the right leg in front of the left leg at thigh level and pressing the legs firmly against each other. Simultaneously, the subjects were asked to tense the muscles of the legs and buttocks to their best ability, and to maintain this tension during the measurement. The physical counter-manoeuvre was performed while standing on both legs, distributing the body weight equally on both legs. Subjects were instructed to avoid straining during the physical counter-manoeuvre. [START_REF] Krediet | Management of Vasovagal Syncope: Controlling or Aborting Faints by Leg Crossing and Muscle Tensing[END_REF]13] Subjects were then placed in the supine position on a manually controlled tilt table with footboard in such manner that they could stand on the footboard in the head-up tilt position. The subjects were supported with a chest belt to prevent them from falling down during the experiment in case of vasovagal syncope. When presyncopal symptoms occurred the experiment was aborted and the subject was immediately tilted back to the supine position.

During a acclimatization period of minimal twenty minutes in the supine position the measurement apparatus was connected, where after the experimental protocol started with ten minutes in the supine Copyright 2006 The Biochemical Society position in order to establish steady state values. Subjects were subsequently tilted manually within five seconds to 70° head-up tilt. In this position subjects were instructed to relax, standing with the weight of the body equally distributed on both legs. After ten minutes in the head-up tilt position the subjects were asked to perform the physical counter-manoeuvre. The physical counter-manoeuvre was executed within two seconds. The physical counter-manoeuvre was continued for two minutes. Then the subjects were tilted back to the supine position and the experiment was ended.

Measurements

Arterial blood pressure and heart rate were continuously and non-invasively measured using Portapres (TNO, Amsterdam, the Netherlands), a portable blood pressure device. A finger cuff was attached to the middle phalanx of the third finger of the right hand in order to measure the finger arterial blood pressure and heart rate. Finger arterial blood pressure measurements accurately reflect intra-arterial blood pressure changes during orthostatic stress [18]. Data were collected during the experiment at a rate of 200 Hz. A built-in expert system, physiocal, was in operation during the entire experiment to establish and adjust the proper volume clamp set point. Mean arterial blood pressure (MAP) values were derived beat to beat. Heart rate was the inverse of the interbeat interval. Stroke volume (SV) was determined by a three-element model of arterial input impedance, using the Modelflow program, that provides accurate estimates of changes in SV during orthostatic stress [19]. Cardiac output (CO) was calculated as SV times heart rate, and total peripheral resistance was calculated as MAP divided by CO. Monitoring of CO needs calibration against a gold standard if accurate absolute values are required [START_REF] Van Lieshout | Muscle Tensing During Standing : Effects on Cerebral Tissue Oxygenation and Cerebral Artery Blood Velocity[END_REF]12]. Because the Modelflow measure was not calibrated in the present study, SV, CO and total peripheral resistance were expressed as relative change from steady state values in the head-up tilt position [12,20]. Changes in SV, CO and total peripheral resistance were calculated only in the head-up tilt position because there is some debate on using Modelflow with change of posture [21]. At the start of the physical counter-manoeuvre (Time = 0 in figures 2 and 4) Vmean was calculated every 5 seconds for a 20-seconds period and thereafter every 2 seconds. For diameter measurements at five minutes in supine and head-up tilt position, two consecutive longitudinal vessel images were analyzed at the peak-systolic and end-diastolic phase. The diameter of the common femoral artery was calculated as 1/3 • systolic + 2/3 • diastolic diameter [22]. Leg blood flow was calculated with the following formula: (π • r 2 • Vmean) • 60 (r = ½ • diameter of the common femoral artery). Leg blood flow was calculated in the supine position using the supine diameter, and in the upright position using the upright diameter. For the calculations the assumption that the femoral artery diameter did not change is crucial. This assumption was confirmed by a pilot study performed in our laboratory in five healthy young men (22-28 years). Right common femoral artery diameter (in cm) in supine: 9.6 ± 0.6, HUT: 9.8 ± 0.8 and physical counter-manoeuvre: 10.1 ± 0.7 (non-significant differences within the error margin of the ultrasound device (CV = 1.5 % [22])).

Leg vascular conductance was calculated as leg blood flow divided by MAP. We choose to use leg vascular conductance as it is the most appropriate parameter to quantify the functional importance of local responses in the regulation of blood pressure, when large changes in blood flow are present [START_REF] O'leary | Regional vascular resistance vs. conductance: which index for baroreflex responses?[END_REF].

For these calculations, we assumed that central venous pressure was near zero in the supine position and that hydrostatic pressure made an identical contribution to leg venous pressure as to leg arterial pressure.

Clinical Science Immediate Publication. Published on 20 Sep 2006 as manuscript CS20060241

Copyright 2006 The Biochemical Society

Data analysis

Steady state values of MAP, heart rate, SV, CO, total peripheral resistance, Vmean, leg blood flow and leg vascular conductance were averaged over the intervals 7 to 9.5 min in supine and head-up tilt position and 1.5 to 2 min in head-up tilt position combined with the physical counter-manoeuvre. SV, CO and total peripheral resistance in the head-up tilt position combined with the physical countermanoeuvre (interval 1,5 to 2 min) were also expressed as percentage change from steady state values in the head-up tilt position.

Statistical analyses

Results are expressed as mean ± SEM or as mean (95%CI). Differences in steady state values of MAP, heart rate, Vmean, leg blood flow and leg vascular conductance between the supine and head-up tilt position and differences in MAP, heart rate, SV, CO, total peripheral resistance, Vmean, leg blood flow and leg vascular conductance between the head-up tilt position and head-up tilt position combined with the physical counter-manoeuvre were tested with a two-tailed paired Student's t-test.

Differences in diameter of the common femoral artery between supine and the head-up tilt position were tested with a two-tailed paired Students t-test. Gender differences in leg blood flow and leg vascular conductance between the head-up tilt position and head-up tilt position combined with the physical counter-manoeuvre were tested with a two-tailed unpaired Student's t-test. A 2-sided probability value of < 0.05 was considered to be statistically significant.

Clinical Science Immediate Publication. Published on 20 Sep 2006 as manuscript CS20060241

Copyright 2006 The Biochemical Society

Results

Four subjects, one male and three females, could not complete the protocol, due to presyncopal symptoms, and had to be returned to the supine position before the physical counter-manoeuvre could be performed. This left thirteen subjects for analysis, eight males and five females. General characteristics of the thirteen subjects are shown in table 1.

Central parameters

A significant increase in heart rate (24 bpm (20 to 29)) (mean (95%CI)) was seen between the supine and head-up tilt position with no change in MAP (1 mmHg (-2 to 4). (Table 2, Figure 1). An immediate rise in MAP, SV and CO and a decline in total peripheral resistance were seen at the start of the physical counter-manoeuvre (Figure 1). After the rapid initial changes significant increases in steady state values were seen in MAP (13 mmHg (8 to 18)), SV (28 % (17 to 39)) and CO (21 % (10 to 31)), a significant decrease in heart rate (-5 bpm (-10 to -1)), but no change in total peripheral resistance (-3 % (-12 to 6)) (Table 2, Figure 1).

Peripheral parameters

A significant increase in diameter of the right common femoral artery (0.03 cm (0.00 to 0.07)) was seen between the supine and head-up tilt position, while significant decreases were seen in Vmean (-4.4 cm/s (-9.3 to -1.6)), leg blood flow (-165 ml/min (-291 to -30)) and leg vascular conductance (-1.8 (-3.7 to -0.2)) (Table 2, Figure 2 and3). An immediate rise in leg blood flow and leg vascular conductance in the right, actively crossed, leg was seen at the start of the physical counter-manoeuvre (Figure 2). The physical counter-manoeuvre in the head-up tilt position resulted in significant increases in steady state values of Vmean (7.4 cm/s (1.8 to 15.5)), leg blood flow (325 ml/min (82 to 691)) and leg vascular conductance (2.9 AU (0.8 to 6.1)) in the right common femoral artery (Table 2,

Discussion

The major findings of this study are that the physical counter-manoeuvre, leg crossing combined with muscle tensing, enhances leg blood flow and leg vascular conductance. Concurrently, the physical counter-manoeuvre increases mean arterial blood pressure and cardiac output with an unchanged total peripheral resistance. Thus the hypothesis that leg blood flow would increase during the physical counter-manoeuvre could be confirmed, however the increase in leg vascular conductance was not hypothesised.

Haemodynamic effects

The head-up tilt position causes a gravitational displacement of blood from the thorax to the venous vascular beds of the legs, buttock and abdomen, known as 'venous pooling'. SV decreases resulting in a decrease in CO and MAP [START_REF] Rowell | Reflex control during orthostasis[END_REF][START_REF] Van Lieshout | Muscle Tensing During Standing : Effects on Cerebral Tissue Oxygenation and Cerebral Artery Blood Velocity[END_REF][START_REF] Harkel | Effects of leg muscle pumping and tensing on orthostatic arterial pressure: a study in normal subjects and patients with autonomic failure[END_REF][START_REF] Arbeille | Cardiovascular echographic and Doppler parameters for the assessment of orthostatic intolerance[END_REF]25]. In order to maintain sufficient CO, sympathetic activation results in an increase in peripheral vasoconstriction and heart rate [START_REF] Rowell | Reflex control during orthostasis[END_REF][START_REF] Van Lieshout | Muscle Tensing During Standing : Effects on Cerebral Tissue Oxygenation and Cerebral Artery Blood Velocity[END_REF][START_REF] Harkel | Effects of leg muscle pumping and tensing on orthostatic arterial pressure: a study in normal subjects and patients with autonomic failure[END_REF][START_REF] Arbeille | Cardiovascular echographic and Doppler parameters for the assessment of orthostatic intolerance[END_REF][25][26][27]]. In the present study, no change in MAP with an increase in heart rate was seen between supine and the head-up tilt position (Table 2, Figure 1) and leg blood flow and leg vascular conductance decreased (Table 2, Figure 23).

Supine and head-up tilt leg blood flow values found in this study are in agreement with previous studies [START_REF] Rowell | Reflex control during orthostasis[END_REF][START_REF] Arbeille | Cardiovascular echographic and Doppler parameters for the assessment of orthostatic intolerance[END_REF][26][27][START_REF] Leyk | Leg blood flow during slow head-down tilt with and without leg venous congestion[END_REF].

In parallel with previous studies [START_REF] Van Lieshout | Muscle Tensing During Standing : Effects on Cerebral Tissue Oxygenation and Cerebral Artery Blood Velocity[END_REF][START_REF] Harkel | Effects of leg muscle pumping and tensing on orthostatic arterial pressure: a study in normal subjects and patients with autonomic failure[END_REF] we documented that leg crossing with muscle tensing during head-up tilt enhanced SV as well as CO and MAP, while heart rate decreased (Table 2, Figure 1). We attribute the increase in CO to the fact that leg crossing with muscle tensing raises intramuscular pressure of the skeletal muscles in the legs, which causes mechanical compression of the venous vascular beds. This will counteract the 'venous pooling' and translocate venous blood back to the thorax, resulting in an increase in cardiac filling, SV and CO [5-9, 12, 13]. This translocation of venous blood occurs in the first 30 seconds after the onset of the physical counter-manoeuvre [20]. This study is the first to report that, leg crossing with muscle tensing during head-up tilt increases leg blood flow (Table 2, Figure 2 and3). Several factors could be involved. First, blood flow increases instantly at the onset of dynamic exercise [29] and peaks within thirty seconds [20, [START_REF] Sheriff | Muscle pump function during locomotion: mechanical coupling of stride frequency and muscle blood flow[END_REF][31][START_REF] Delp | Control of skeletal muscle perfusion at the onset of dynamic exercise[END_REF] (Figure 2). However, in the present study the subjects did not perform sustained dynamic exercise but maintained isometric muscle tensing after the dynamic initial bout. Isometric exercise also increases blood flow by dilatation of the vasculature, but the dilatation is less pronounced compared to dynamic exercise. in A second explanation may be that leg blood flow increases as a result of an increase in the arteriovenous pressure gradient (P arterial-P venous) due to venous emptying and increase in MAP.

Hydrostatic pressure, caused by the upright position, is can be assumed to be equal on the venous and arterial side of the leg circulation. Therefore, leg blood flow (pressure difference divided by resistance) will increase due to an increase in MAP and a decrease in venous pressure [START_REF] Harkel | Effects of leg muscle pumping and tensing on orthostatic arterial pressure: a study in normal subjects and patients with autonomic failure[END_REF]29,31].

Thirdly, as a result of the instant venous emptying induced by leg crossing with muscle tensing, withdrawal of the veno-arteriolar axon 'reflex' could occur. If so, the consequent increase in leg vascular conductance [START_REF] Leyk | Leg blood flow during slow head-down tilt with and without leg venous congestion[END_REF][START_REF] Leyk | Early leg blood flow adjustment during dynamic foot plantarflexions in upright and supine body position[END_REF][START_REF] Tschakovsky | Venous emptying mediates a transient vasodilation in the human forearm[END_REF] will contributed to the increase in leg blood flow.

A fourth possible explanation is related to baroreflex activity. In response to a rise in MAP upon leg crossing with muscle tensing arterial baroreceptor activity increases, resulting in a decrease in heart rate, CO and total peripheral resistance [35]. In the present study, the physical counter-manoeuvre caused an increase in MAP with a slight decrease in heart rate. Total peripheral resistance, however, did not change (table 2, figure 1). If baroreceptor activity explains the increase in leg blood flow during leg crossing with muscle tensing, by causing a reflex increase in leg vascular conductance, then this should have been reflected in a decrease in total peripheral resistance. Leg vascular conductance increases about 2.5 fold, when the steady state values of head-up tilt with and without the physical counter manoeuvre are compared, however, the decrease in peripheral resistance is not reflected in the total peripheral resistance change between head-up tilt and head-up tilt with the physical counter-manoeuvre (Table 2). Total peripheral resistance is a computed central cardiovascular parameter (MAP divided by CO). It expresses total body peripheral resistance, which is the sum of vasodilatation in the active muscles of the legs and apparently vasoconstriction in other areas of the body, such as the nonworking muscles, kidneys and splanchnic vascular bed.

Gender differences

In accordance with the study of van Dijk et al. [12], who reported no gender differences in central haemodynamic effects, except for a larger increase in systolic blood pressure and pulse pressure in male individuals, when a physical counter-manoeuvre was applied in the head-up tilt position in patients with vasovagal syncope, we found no gender differences in peripheral haemodynamic effects, such as leg blood flow and leg vascular conductance changes induced by the physical countermanoeuvre (Figure 4). However, we must take the small sample size (male n = 8, female n = 5) into account.

Limitations

Doppler ultrasound measurements were performed on the right common femoral artery, the leg that was actively crossed over the left leg. One may argue that the results obtained in this study do not represent left leg peripheral haemodynamics directly. The physical counter-manoeuvre encompassed crossing the right leg in front of the left leg at thigh level and pressing them firmly against each other.

Simultaneously, the muscles of both legs and buttocks were tensed. The pressure applied is assumed to be similar in both legs resulting in partial compression of the thigh vascular bed. Although the anatomical site of pressure is different between both legs, i.e. right leg: medial-dorsal and left leg: For calculation of the leg blood flow during the physical counter-manoeuvre we used the diameter of the common femoral artery in the head-up tilt position. We assumed that the diameter would not change during the entire experiment [START_REF] Arbeille | Cardiovascular echographic and Doppler parameters for the assessment of orthostatic intolerance[END_REF]. Although we found a significant difference in diameter of the common femoral artery between supine and head-up tilt, this difference is within the error margin of the ultrasound method used (CV = 1.5 % [22]). It is not likely that the physical counter-manoeuvre did affect the diameter of the common femoral artery, because 1) the crossing of the legs occurred on thigh level and the ultrasound measurements were performed well above this level in the inguinal area and 2) to affect the diameter of a large conduit artery you will need a very high pressure and still it is probably more a deforming of than an actual change in diameter.

We studied healthy, normotensive subjects. However, physical counter-manoeuvres are usually advised to patients with orthostatic intolerance [START_REF] Van Lieshout | Physical manoeuvres for combating orthostatic dizziness in autonomic failure[END_REF][START_REF] Van Lieshout | Syncope, cerebral perfusion, and oxygenation[END_REF][START_REF] Wieling | Nonpharmacological treatment of reflex syncope[END_REF][12][13]. Patients with orthostatic intolerance show similar initial central and peripheral haemodynamic effects from supine to the upright posture, compared with healthy subjects [START_REF] Krediet | Management of Vasovagal Syncope: Controlling or Aborting Faints by Leg Crossing and Muscle Tensing[END_REF][START_REF] Harkel | Effects of leg muscle pumping and tensing on orthostatic arterial pressure: a study in normal subjects and patients with autonomic failure[END_REF]12,13,25,[36][START_REF] Wieling | Dynamics of circulatory adjustments to head-up tilt and tilt-back in healthy and sympathetically denervated subjects[END_REF][START_REF] Chandler | Haemodynamic responses during head-up tilt and tilt reversal in two groups with chronic autonomic failure: pure autonomic failure and multiple system atrophy[END_REF]. These patients show a drop in CO before the onset of syncope, starting at the onset of the prodromal symptoms. If a physical counter-manoeuvre is applied at the moment that prodromal symptoms occur, as is advised, the central haemodynamic effects are similar to healthy subjects [START_REF] Harkel | Effects of leg muscle pumping and tensing on orthostatic arterial pressure: a study in normal subjects and patients with autonomic failure[END_REF]13]. We therefore believe that the changes in peripheral parameters in the present study would also apply for patients with orthostatic intolerance.

Conclusion

The present study demonstrates that the physical counter-manoeuvre of leg crossing combined with muscle tensing clearly enhances leg blood flow and at the same time elevates mean arterial blood pressure. The largest and immediate effect seems to be achieved by the compression of the venous vascular bed at the onset of the physical counter-manoeuvre, translocating blood back to the thorax, simultaneously with this effect an enhanced arterio-venous pressure gradient and possibly withdrawal of the veno-arterial reflex may occur, explaining the observed increase in leg blood flow. Copyright 2006 The Biochemical Society

  Changes in Modelflow CO during leg crossing with muscle tensing are in excellent agreement with CO changes measured by inert gas breathing [12]. Mean red blood cell velocity (Vmean) and systolic and diastolic vessel diameter of the right common femoral artery were measured with a Doppler ultrasound device (Megas, ESAOTE, Firenze, Italy) Clinical Science Immediate Publication. Published on 20 Sep 2006 as manuscript CS20060241 Copyright 2006 The Biochemical Society with a 5-7.5 MHz broadband linear array transducer. Doppler ultrasound measurements were performed on the right leg, which is the actively crossed leg. The sample volume was placed in the middle of the vessel, ~ 2 cm proximal of the bifurcation of the common femoral artery into the deep and superficial femoral artery. The angle of inclination for the red blood cell velocity measurements was consistently below 60°, which is online visible on the screen, and the vessel area was adjusted parallel to the transducer. A single analyst manually traced the Doppler waveforms thereafter. Vmean was calculated as the average of 10 Doppler waveforms every 15 seconds in supine and HUT position.

Figure 2

 2 Figure 2 and 3).

10

 10

Femoral

  artery blood flow increases slightly during isometric contractions of the quadriceps muscle at 5 to 50 percent of maximal voluntary contraction (MVC) [15-17]. Sjogaard et al, reported that the increase in blood flow fell with increasing MVC, due to the increase in intramuscular pressure [16]. MVC was not measured in the present study, but young healthy individuals can persist 25 to 50 percent of MVC during a two-minute period before fatigue occurs. Femoral artery blood flow increases during this period by about 40 % [15-17]. Hence, only a minor part of the increase in leg blood flow in the present study (> 200% increase) could be explained by maintained muscle tensing.

Clinical

  Science Immediate Publication. Published on 20 Sep 2006 as manuscript CS20060241 Copyright 2006 The Biochemical Society Finally, central command, present at the start and during exercise, could have contributed to a vasodilation during the physical counter-manoeuvre.

Clinical

  Science Immediate Publication. Published on 20 Sep 2006 as manuscript CS20060241 Copyright 2006 The Biochemical Society medial-ventral, the results obtained for the right common femoral artery likely apply for the left common femoral artery as well.

Figure 1 .

 1 Figure 1. Central haemodynamic effects of leg crossing combined with muscle tensing in the head-up tilt position. Graphs represent mean ± SEM. n = 13. Mean arterial blood pressure (MAP), heart rate

Figure 2 .

 2 Figure 2. Peripheral haemodynamic effects of leg crossing combined with muscle tensing in the headup tilt position. Values represent mean ± SEM. n = 13. Leg blood flow (upper graph), and leg vascular

Figure 3 .

 3 Figure 3. Steady state values of leg blood flow (upper plot) and leg vascular conductance (lower plot)

Figure 4 .

 4 Figure 4. Leg vascular conductance effect of leg crossing combined with muscle tensing in the headup tilt position for male (▲, n = 8) and female (○, n = 5) subjects. Values represent mean ± SEM. The

Clinical Science Immediate Publication. Published on 20 Sep 2006 as manuscript CS20060241

Clinical Science Immediate Publication. Published on 20 Sep 2006 as manuscript CS20060241 Copyright 2006 The Biochemical Society van

  Dijk, N., de Bruin, I.G.J.M., Gisolf, J., et al. (2005) Hemodynamic effects of leg crossing and skeletal muscle tensing during free standing in patients with vasovagal syncope. J Appl Physiol

	98, 584-590
	13	Krediet, C.T.P., de Bruin, I.G.J.M., Ganzeboom, K.S., Linzer, M., van Lieshout, J.J. and
	Wieling, W. (2005) Leg crossing, muscle tensing, squatting, and the crash position are effective
	against vasovagal reactions solely through increases in cardiac output. J Appl Physiol 99, 1697-1703
	14	Brignole, M., Croci, F., Menozzi, C., et al. (2002) Isometric arm counter-pressure maneuvers
	to abort impending vasovagal syncope. J Am Coll Cardiol 40, 2053-2059
	15	Wesche, J. (1986) The time course and magnitude of blood flow changes in the human
	quadriceps muscles following isometric contraction. J Physiol 377, 445-462
	16	Sjogaard, G., Savard, G. and Juel, C. (1988) Muscle blood flow during isometric activity and
	its relation to muscle fatigue. Eur J Appl Physiol 57, 327-335
	17	Gaffney, F.A., Sjogaard, G. and Saltin, B. (1990) Cardiovascular and metabolic responses to
	static contraction in man. Acta Physiol Scand 138, 249-258
	18	Imholz, B.P.M., Wieling, W., van Montfrans, G.A. and Wesseling, K.H. (1998) Fifteen years
	experience with finger arterial pressure monitoring:: assessment of the technology. Cardiovasc Res
	38, 605-616
	19	Harms, M.P., Wesseling, K.H., Pott, F., Jenstrup, M., van Goudoever, J. and Secher, N.H.
	(1999) Continuous stroke volume monitoring by modelling flow from non-invasive measurement of
	arterial pressure in humans under orthostatic stress. Clin Sci 97, 291-301
	20	Wieling, W., Harms, M.P., ten Harkel, A.D.J., van Lieshout, J.J. and Sprangers, R.L.H. (1996)
	Circulatory response evoked by a 3 s bout of dynamic leg exercise in humans. J Physiol 494, 601-611
	21	Bogert, L.W.J. and van Lieshout, J.J. (2005) Non-invasive pulsatile arterial pressure and
	stroke volume changes from the human finger. Exp Physiol 90, 437-446
	22	de Groot, P.C.E., Poelkens, F., Kooijman, M. and Hopman, M.T.E. (2004) Preserved flow-
	mediated dilation in the inactive legs of spinal cord-injured individuals. Am J Physiol Heart Circ
	Physiol 287, H374-380

Clinical Science Immediate Publication. Published on 20 Sep 2006 as manuscript CS20060241 Copyright 2006 The Biochemical Society

Table Legends Table 1 .

 Legends1 General characteristics of the subjects. Values represent mean ± SEM. MAP, mean arterial blood pressure. * ,significantly different from male subjects.

Table 2 .

 2 Steady state values of central and peripheral haemodynamic parameters during supine, headup tilt and head-up tilt with physical counter-manoeuvre. n = 13. Values represent mean ± SEM. Stroke volume, cardiac output and total peripheral resistance are presented as absolute values. MAP, mean arterial blood pressure. AU, arbitrary units. Vmean, mean red blood cell velocity. *, significantly different from supine. †, significantly different from head-up tilt.

	Clinical

Science Immediate Publication. Published on 20 Sep 2006 as manuscript CS20060241

Table 1 .

 1

	All subjects	Male subjects	Female subjects
	(n = 13)	(n = 8)	(n = 5)

Clinical Science Immediate Publication. Published on 20 Sep 2006 as manuscript CS20060241 Copyright 2006 The Biochemical Society

Table 2 .

 2

		Supine	Head-up tilt	Leg crossing
	Central parameters
	MAP, mmHg	90.4 ± 3.2	89.3 ± 2.5	102.5 ± 3.4 †
	Heart rate, bpm	67.3 ± 2.3	91.8 ± 3.2*	86.4 ± 3.3 †
	Stroke volume, ml	92.4 ± 6.6	65.3 ± 5.1	82.6 ± 5.6
	Cardiac output, l/min	6.12 ± 0.47	5.93 ± 0.52	7.02 ± 0.50
	Total peripheral resistance, AU	0.960 ± 0.089	0.991 ± 0.089	0.941 ± 0.081
	Peripheral parameters
	Diameter femoral artery, cm	0.95 ± 0.04	0.98 ± 0.04*
	Vmean, cm/s	7.9 ± 0.8	3.5 ± 0.3*	10.8 ± 1.2 †
	Leg blood flow, ml/min	318.5 ± 25.2	153.7 ± 12.8*	478.7 ± 57.4 †
	Leg vascular conductance, AU	3.58 ± 0.32	1.75 ± 0.16*	4.60 ± 0.48 †

Clinical Science Immediate Publication. Published on 20 Sep 2006 as manuscript CS20060241 Copyright 2006 The Biochemical Society

	Figure 1
			Supine				Head-up tilt					Leg crossing
	110
	95
	80 mmHg	MAP
	7	7,5	8	8,5	9	9,5	7	7,5	8	8,5	9	9,5	0	0,1	0,2	0,3	1,5	1,6	1,7 Time (min) 1,8	1,9	2
	120
	80
	40 bpm	HR
	7	7,5	8	8,5	9	9,5	7	7,5	8	8,5	9	9,5	0	0,1	0,2	0,3	1,5	1,6	1,7 Time (min) 1,8	1,9	2
	180
	120
	60	SV
	7	7,5	8	8,5	9	9,5	7	7,5	8	8,5	9	9,5	0	0,1	0,2	0,3	1,5	1,6	1,7 Time (min) 1,8	1,9	2
	180
	120
	60 %bl	CO
	7	7,5	8	8,5	9	9,5	7	7,5	8	8,5	9	9,5	0	0,1	0,2	0,3	1,5	1,6	1,7 Time (min) 1,8	1,9	2
	180
	120
	60 %bl	TPR
	7	7,5	8	8,5	9	9,5	7	7,5	8	8,5	9	9,5	0	0,1	0,2	0,3	1,5	1,6	1,7 Time (min) 1,8	1,9	2

%bl Clinical Science Immediate Publication. Published on 20 Sep 2006 as manuscript CS20060241 Copyright 2006 The Biochemical Society

Acknowledgements

We would like to acknowledge the participation of all subjects in this study. In addition, we would like to thank Bregina Kersten and Jos Evers for assistance during the experiments. Clinical Science Immediate Publication. Published on 20 Sep 2006 as manuscript CS20060241 Copyright 2006 The Biochemical Society

counter-manoeuvre. AU, arbitrary units. Time = 0 indicates the start of the physical countermanoeuvre. Note the time scale is not continuous.