Maria Klaude
email: maria.klaude@ki.se

Katarina Fredriksson

Inga Tjäder

Folke Hammarqvist

Bo Ahlman

Olav Rooyackers

Jan Wernerman

Proteasome proteolytic activity in skeletal

Keywords: Protein degradation, Muscle wasting, Sepsis, Multiple organ dysfunction

come

Introduction

Patients with septic shock in the intensive care unit are prone to develop muscle wasting, a condition that is mainly manifested in skeletal muscle. The net loss of muscle proteins could either depend on decreased protein synthesis or an increase in protein degradation or a combination of both. Despite the progressive loss of muscle proteins during critical illness, fractional protein synthesis rate in skeletal muscle is on average within the range of healthy subjects [START_REF] Essen | Tissue protein synthesis rates in critically ill patients[END_REF][START_REF] Gamrin | Protein sparing effect in skeletal muscle of growth hormone treatment in critically ill patients[END_REF][START_REF] Tjader | Effects on skeletal muscle of intravenous glutamine supplementation to ICU patients[END_REF]. This indicates that muscle wasting in these patients is mainly caused by an increase in protein degradation.

The majority of intracellular proteins, including myofibrillar proteins in muscle, are degraded via the ubiquitin proteasome pathway [START_REF] Jagoe | What do we really know about the ubiquitinproteasome pathway in muscle atrophy?[END_REF]. Most proteins degraded by this pathway are first marked with a polyubiquitin chain in order to be degraded by the 26S proteasome. The 26S proteasome consists of two 19S regulatory domains and a central 20S protease core. The proteolytic activity comprises two chymotrypsin-like, two trypsin-like and two caspase-like active sites. The chymotrypsin-like activity is the rate-limiting step and initially cleaves the protein thereby stimulating the caspase-like activity [START_REF] Kisselev | Proteasome active sites allosterically regulate each other, suggesting a cyclical bite-chew mechanism for protein breakdown[END_REF]. Catabolic conditions have been associated with an increase in mRNA expression of several subunits of this pathway as well as an increase in the proteasome proteolytic activity. Increased levels of mRNA encoding ubiquitin and subunits of the 20S proteasome have been found in animal models for muscle wasting caused by fasting and denervation [START_REF] Medina | Increase in levels of polyubiquitin and proteasome mRNA in skeletal muscle during starvation and denervation atrophy[END_REF], sepsis [START_REF] Hobler | Activity and expression of the 20 S proteasome are increased in skeletal muscle during sepsis[END_REF], diabetes [START_REF] Price | Muscle wasting in insulinopenic rats results from activation of the ATP dependent ubiquitin-proteasome pathway by a mechanism including gene transcription[END_REF] and glucocorticoid treatment [START_REF] Auclair | Activation of the ubiquitin pathway in rat skeletal muscle by catabolic doses of glucocorticoids[END_REF]. Also studies of patients with different catabolic diseases such as trauma [10][START_REF] Biolo | Contribution of the ubiquitin-proteasome pathway to overall muscle proteolysis in hypercatabolic patients[END_REF], cancer [START_REF] Williams | The expression of genes in the ubiquitin-proteasome proteolytic pathway is increased in skeletal muscle from patients with cancer[END_REF][START_REF] Khal | Increased expression of proteasome subunits in skeletal muscle of cancer patients with weight loss[END_REF] and sepsis [START_REF] Tiao | Sepsis is associated with increased mRNAs of the ubiquitin-proteasome proteolytic pathway in human skeletal muscle[END_REF] have revealed increased mRNA levels of ubiquitin and proteasome subunits. The proteolytic activity of the proteasome can be studied in vitro in muscle extracts incubated with fluorogenic peptide substrates. In animal experiments increased activities were found after induction of sepsis or burn injury [START_REF] Hobler | Activity and expression of the 20 S proteasome are increased in skeletal muscle during sepsis[END_REF][START_REF] Fang | Burn injury upregulates the activity and gene expression of the 20S proteasome in rat skeletal muscle[END_REF]. Also in humans increased proteasome proteolytic activities were found in abdominal muscle from cancer patients [START_REF] Bossola | Increased muscle proteasome activity correlates with disease severity in gastric cancer patients[END_REF] and by our group in leg muscle from patients with sepsis and multiple organ failure [START_REF] Klaude | An assay of microsomal membrane-associated proteasomes demonstrates increased proteolytic activity in skeletal muscle of intensive care unit patients[END_REF].

In septic shock with a prolonged critical illness, the depletion of skeletal muscle is of increasing importance for morbidity and mortality. The majority of patients with septic shock treated in the ICU suffer from respiratory dysfunction and need mechanical ventilation. Many of these patients have difficulties in being weaned from the ventilator, thus prolonging the ICU treatment and increasing the incidence of pneumonia [START_REF] Tobin | Respiratory muscle dysfunction in mechanically-ventilated patients[END_REF]. The underlying causes of weaning difficulties might be attributed to muscle weakness due to atrophy. Today there are no efficient prophylactics or treatment for the muscle depletion although a number of strategies have been suggested over the years. The understanding of the underlying biochemical and molecular mechanisms for muscle protein degradation in catabolic patients is still limited. To be able to better predict and evaluate the outcome of therapeutic efforts for critically ill patients, it is therefore necessary to develop experimental approaches that increase our understanding of how the proteolytic machinery is regulated in the cell.

The aim of the present study was to measure the proteasome proteolytic activity simultaneously in respiratory and leg muscles in patients with septic shock requiring mechanical ventilation in comparison to healthy controls. In earlier studies we observed a large scatter of both protein synthesis rates and proteasome activity in leg skeletal muscle in septic patients as compared to control subjects [START_REF] Essen | Tissue protein synthesis rates in critically ill patients[END_REF][START_REF] Gamrin | Protein sparing effect in skeletal muscle of growth hormone treatment in critically ill patients[END_REF][START_REF] Tjader | Effects on skeletal muscle of intravenous glutamine supplementation to ICU patients[END_REF][START_REF] Klaude | An assay of microsomal membrane-associated proteasomes demonstrates increased proteolytic activity in skeletal muscle of intensive care unit patients[END_REF]. So a second aim of the present study was to elucidate the variation of proteasome activity in between duplicate muscle biopsy specimens and in between the individual subjects.

Materials and methods

Patients:

Two different experiments were performed in this study, both consisting of one group of patients with septic shock admitted to the general intensive care unit at Karolinska University Hospital Huddinge and another group of metabolically healthy, age and sex matched control patients undergoing elective surgery. In experiment II the control subjects were recruited at Ersta Hospital Stockholm. The study protocols conformed to the ethical guidelines of the 1975 Declaration of Helsinki and were approved by the Ethical Committee of Karolinska Institutet, Stockholm, Sweden. All patients, or for the ICU patients close relatives, gave informed consent to participate in the studies after receiving both oral and a standardised written information approved by the Ethical Committee.

Experiment I included ICU patients (n=10) with septic shock at admittance requiring mechanical ventilation and control patients (n=10) undergoing elective surgery. Muscle biopsies were taken from leg muscle and external respiratory muscle. Pre-existing neuromuscular disease, COPD, long-term pre-ICU corticosteroid treatment or severe coagulopathies not enabling muscle biopsies were exclusion criteria. All ICU patients suffered from respiratory dysfunction and multiple organ failure as indicated by the SOFA scores (Sepsis-related Organ Failure Assessment) [START_REF] Vincent | The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction/failure. On behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive Care Medicine[END_REF] at the day of study (Table 1). All patients had sepsis according to the Bone criteria [START_REF] Bone | Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee[END_REF]. All patients were sedated with propofol together with intermittent doses of analgesics. Mechanical ventilation was pressure support in 8 and pressure control in 2 patients. All patients were circulatory stable at the time of study and received low rate infusion of vasopressor drugs, if any. Three of the patients did not receive any glucocorticoid treatment, 4 of them received a substitution dose and 3 a therapeutic dose of short duration. Nutrition was given according to the routines of the unit with a target of 20-25 kcal/kg/24 h. Six patients received enteral nutrition, 3 received combined enteral and parenteral nutrition and one received parenteral nutrition only. Both Copyright 2006 The Biochemical Society from both the left and right leg. Patients with severe liver failure or undergoing dialysis or with impaired coagulation, not enabling muscle biopsies, were excluded from the study. None of the patients had a long-term, pre-ICU systemic corticosteroid treatment. All patients had sepsis according to the Bone criteria [START_REF] Bone | Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee[END_REF]. Characteristics of the ICU patients are given in table 1. According to the SOFA scores all patients except one had multiple organ failure. All patients were sedated with propofol together with intermittent doses of analgesics and required ventilator treatment. At the time of study all patients were circulatory stabilised, although all but three patients required vaso-pressor and/or inotropic support. Intravenous short-term corticosteroid treatment was given to four patients and another six patients were given corticosteroid substitution therapy. Antibiotics were given to all patients and nine patients were treated with more than one antibiotic. Anti fungal therapy was given to four patients. At the time of study, three of the patients had an indwelling thoracic epidural catheter with continuous infusion of local anaesthetics and short acting opioids. Total parental nutrition, enteral nutrition or combinations were given to 15 of the patients and the two remaining patients received 50 mg/ml and 100 mg/ml of glucose respectively. All patients received intravenous insulin to keep blood glucose between 4-8 mmol/L. Four of the patients did not survive (patient No 3, 12, 13, and 17) but died 40 days, 3 months, 2 weeks and 1 week respectively after the study. The control group was entirely separate from that in experiment I and consisted of 9 males and one female patient with a median age of 70 years (range 48-76).

They were all undergoing elective surgery for ventral hernia repair except for one male patient undergoing laparoscopic cholecystectomy.

Muscle biopsies: Leg muscle biopsies were taken percutaneously from the vastus lateralis using a Bergstrom needle. The biopsies were taken using local anaesthesia confined to the skin and fascia only. In the control patients, biopsies were taken just after induction of anaesthesia, but before surgery was started. In experiment I, the muscle covering the rib, mainly the anterior serratus muscle, was identified and used for sampling in order to prevent complications like pneumothorax and bleeding. The anterior serratus is considered as an accessory muscle for respiration [START_REF] Cerqueira | Electromyographic study of the pectoralis major, serratus anterior and external oblique muscles during respiratory activity in humans[END_REF]. Biopsies were taken by a surgeon through a 3-5 cm long incision at the level of the 6 th rib in the anterior axillar line. The biopsy was taken using local anaesthesia confined to the skin and subcutaneous tissue without infiltration in the muscle. Local anaesthesia was used in all patients, both the ICU patients with sedation and in the control patients preoperatively but after induction of general anaesthesia. A surgical biopsy of 50-200 mg of wet weight was taken. The biopsy site was sutured keeping attention to prevent bleeding. The incision was finally closed using intracutaneous technique. No complications were seen with regard to this technique. All biopsies were weighed within three minutes, frozen in liquid nitrogen and thereafter stored at -80° C until analysis.

Muscle biopsies were homogenised in a buffer (50 mM Tris-HCl pH 7.2, 1 mM EDTA, 100 mM KCl, 5 mM MgCl 2 1.8 mM ATP) to obtain a 5 % homogenate and then centrifuged at 700 × g for 10 min to remove cell debris. The resulting supernatant was centrifuged for 10 min at 15,000 × g. Glycerol was added to this supernatant (final concentration 10 % v/v) which was thereafter frozen in liquid nitrogen and stored at -80°C until analysis.

Haemoglobin concentrations in the homogenates were estimated by a Haemometer (Zeiss).

Assay of proteasome activity: Proteasome chymotrypsin-like activity was measured using the fluorogenic peptide substrate succinyl-leu-leu-val-tyr-7-amido-4-methylcoumarin (LLVY) (Sigma, St Louis, MO, USA). Triplicates of the 15,000 × g supernatant (10 µl, 10-20 µg of protein) were incubated in 50 µl of buffer (50 mM Tris-HCl pH 7.5, 1 mM ATP, 5 mM MgCl 2 , 1 mM DTT and 150 µM LLVY). In parallel incubations leupeptin or MG115 were added to the incubation system to a final concentration of 100 µM respectively (Both from Sigma, St Louis, MO, USA). As blank, LLVY was incubated in the absence of muscle extract. After incubation at 37°C for 45 min the reaction was stopped by adding 1 ml of 100 mM sodium acetate buffer, pH 4.3. Standard curves were prepared using 7-amino-4methylcoumarin (AMC) (Sigma, St Louis, MO, USA). In experiment I the fluorescence was measured at 342 nm excitation and 442 nm emission wavelengths with a Perkin-Elmer LS 50B and in experiment II at 380 nm excitation and 460 nm emission with a FLUOstar OPTIMA (BMG Labtechnologies) spectrofluorometer. The proteolytic activity was expressed as pmol of released AMC per µg of protein and min. In all experiments we chose to measure the chymotryptic activity since this is the rate limiting step of the protease activities in the proteasome [START_REF] Kisselev | Proteasome active sites allosterically regulate each other, suggesting a cyclical bite-chew mechanism for protein breakdown[END_REF] and also the peptide substrate used has been found to be the most proteasome specific substrate [START_REF] Rodgers | Assessment of proteasome activity in cell lysates and tissue homogenates using peptide substrates[END_REF].

Statistical methods: Students t-test was used to compare the proteasome activity between the ICU patients and their respective control groups. Values are given as means ± SD.

Results

In experiment I the proteolytic activity of proteasomes was measured in respiratory and leg muscles from patients with sepsis and control patients. In the respiratory muscle the proteasome activity was 30 % higher in the septic patients as compared to the activity in the control patients (0.24 ± 0.07 versus 0.18 ± 0.04 pmol/µg protein × min, P=0.04) (Fig 1). Also the leg muscle showed a similar pattern with a 45 % higher activity in the septic patients (0.29 ± 0.10 versus 0.20 ± 0.05 pmol/µg protein × min, P=0.02) (Fig 1).

In experiment II the variability of proteasome activity between individuals and between biopsy specimens within an individual was assessed in one group of patients with sepsis and one group of control patients. In accordance with experiment I, the scatter of proteasome activity between individuals was larger in the group of septic patients than in the controls (Fig 2). Biopsies were taken from the left and right leg of each subject to avoid interference from the 1 st biopsy taken and be able to take the muscle biopsies from the same position in the leg.

Compared to the control group the mean proteasome activity in the patients with sepsis was 0.16 ± 0.06 versus 0.10 ± 0.04 pmol/µg protein × min (P=0.003) in the left leg and 0.16 ± 0.06 versus 0.11 ± 0.02 pmol/µg protein × min (P=0.005) in the right leg (Fig 2). The intra individual variation in proteasome activity between the two biopsies taken from the legs was quite high in several of the subjects in both septic and control patients (Fig 3). Each individual muscle extract was assayed for proteasome activity in triplicates. The mean difference in activity between duplicate biopsies was 44 % in the septic patients and 52 % in the controls whereas the mean difference between the highest and lowest activity values in the triplicates was 17 % and 24 % respectively. This means that the difference in activity between biopsies cannot be explained by the variation in the method. The presence of blood in the muscle extracts differed between biopsies as judged by heamometer measurements which might contribute to the variation between the duplicate biopsies since proteasome activity was expressed per protein. In the septic patients haemoglobin concentration correlated negatively with the proteasome activity (r=-0.476, P<0.01, n=34) whereas no such correlation was found in the controls (r=0.250, NS, n=20).

All determinations of the proteasome proteolytic activity were performed in three parallel incubations: in the presence of the substrate LLVY only, with addition of the lysosomal inhibitor leupeptin or with addition of the proteasome inhibitor MG115. Leupeptin did not Copyright 2006 The Biochemical Society influence the proteasome activity whereas MG115 almost completely inhibited the activity during incubation, a pattern that was the same for all biopsies from both patients with sepsis and controls (Fig 4). This shows that the proteolytic activity measured in the assay represents the proteasome activity only.

Discussion

Catabolic conditions including sepsis, trauma, and cancer are associated with loss of skeletal muscle tissue [START_REF] Gamrin | Longitudinal changes of biochemical parameters in muscle during critical illness[END_REF]. In the present study two groups of ICU patients with sepsis and multiple organ failure were found to have increased proteasome proteolytic activity in both leg and respiratory muscles. Compared to control groups the mean proteasome activity of ICU patients was 30 % higher in the respiratory and 45-55 % higher in the leg muscle.

From the results in the present study it is not possible to ascertain whether the increased activity is a result of an increased number of proteasomes in the muscle cell or of an increased intrinsic proteolytic activity of the proteasomes. In a recent study of cancer patients with weight loss concomitant increases in mRNA levels and protein levels for the proteasome subunits C2 and C5 were found [START_REF] Khal | Increased expression of proteasome subunits in skeletal muscle of cancer patients with weight loss[END_REF]. Yet, other studies have failed to show a correlation between mRNA expression and translation products. After mitogen-induced proliferation of human T-lymphocytes, increased levels of mRNA for proteasome subunits C2, C3 and C5 were detected but no increase in the cellular content of proteasomes could be seen [START_REF] Schimbara | Regulation of gene expression of proteasomes (multi-protease complexes) during growth and differentiation of human hematopoietic cells[END_REF]. Also in a rat model of sepsis no correlation between mRNA expression and proteasome content in skeletal muscle was seen [START_REF] Hobler | Activity and expression of the 20 S proteasome are increased in skeletal muscle during sepsis[END_REF]. In mammalian cells the 20S proteasomes exist in several different forms which exhibit different proteolytic activities. Six different forms of proteasome subtypes have been separated from rat skeletal muscle [START_REF] Dahlmann | Subtypes of 20S proteasomes from skeletal muscle[END_REF]. After induction of diabetes mellitus a redistribution of proteasome subtypes was found in rat muscle [START_REF] Merforth | Alteration of 20S proteasome-subtypes and proteasome activator PA28 in skeletal muscle of rat after induction of diabetes mellitus[END_REF]. So the increase of proteasome activity seen in the ICU patients could be an effect of an increased number of proteasomes in the muscle cells or also be due to a redistribution of proteasome subtypes exhibiting a higher intrinsic proteolytic activity or maybe a combination of both.

Patients with sepsis and multiple organ failure usually suffer from respiratory impairment and therefore need mechanical ventilation. As many as 20 % of these patients, experience difficulties in weaning from the ventilator [START_REF] Tobin | Respiratory muscle dysfunction in mechanically-ventilated patients[END_REF]. Atrophy of respiratory muscles is thought to be one of the underlying causes of the development of respiratory difficulties. In a group of patients with mild to moderate chronic obstructive lung disease a reduction in diaphragm myosin heavy chain content together with an increase in ubiquitin-protein conjugation in diaphragm muscle homogenates indicated an enhanced protein degradation [START_REF] Ottenheijm | Diaphragm dysfunction in chronic obstructive pulmonary disease[END_REF]. On the other hand mechanical ventilation alone seems to induce respiratory muscle atrophy and thus may contribute to prolonged ventilation treatment. Studies of the diaphragm from ventilated rats show a decrease in myofibrillar protein and an increase in proteasome activity [START_REF] Shanely | Mechanical ventilationinduced diaphragmatic atrophy is associated with oxidative injury and increased proteolytic activity[END_REF][START_REF] Betters | Trolox attenuates mechanical ventilation-induced diaphragmatic dysfunction and proteolysis[END_REF]. Here Copyright 2006 The Biochemical Society we show that also patients with sepsis-induced multiple organ failure and needing mechanical ventilation, in addition to general muscle wasting, also had an increased proteasome activity in respiratory muscles. However, the muscle used in this study, serratus anterior, is an accessory respiratory muscle and results might be different in the main respiratory muscles i.e. diaphragm and intercostal muscles.

In experiment II the inter-and intra-individual variation of proteasome activity was investigated in a group of septic patients and a control group. The greater variation of proteasome activity in between septic patients, as compared to controls, may be dependent on the size of muscle mass by the onset of disease. One theory is that muscle wasting might be beneficial during sepsis supplying splanchnic organs and immune cells with increased amounts of amino acids released from skeletal muscle. If muscle mass is small a proportionally larger amount of muscle might have to be degraded in order to provide sufficient amounts of amino acids for other organs. It is known that lean body mass decrease by age, however, no correlation was found between age of the ICU patients and proteasome activity (r=-0.045, NS). Nor was there any correlation between proteasome activity and SOFA scores at study day (r=-0.286, NS). When looking upon length of ICU stay a correlation was found (r=0.621, P<0.01), but two patients that stayed 42 and 35 days respectively was the determining factor with their high activity. This correlation was not found in the rest of the 15 patients that had stayed between 1 to 7 days in the ICU(r=0.276, NS). Some of the patients received short-term glucocorticoid treatment at different doses and glucocorticoids are known mediators of muscle proteolysis [START_REF] Auclair | Activation of the ubiquitin pathway in rat skeletal muscle by catabolic doses of glucocorticoids[END_REF]. However, we found no correlation between proteasome activity and glucocorticoid dose when comparing patients with no treatment, low-dose substitution therapy and high-dose treatment (r=-0.132, NS). Of course ICU patients with sepsis have a great variety of underlying diseases that probably contributed to the large variation.

Fractional protein synthesis of total protein in leg muscle varies 6 % between duplicate biopsy specimens [START_REF] Tjader | Reproducibility in skeletal muscle protein synthesis rate in intensive care patients[END_REF]. The variation of proteasome activity between the two biopsy specimens in experiment II was significantly larger. It can probably not be explained by skewed distribution of fibre type between biopsies, since human leg muscle consists of a mixture of slow and fast fibre types that are evenly distributed within vastus lateralis [START_REF] Serrano | Immunolabelling, histochemistry and in situ hybridisation in human skeletal muscle fibres to detect myosin heavy chain expression at the protein and mRNA level[END_REF][START_REF] Lexell | Distribution of different fibre types in human skeletal muscles[END_REF]. The presence of blood in the biopsies can to some extent explain the result, at least in the septic patients, where high haemoglobin concentration in the homogenates correlated negatively Copyright 2006 The Biochemical Society with proteasome activity. However, no such correlation was found in the control patients. In both experiments, in the present study, we did not have access to large enough biopsy specimens and therefore decided to measure the proteasome activity in crude homogenate fractions (15,000 × g supernatants). Probably the variation between duplicate biopsies would decrease if activity is measured in fractions of proteasomes isolated by sequential ultra centrifugation as we did in an earlier study [START_REF] Klaude | An assay of microsomal membrane-associated proteasomes demonstrates increased proteolytic activity in skeletal muscle of intensive care unit patients[END_REF] but this method requires muscle biopsies of at least 70 mg. Nevertheless, in spite of the large variation between duplicate biopsies in both groups of study subjects in the present study, it was still possible to detect a statistically significant difference in proteasome activity between the septic patients and their controls.

Thus, proteasome activity measurements in crude extracts still could be an option in studies with limited size of biopsy specimens.

In conclusion, our results showed that proteasome activity was significantly increased in skeletal muscle during sepsis and multiple organ failure and we showed for the first time that both respiratory and leg muscles were affected. The increased proteasome activity in respiratory muscle indicates an accelerated protein degradation that may contribute to the respiratory dysfunction in patients that are treated in the intensive care unit. Since protein synthesis rate in skeletal muscle of these patients is not changed, the increased proteasome activity supports the hypothesis that muscle wasting during severe catabolic conditions like sepsis and multiple organ failure is the result of increased protein degradation.

  enteral and parenteral nutrition contained glutamine. Three of the patients did not survive. Patient No 5 died after one week and patients No 4 and 8 two months after the study. The control group consisted of 3 women and 7 men with a median age of 67 years (range 45-87) undergoing elective surgery for hernia repair, ilestomy closure, recurrent diverticulitis, or colorectal resection. One control patient had a malignant disease without evident spread. Experiment II included ICU patients with septic shock at admittance (n=17) and control patients (n=10) undergoing elective surgery. A muscle biopsy from vastus lateralis was taken Clinical Science Immediate Publication. Published on 22 Nov 2006 as manuscript CS20060265

Clinical

  Science Immediate Publication. Published on 22 Nov 2006 as manuscript CS20060265

Figure 1 :

 1 Figure 1: Proteasome proteolytic activity in muscle extracts from patients with sepsis (n=10) and controls (n=10). Biopsies were taken from respiratory and leg muscles. Each circle represents an individual subject. Horizontal lines indicate mean values. * Significantly different from mean control value P<0.05, Students t-test.

Figure 2 :

 2 Figure 2: Proteasome proteolytic activity in muscle extracts from patients with sepsis (n=17) and controls (n=10). Muscle biopsies were taken from the left and right leg. Each circle represents an individual subject. Horizontal lines indicate mean values. * Significantly different from mean control value P<0.01, Students t-test.

Figure 3 :

 3 Figure 3: Proteasome proteolytic activity in muscle extracts from the left and right leg in individual septic patients (1-17) and controls (1-10). Black bars represent the left leg and grey bars represent the right leg.

Figure 4 :

 4 Figure 4: Proteasome proteolytic activity in muscle extracts in the absence (LLVY) and presence of lysosomal (Leup) or proteasome (MG115) inhibitors expressed as percentage of LLVY value. Calculations were made from mean values of all subjects in Experiments I and II.

  Figure 1

Clinical Science Immediate Publication. Published on 22 Nov 2006 as manuscript CS20060265

Clinical Science Immediate Publication. Published on 22 Nov 2006 as manuscript CS20060265

Acknowledgements

The authors would like to thank Viveka Gustavsson at Karolinska University Hospital Huddinge, Ann-Sofie Andersson and Nina Johansson at Ersta Hospital for excellent nursing assistance.


Tom S Davies

Michael P Frenneaux

Ross I Campbell

Michael J White

Human arterial responses to isometric exercise: the role of the muscle metaboreflex

Keywords: arteries, exercise, sympathetic nervous system, Arterial stiffness and the muscle metaboreflex

The effects of exercise on the distensibility of large and medium sized arteries are poorly understood, but can be attributed to a combination of local vasodilator effects of exercise opposed by sympathetic vasoconstrictor tone. We sought to examine this relationship at the conduit artery level, with particular reference to the role of the sympathoexcitatory muscle metaboreflex. The effect of maintained muscle metaboreflex activation on a previously passive or exercised limb femoral artery was investigated. 10 healthy volunteers performed 2 minutes of isometric ankle plantar-flexion at 40% MVC, in conjunction with 2 minutes of either non-ischemic isometric handgrip (HG, control condition) or ischemic handgrip (IHG) at 40% MVC. IHG was followed by 2 minutes of post exercise circulatory occlusion (PECO) to maintain muscle metaboreflex activation. Femoraltibial pulse wave velocity (FTPWV) was measured in the exercised or contralateral limb at baseline and immediately following calf exercise. Blood pressure (BP) and heart rate (HR) were measured continuously throughout. In the HG condition, BP and HR returned promptly to baseline post exercise whilst exercised leg FTPWV was decreased (less stiff) by 0.6m/s (P<0.05) and non-exercised leg PWV was not changed from baseline. PECO caused a sustained increase in BP but not HR in the IHG condition. Contralateral leg PWV increased (stiffened) during PECO by 0.9m/s (P<0.05), whereas exercised limb FTPWV was not changed from baseline.

Muscle metaboreflex activation causes a systemic stiffening of the arterial tree, which can overcome local exercise induced decreases in arterial PWV.

Introduction

Changes in microvascular tone during exercise and the regulatory mechanisms responsible for these changes have been well described. These mechanisms ensure diversion of blood flow to exercising skeletal muscle [START_REF] Buckwalter | The Paradox of Sympathetic Vasoconstriction in Exercising Skeletal Muscle[END_REF]. In contrast, the effect of exercise on large artery function has not been extensively investigated. Large artery stiffness determines central aortic systolic and pulse pressure via the Windkessel effect and via effects on the timing of reflected waves to the central aorta [START_REF] Nichols | McDonald's Blood Flow in Arteries[END_REF]. Large artery distensibility declines with age, resulting in an increase in systolic and pulse pressures [START_REF] Benetos | Influence of age, risk factors, and cardiovascular and renal disease on arterial stiffness: clinical applications[END_REF]. The systolic hypertension resulting from these changes is a potent risk factor for cardiovascular disease (including myocardial infarction, heart failure and stroke) [START_REF] Safar | Current Perspectives on Arterial Stiffness and Pulse Pressure in Hypertension and Cardiovascular Diseases[END_REF]. Until recently large artery function was thought to relate virtually entirely to the structural characteristics of the vessel wall, reduced distensibility with age being a consequence of loss of elastin and collagen cross-linking [START_REF] Cameron | The relationship between arterial compliance, age, blood pressure and serum lipid levels[END_REF].

Over recent years there has been an increasing recognition that changes in the tone of the vascular smooth muscle in large arteries also influences their distensibility [START_REF] Kingwell | Spontaneous running increases aortic compliance in Wistar-Kyoto rats[END_REF][START_REF] Matsuda | Effects of exercise training on biochemical and biomechanical properties of rat aorta[END_REF][START_REF] Koutsis | Effects of an endurance training programme on the passive and noradrenaline-activated compliances of rat aorta[END_REF]. This tone is determined by endothelial, humoral and neural factors [START_REF] Ramsey | Endothelial Control of Arterial Distensibility Is Impaired in Chronic Heart Failure[END_REF]. Changes in large artery distensibility may have considerable impact on cardiac performance during exercise. Acute increases in impedance are known to cause an acute impairment of LV active relaxation, and it has been proposed that this may be an important mechanism responsible for the syndrome of Heart Failure with Preserved Left Ventricular Ejection Fraction [START_REF] Kawaguchi | Combined Ventricular Systolic and Arterial Stiffening in Patients With Heart Failure and Preserved Ejection Fraction: Implications for Systolic and Diastolic Reserve Limitations[END_REF][START_REF] Kass | Ventricular Arterial Stiffening: Integrating the Pathophysiology[END_REF].

In the present study we investigated the role of the skeletal muscle metaboreflex in controlling femoral-tibial arterial distensibility in the exercised and non exercised leg in healthy volunteers following isometric exercise. Previous studies have shown that the skeletal metaboreflex contributes to the vasoconstriction in non-exercising microvascular beds and restrains the microvascular dilation seen in exercising limbs [START_REF] Hansen | Muscle metaboreflex triggers parallel sympathetic activation in exercising and resting human skeletal muscle[END_REF]. However the impact of this reflex on large artery distensibility is currently unknown.

Isometric exercise lends itself to this type of study because the influence of the metaboreflex on cardiovascular function can be readily controlled [START_REF] Fisher | Muscle afferent contributions to the cardiovascular response to isometric exercise[END_REF]. It has long been established [START_REF] Alam | Observations in man upon a blood pressure raising reflex arising from the voluntary muscles[END_REF] that this reflex can be evaluated using the technique of post exercise circulatory occlusion (PECO), which traps metabolites in a previously exercised muscle and sustains the symapthoexcitatory stimulus at the same levels seen during exercise [START_REF] Victor | Reflex stimulation of sympathetic outflow during rhythmic exercise in humans[END_REF][START_REF] Fisher | Decreased muscle sympathetic nerve activity does not explain increased vascular conductance during contralateral isometric exercise in humans[END_REF]. Using this technique to manipulate systemic sympathoexcitation in a controlled fashion, whilst local Copyright 2006 The Biochemical Society dilator factors are still active in a previously exercised limb [START_REF] Kingwell | Arterial compliance increases after moderate-intensity cycling[END_REF][START_REF] Naka | Arterial distensibility: acute changes following dynamic exercise in normal subjects[END_REF][START_REF] Sugawara | The effects of low-intensity single-leg exercise on regional arterial stiffness[END_REF][START_REF] Sugawara | Effects of nitric oxide synthase inhibitor on decrease in peripheral arterial stiffness with acute low-intensity aerobic exercise[END_REF] allows their respective contributions to conduit artery function to be revealed. Additionally isometric exercise allows assessment of conduit artery function to be made immediately post-exercise (within 1 minute) using non invasive, pulse wave velocity (PWV) measures. This rapid assessment is not possible following dynamic exercise because of inevitable temporal delays and movement artefacts upon cessation of this exercise mode [START_REF] Kingwell | Arterial compliance increases after moderate-intensity cycling[END_REF][START_REF] Naka | Arterial distensibility: acute changes following dynamic exercise in normal subjects[END_REF][START_REF] Sugawara | The effects of low-intensity single-leg exercise on regional arterial stiffness[END_REF][START_REF] Sugawara | Effects of nitric oxide synthase inhibitor on decrease in peripheral arterial stiffness with acute low-intensity aerobic exercise[END_REF].

In the present study subjects performed a standardised level of ischemic handgrip exercise to evoke a reproducible level of systemic sympathetic vasoconstrictor drive and this was sustained with subsequent forearm PECO [START_REF] Victor | Sympathetic nerve discharge is coupled to muscle cell pH during exercise in humans[END_REF][START_REF] Fadel | Reflex sympathetic activation during static exercise is severely impaired in patients with myophosphorylase deficiency[END_REF]. We investigated the effect of simultaneous one legged isometric calf plantar flexor exercise on arterial stiffness immediately following the combined handgrip and leg exercise and during recovery with PECO. This was achieved by measuring PWV in the femoral-tibial arterial segment of the exercised and contralateral limbs. As a control, these PWV measures were repeated while subjects recovered from nonischemic handgrip without forearm PECO.

We hypothesised that in the control condition calf exercise would cause local arterial vasodilatation and therefore decrease femoral-tibial PWV in the active limb but not in the inactive limb. With the addition of sympathoexcitation, caused by forearm PECO, we hypothesised that passive limb femoral-tibial PWV would increase above resting levels.

Finally, we hypothesised that this systemic sympathoexcitation would override local vasodilator responses in the active limb and attenuate the decrease in PWV seen following calf exercise. Methods Subjects 10 subjects (5 male, 5 female) aged 22 ± 1 yr (mean ± standard error of the mean, SEM) volunteered to participate in this study. None smoked, were hypertensive (blood pressure >140/80mmHG), or were taking medication. All subjects gave informed written consent and were habituated with the experimental procedures, which were approved by the local ethics committee and conformed to the Declaration of Helsinki (2002). Subjects were asked to refrain from consuming food and caffeine in the three hours preceding the experiments. Before each trial, the subject rested for 10min in supine position to establish a stable baseline.

Each protocol began with 10min of baseline recordings after which the subject performed 2mins of isometric ankle plantar flexion at 40% MVC, in conjunction with 2mins of either non-ischemic isometric handgrip (HG, control condition) or ischemic handgrip (IHG) at 40% MVC. IHG was followed by 2mins PECO to maintain metaboreflex activation. PWV in the exercised or contralateral femoral-tibial arterial segment was measured at baseline and for 5mins immediately following calf exercise. A schematic diagram of the experimental protocol is shown in Figure 1.

Measured Variables

Continuous beat-to-beat BP was assessed using a non-invasive, bio-impedance monitor (Task Force® Monitor) at the middle finger of the left hand. A standard 3-lead electrocardiogram (ECG) was used to measure heart rate continuously during baseline, exercise and recovery periods. The monitor sampled at a rate of 1000Hz. Femoral-Tibial PWV (FTPWV) was measured simultaneously and non-invasively by oscillometry (time resolution +/-2ms; QVL P84, SciMed, Bristol, UK) using a method developed to identify waveforms by their early phase in order to improve wave recognition and timing. This technique has been shown to give within day (minute to minute variation) of 2.2% and between day reproducible (10 minute averaged) of 5.6% [START_REF] Naka | Arterial distensibility: acute changes following dynamic exercise in normal subjects[END_REF]. Non-occlusive cuffs were placed over the mid thigh (femoral) and ankle (tibial), and connected to computerised pressure transducers by non-compliant tubing. The cuffs were inflated to 65-70mmHg and pulse pressure waveforms caused by volume displacement were obtained from each cuff.

Waveforms were characterised by a computer programme with respect to time at 30, 40 and Copyright 2006 The Biochemical Society 50% of peak pressure along their ascending phase. The programme was designed to discard ectopic beats and abnormally shaped waveforms. The transit time between cuffs was calculated using an average of the three points (30, 40 and 50%), for 15 continuous beats.

PWV was derived as the distance between the proximal edges of each cuff (measured around the flexed knee joint) divided by pulse transit time. It is important to note that neither cuff or leg was allowed to move during either protocol. HR and BP during corresponding periods of PWV measurement (1min) were calculated from the Task Force data.

Average values for cardiovascular variables and PWV were calculated over the whole 10 minute baseline period and each successive minute of the 5 minute recovery period. In addition average values for blood pressure and heart rate at the end of the exercise period (last 10 seconds of exercise) were also calculated.

Statistical Analysis

All values are expressed as mean ± SEM. Statistical analysis was performed using repeated measures ANOVA and post hoc analysis using paired t-tests with Bonferroni correction.

Significance levels were set at P<0.05.

Results

Control Condition

Table 1 shows average heart rate and blood pressure values for the baseline, end exercise, and recovery phases of the trials performed to examine PWV in the previously exercised and contralateral limbs. Heart rate and blood pressure increased markedly during exercise and to the same extent in both trials, and in both trials heart rate and blood pressure returned promptly to baseline levels within the first minute of recovery and remained at these levels thereafter.

FTPWV data is shown in Figure 2. It is clear that immediately following combined calf and handgrip exercise, when blood pressure had recovered to baseline, FTPWV in the previously exercised leg had declined (p<0.05) by 0.6m/s, 0.6m/s and 0.5m/s relative to baseline, during minutes 1, 2 and 3 respectively. By minute 4 of recovery FTPWV had returned to baseline levels. In contrast, in the contralateral leg FTPWV was not significantly changed from baseline after the combined calf and handgrip exercise.

IHG Condition

During exercise, heart rate and blood pressure increased in both trials. During PECO, heart rate returned to baseline, whilst blood pressure remained significantly elevated in both IHG trials. Following PECO, during the 3rd minute of recovery, blood pressure returned to baseline and in both trials remained unchanged thereafter.

During the two minutes of PECO which followed combined calf and ischaemic handgrip exercise and when blood pressure was markedly elevated above baseline, FTPWV in the exercised limb was not significantly changed from baseline (p=0.1) Upon release of PECO and recovery of blood pressure to resting levels, FTPWV remained statistically indistinguishable from baseline.

In the contralateral limb, FTPWV was initially increased during PECO (p<0.05) by 0.8m/s and 0.9m/s, in the first and second minutes, respectively, of recovery. It then returned to baseline upon release of the occlusion. The increase in FTPWV in this limb during PECO was in complete contrast to the unchanged FTPWV values seen in the exercised leg during PECO. It also contrasts with unaltered FTPWV in the contralateral limb in the absence of PECO in the control condition.

Discussion

The present study investigated the acute effects of isometric exercise on large (conduit) artery distensibility. Immediately after exercise PWV was measured in the contralateral or exercised femoral artery to examine the influence of systemic and local factors on arterial function. In addition, the ability of exercise induced sympathoexcitation, mediated by muscle metaboreflex activation, to alter these responses was investigated.

Femoral Artery Responses to Isometric Calf Exercise

In the absence of PECO, required to sustain sympathoexcitation and elevated BP, arterial distensibility of the exercised limb was augmented for three minutes following calf exercise. This data is in accordance with all aerobic exercise studies to date [START_REF] Kingwell | Arterial compliance increases after moderate-intensity cycling[END_REF][START_REF] Naka | Arterial distensibility: acute changes following dynamic exercise in normal subjects[END_REF][START_REF] Sugawara | The effects of low-intensity single-leg exercise on regional arterial stiffness[END_REF][START_REF] Sugawara | Effects of nitric oxide synthase inhibitor on decrease in peripheral arterial stiffness with acute low-intensity aerobic exercise[END_REF]. One idea used to explain the increase in arterial distensibility post exercise is that it is caused by exercise-related regional factors. Although these factors have yet to be fully elucidated, local vasodilator mechanisms involving the muscle vascular endothelium and limited retrograde travel of dilator signals have been postulated [START_REF] Kingwell | Arterial compliance increases after moderate-intensity cycling[END_REF][START_REF] Naka | Arterial distensibility: acute changes following dynamic exercise in normal subjects[END_REF][START_REF] Sugawara | The effects of low-intensity single-leg exercise on regional arterial stiffness[END_REF][START_REF] Sugawara | Effects of nitric oxide synthase inhibitor on decrease in peripheral arterial stiffness with acute low-intensity aerobic exercise[END_REF] Copyright 2006 The Biochemical Society vasodilatation has occurred then flow in the femoral artery will increase and therefore cyclic wall stress on its endothelium will follow. This is a well known vasodilator stimulus [START_REF] Pyke | The relationship between shear stress and flow-mediated dilatation: implications for the assessment of endothelial function[END_REF] which would facilitate the increased muscle blood flow. Therefore the observed increase in distensibility immediately post exercise in the control condition could be attributed to endothelium dependent, vasodilation. An alternative idea to explain post exercise increases in arterial distensibility is that an exercise driven increase in sympathetic vasoconstrictor tone is decreased at this time (see below). Certainly systemic sympathoexcitation caused by muscle metaboreflex activation during the handgrip and calf exercise would be expected to oppose vasodilatation during exercise [START_REF] Hansen | Muscle metaboreflex triggers parallel sympathetic activation in exercising and resting human skeletal muscle[END_REF][START_REF] Victor | Sympathetic nerve discharge is coupled to muscle cell pH during exercise in humans[END_REF][START_REF] Fadel | Reflex sympathetic activation during static exercise is severely impaired in patients with myophosphorylase deficiency[END_REF]. However, rapid wash out of the metabolites on cessation of exercise removes this reflex response and sympathetic activity quickly returns to baseline, as is supported by the rapid recovery of BP in the control experiments. Finally, the fact that no change was seen in FTPWV in the contralateral limb following exercise, even though it would have experienced the same increased level of sympathetic drive during exercise as the exercise limb [START_REF] Hansen | Muscle metaboreflex triggers parallel sympathetic activation in exercising and resting human skeletal muscle[END_REF], provides further support for the idea that the decrease in PWV in the exercise limb is likely dominated by local factors.

The Effects of Metaboreflex Activation

Sympathetic nerve activity has been reported to exert a tonic stiffening influence on the arterial walls [START_REF] Gerova | Range of the sympathetic control of the dog femoral artery[END_REF][START_REF] Dobrin | Vascular mechanics[END_REF]. Arterial distensibility can be reduced by an increased sympathetic drive, as shown by the effects on the radial and carotid arteries during cold-pressor and mental arithmetic tests [START_REF] Boutouyrie | Sympathetic activation decreases medium-sized arterial compliance in humans[END_REF], and smoking [START_REF] Failla | Effects of cigarette smoking on carotid and radial artery distensibility[END_REF], i.e. manoeuvres that cause sympathetic activation. A period of circulatory occlusion following isometric exercise has been widely used to maintain muscle metaboreflex activation and cause sympathoexcitation [START_REF] Wallin | Sympathetic outflow to resting muscles during static handgrip and postcontraction muscle ischemia[END_REF]. PECO entraps the metabolic results of exercise and therefore sustains afferent feedback from the exercised muscle [START_REF] Alam | Observations in man upon a blood pressure raising reflex arising from the voluntary muscles[END_REF]. Muscle metaboreflex activation typically evokes parallel sympathetic activation in exercising and resting human skeletal muscle [START_REF] Hansen | Muscle metaboreflex triggers parallel sympathetic activation in exercising and resting human skeletal muscle[END_REF] that maintains or elevates arterial pressure [START_REF] Lind | The Circlatory Effects of Sustained Voluntary Muscle Contraction[END_REF].

In the present study, elevated sympathetic outflow was associated with a stiffening of the arterial tree, as observed in the increased FTPWV of the contralateral limb femoral artery during PECO. The decrease in femoral artery distensibility in the contralateral limb during PECO, mirroring the BP rise at this time, is consistent with sustained muscle metaboreflexmediated vasoconstriction. This data, to our knowledge, is the first to show a stiffening of the large conduit arteries during muscle metaboreflex activation. As the muscles were relaxed Copyright 2006 The Biochemical Society and there was no intention to perform exercise, we can rule out any contribution from muscle mechanoreflex or central command.

Local Dilatory Mechanisms Vs Increased Sympathetic Outflow

The interaction between functional vasodilation and sympathetic vasoconstriction and the phenomenon of "functional sympatholysis" has long driven studies of blood flow control [START_REF] Buckwalter | The Paradox of Sympathetic Vasoconstriction in Exercising Skeletal Muscle[END_REF][START_REF] Remensnyder | Functional sympatholysis during muscular activity. Observations on influence of carotid sinus on oxygen uptake[END_REF][START_REF] Hansen | Differential Sympathetic Neural Control of Oxygenation in Resting and Exercising Human Skeletal Muscle[END_REF][START_REF] Tschakovsky | Is sympathetic neural vasoconstriction blunted in the vascular bed of exercising human muscle?[END_REF][START_REF] Dinenno | Blunted sympathetic vasoconstriction in contracting skeletal muscle of healthy humans: is nitric oxide obligatory?[END_REF]. Of particular interest to the present study are recent findings that the interaction between sympathetic nerve activity and vasodilatory stimuli seems to vary with vessel branch order [START_REF] Haug | Sympathetic neural inhibition of conducted vasodilatation along hamster feed arteries: complementary effects of {alpha}1-and {alpha}2-adrenoreceptor activation[END_REF]. The present study measures femoral-tibial arterial PWV between the mid thigh and ankle. As subjects performed isolated calf exercise these measures will provide information about the artery passing through the exercising muscle and also the conduit artery (mid femoral) proximal to the exercising muscle itself.

The ability of muscular contractions to limit the amount of vasoconstriction appears to be a local regulatory mechanism, particularly arsing in the arterioles embedded within active muscle fibres, to ensure adequate blood flow and oxygen delivery to the contracting muscle [START_REF] Anderson | Differential sensitivity of arteriolar alpha 1-and alpha 2-adrenoceptor constriction to metabolic inhibition during rat skeletal muscle contraction[END_REF][START_REF] Vanteeffelen | Interaction between sympathetic nerve activation and muscle fibre contraction in resistance vessels of hamster retractor muscle[END_REF]. Further upstream however, vasoconstriction is sustained, as sympathetic nerve activity seems able to impair ascending vasodilation of the feed arteries [START_REF] Haug | Sympathetic neural inhibition of conducted vasodilatation along hamster feed arteries: complementary effects of {alpha}1-and {alpha}2-adrenoreceptor activation[END_REF][START_REF] Anderson | Differential sensitivity of arteriolar alpha 1-and alpha 2-adrenoceptor constriction to metabolic inhibition during rat skeletal muscle contraction[END_REF][START_REF] Vanteeffelen | Interaction between sympathetic nerve activation and muscle fibre contraction in resistance vessels of hamster retractor muscle[END_REF][START_REF] Folkow | Loci of neurogenic and metabolic effects on precapillary vessels of skeletal muscle[END_REF]. Data from the present study further extends this idea to the large conduit artery level, as following exercise with PECO; metaboreflex activation abolished the increase in distensibility that was seen in the femoral-tibial arterial segment of the exercised limb in the control condition. If this observation can be extrapolated to exercise conditions, then it suggests that the proximal branches of the network serve as key sites for regulating the distribution of cardiac output and the maintenance of arterial pressure.

The Potential Influence of Blood Pressure Changes on PWV

Arterial compliance is reduced with a rise in mean arterial pressure (MAP) [START_REF] Liu | Estimation of total arterial compliance: an improved method and evaluation of current methods[END_REF]. Therefore changes in arterial compliance occurring in the presence of MAP changes may simply be a consequence of the nonlinearity of the vascular pressure-volume relationship rather than an intrinsic change in vessel wall properties [START_REF] Liu | Estimation of total arterial compliance: an improved method and evaluation of current methods[END_REF]. It is therefore difficult to exclude the possibility that the rise in MAP seen during PECO will account for any increases seen in PWV during this period. However, despite similar MAP increases during both IHG protocols, the PWV responses of exercised and contralateral femoral arteries were markedly different. It is therefore unlikely that the arterial responses observed were entirely BP dependent.

Method of PWV Measurement

Pulse transit time measurement critically depends on accurately identifying potential changes in the waveform. Conventionally this is achieved by measuring the timing of the foot of the wave. The present technique, which characterises and times the waveform by averaging 3 points along the first 50% of its rising phase during which its upstroke should be relatively uncontaminated by reflected waves and wave propagation [START_REF] Mcdonald | Regional pulse wave velocity in the arterial tree[END_REF], and is likely to bring advantage over techniques relying on the identification of a single point [START_REF] Naka | Arterial distensibility: acute changes following dynamic exercise in normal subjects[END_REF]. PWV measured with the present technique has been shown to be sensitive to acute changes of vascular tone, independently of any associated change in blood pressure. PWV in the upper limb, for example, is decreased or increased ~10% by local intra-arterial acetylcholine or NGmonomethyl-L-arginine, whereas lower limb PWV, heart rate, and blood pressure are unchanged [START_REF] Ramsey | Endothelial Control of Arterial Distensibility Is Impaired in Chronic Heart Failure[END_REF][START_REF] Kinlay | Endothelium-Derived Nitric Oxide Regulates Arterial Elasticity in Human Arteries In Vivo[END_REF][START_REF] Naka | Changes in pulse wave velocity in large arteries are NO-mediated (Abstract)[END_REF]. It is acknowledged, that a variety of other techniques exist which can be employed to assess the mechanical properties of arteries, e.g. the windkessel 'area method' approach of Liu et al. [START_REF] Liu | Estimation of total arterial compliance: an improved method and evaluation of current methods[END_REF] referred to above. However it is difficult to compare the different indices produced by these methods with changes in PWV. Nevertheless, in the present study PWV was measured over the same defined sections of the femoral-tibial arterial segment before and immediately after exercise. The results therefore provide evidence of relative arterial distensibility changes, and as such the technique provides an objective, sensitive but robust and reproducible measure of acute changes in arterial distensibility as influenced by changes in vascular tone [START_REF] Naka | Arterial distensibility: acute changes following dynamic exercise in normal subjects[END_REF]

Conclusions

Isometric exercise induced an acute, local augmentation in large artery distensibility.

Increased sympathetic outflow during muscle metaboreflex activation causes a systemic stiffening of the arterial tree. This sympathetically induced stiffening is able to overcome the local increase in distensibility in an exercised artery.

  four experiments in a randomised manner. Subjects lay supine in a custom built isometric dynamometer designed to measure ankle plantar flexion force. The subjects were positioned with the right knee flexed by 50 deg, the ankle flexed at 90 deg, and the foot strapped to a footplate. Straps were aligned around the ankle in order to minimise lifting of the heel away from the footplate when performing plantar flexor exercise. Isometric handgrip exercise was performed with the right hand on a Lafayette hand dynamometer. Maximal voluntary force (MVC) was determined prior to each study, with each exercise mode.

Clinical

  Science Immediate Publication. Published on 6 Dec 2006 as manuscript CS20060276

Clinical

  Science Immediate Publication. Published on 6 Dec 2006 as manuscript CS20060276 Copyright 2006 The Biochemical Society Furthermore in the control condition, exercised leg FTPWV significantly decreased despite unchanged BP during the recovery period. Changing pressure cannot thus be wholly responsible for the observed changes in FTPWV seen during these experiments. The PWV responses thus appear to reflect acute changes in vascular tone, which occur during recovery from exercise.

Figure 2 .

 2 Figure 2.

Figure 2 .

 2 Figure 2. FTPWV in exercised and contralateral limbs following calf and handgrip exercise (top) or following calf and ischemic handgrip exercise with PECO (IHG) (bottom). Dashed vertical lines indicate PECO period. * indicates significantly different from respective baseline value.

Clinical Science Immediate Publication. Published on 6 Dec 2006 as manuscript CS20060276

Clinical Science Immediate Publication. Published on 6 Dec 2006 as manuscript CS20060276

  . Once local intramuscular

Clinical Science Immediate Publication. Published on 6 Dec 2006 as manuscript CS20060276

Clinical Science Immediate Publication. Published on 6 Dec 2006 as manuscript CS20060276 Copyright 2006 The Biochemical Society TablesTable 1 .

 1 Blood pressure and heart rate changes following combined calf and handgrip exercise (control conditions) or following calf and ischemic handgrip with PECO (IHG conditions).

	Baseline

End Ex Post 1 Post 2 Post 3 Post 4 Post 5 SBP (mmHg)

	Exercised	116±4	148±6* 123±4 117±4 115±5 113±5 112±5
	Contralateral	119±3	149±9* 130±8 126±6 125±4 125±3 126±4
	Contralateral (IHG) 121±3	152±7* 138±5* 135±4* 125±3 121±3 121±3
	Exercised (IHG)	120±3	163±7* 146±6* 144±6* 132±6 127±6 124±5
	DBP (mmHg)
	Exercised	67±2	92±3*	66±3	63±2	61±3 59±3 59±3
	Contralateral	65±2	89±3*	71±3	69±3	68±2 68±3 70±3
	Contralateral (IHG) 70±2	96±3*	79±3* 80±2* 70±2 67±2 67±2
	Exercised (IHG)	65±2	99±7*	80±4* 81±4* 70±5 66±4 65±4
	HR (bpm)
	Exercised	64±3	87±4*	71±3	62±3	61±3 60±3 60±3
	Contralateral	64±5	86±6*	72±5	62±5	60±5 61±5 61±4
	Contralateral (IHG) 65±4	90±4*	71±5	62±4	62±3 60±3 60±3
	Exercised (IHG)	64±3	88±4*	72±4	65±4	63±4 62±3 59±3
	SBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate;

* indicates significantly different from respective baseline value.

Clinical Science

Immediate Publication. Published on 6 Dec 2006 as manuscript CS20060276 Copyright 2006 The Biochemical Society Figures Figure 1.

  Schematic of experimental protocol. HG, handgrip; IHG, ischemic handgrip; PECO, post exercise circulatory occlusion.

	Figure 1. Clinical	FPWV measurement IHG condition: Control condition: Baseline Baseline	HG Calf Ex. IHG Calf Ex. 2 mins	PECO 2 mins FPWV measurement Recovery Recovery

Science Immediate Publication. Published on 6 Dec 2006 as manuscript CS20060276 Copyright 2006 The Biochemical Society