Showing papers in "Acta Physiologica Scandinavica in 2003"

Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research

Abstract: Electroporation designates the use of short high-voltage pulses to overcome the barrier of the cell membrane. By applying an external electric field, which just surpasses the capacitance of the cell membrane, transient and reversible breakdown of the membrane can be induced. This transient, permeabilized state can be used to load cells with a variety of different molecules, either through simple diffusion in the case of small molecules, or through electrophoretically driven processes allowing passage through the destabilized membrane--as is the case for DNA transfer. Initially developed for gene transfer, electroporation is now in use for delivery of a large variety of molecules: From ions to drugs, dyes, tracers, antibodies, and oligonucleotides to RNA and DNA. Electroporation has proven useful both in vitro, in vivo and in patients, where drug delivery to malignant tumours has been performed. Whereas initial electroporation procedures caused considerable cell damage, developments over the past decades have led to sophistication of equipment and optimization of protocols. The electroporation procedures used in many laboratories could be optimized with limited effort. This review (i) outlines the theory of electroporation, (ii) discusses factors of importance for optimization of electroporation protocols for mammalian cells, (iii) addresses particular concerns when using electroporation in vivo, e.g. effects on blood flow and considerations regarding choice of electrodes, (iv) describes DNA electrotransfer with emphasis on use in the in vivo setting, and (v) sums up data on safety and efficacy of electroporation used to enhance delivery of chemotherapy to tumours in cancer patients.

Amazing chloride channels: an overview

Abstract: Aim: This review describes molecular and functional properties of the following Cl - channels: the ClC family of voltage-dependent Cl - channels, the cAMP-activated transmembrane conductance regulator (CFTR), Ca 2+ activated Cl - channels (CaCC) and volume-regulated anion channels (VRAC). If structural data are available, their relationship with the function of Cl - channels will be discussed. We also describe shortly some recently discovered channels, including high conductance Cl - channels and the family of bestrophins. We illustrate the growing physiological importance of these channels in the plasma membrane and in intracellular membranes, including their involvement in transepithelial transport, pH regulation of intracellular organelles, regulation of excitability and volume regulation. Finally, we discuss the role of Cl - channels in various diseases and describe the pathological phenotypes observed in knockout mice models.

Sympathetic nerve activity in obstructive sleep apnoea.

Abstract: The mechanisms underlying the link between obstructive sleep apnoea (OSA) and cardiovascular disease are not completely established. However, there is increasing evidence that autonomic mechanisms are implicated. A number of studies have consistently shown that patients with OSA have high levels of sympathetic nerve traffic. During sleep, repetitive episodes of hypoxia, hypercapnia and obstructive apnoea act through chemoreceptor reflexes and other mechanisms to increase sympathetic drive. Remarkably, the high sympathetic drive is present even during daytime wakefulness when subjects are breathing normally and no evidence of hypoxia or chemoreflex activation is apparent. Several neural and humoral mechanisms may contribute to maintenance of higher sympathetic activity and blood pressure. These mechanisms include chemoreflex and baroreflex dysfunction, altered cardiovascular variability, vasoconstrictor effects of nocturnal endothelin release and endothelial dysfunction. Long-term continuous positive airway pressure treatment decreases muscle sympathetic nerve activity in OSA patients. The vast majority of OSA patients remain undiagnosed. Unrecognized OSA may contribute, in part, to the metabolic and cardiovascular derangements that are thought to be linked to obesity, and to the association between obesity and cardiovascular risk. Furthermore, acting through sympathetic neural mechanisms, OSA may contribute to or augment elevated levels of blood pressure in a large proportion of the hypertensive patient population.

Strength and power changes of the human plantar flexors and knee extensors in response to resistance training in old age

Abstract: Aim: The aim of the present study was to assess and compare the improvements of muscle strength and power induced by a 16-week resistive programme in a population of 16 older men aged 65-81 years. Methods: Training was performed three times per week at an intensity of 80% of one repetition maximum (1RM) and consisted of both calf raise and leg press exercises. Before-, during- and after-training, maximum isometric and isokinetic torques, maximum power, 1RM, muscle cross-sectional area (CSA) and electromyographic activity (EMG) of the plantar flexors (PF) and knee extensors (KE) were examined. Results: For the KE and PF, respectively, training resulted in a 29.9 ± 4.4% (mean ± SE) and 21.6 ± 5.4% increase in 1RM (P < 0.001-0.01), a 19.4 ± 4.3 and 12.4 ± 4.7% (P < 0.001-0.05) increase in maximum isometric torque, and a 24.1 ± 6.3 and 33.1 ± 10.9% (P < 0.05) increase in maximum muscle power, calculated from torque-angular velocity curves. The large increase in torque and power was partly accounted by a significant increase in the CSA of the PF (5.0 ± 0.7%) and KE (7.4 ± 0.7%), while no significant changes in integrated EMG activity of vastus lateralis and soleus muscles, and in extrapolated maximum shortening velocity were found. After training, a significant increase in torque/CSA (10.3 ± 4%, P < 0.05) was found for the KE but not for the PF. Conclusion: Hence, hypertrophy cannot alone justify the increase in torque, and other factors, such as an increase in individual fibre-specific tension (in the case of KE), a decrease in antagonist muscles' coactivation, an improved co-ordination and an increased neural drive of the other heads of quadriceps may have contributed to the increments in strength. The significant increase in muscle power seems particularly noteworthy with respect to daily activities involving the displacement of the body over time, namely, the generation of muscle power.

Chemoreflexes--physiology and clinical implications.

Abstract: The chemoreflexes are important modulators of sympathetic activation. The peripheral chemoreceptors located in the carotid bodies respond primarily to hypoxaemia. Central chemoreceptors located in the region of the brainstem respond to hypercapnia. Activation of either the hypoxic or hypercapnic chemoreflex elicits both hyperventilation and sympathetic activation. During apnoea, when the inhibitory influence of stretch of the pulmonary afferents is eliminated, there is a potentiation of the sympathetic response to both hypoxia and hypercapnia. This inhibitory influence of the pulmonary afferents is more marked on the sympathetic response to peripheral compared with central chemoreceptor activation. The arterial baroreflexes also have a powerful inhibitory influence on the chemoreflexes. This inhibition is again more marked with respect to the peripheral compared with central chemoreflexes. In patients with hypertension, there is a marked increase in the sympathetic and ventilatory response to hypoxaemia. During apnoea, with elimination of the inhibitory influence of breathing, the sympathetic response in untreated mild hypertensive patients is strikingly greater than that seen in matched normotensive controls. This potentiated peripheral chemoreflex sensitivity in hypertension may be explained in part by impaired baroreflex function in these patients. Enhanced peripheral chemoreflex sensitivity is also evident in patients with obstructive sleep apnoea. This peripheral chemoreflex enhancement is not explained by obesity, as obese individuals have a selective potentiation of the central chemoreceptors with peripheral chemoreflex responses similar to those seen in lean controls. Increased sensitivity to hypoxaemia has important implications in patients with obstructive sleep apnoea who experience repetitive and severe hypoxaemic stress. Tonic activation of the chemoreflex may also contribute to the high levels of sympathetic activity evident even during normoxic daytime wakefulness in sleep apnoea patients. Administration of 100% oxygen in patients with sleep apnoea results in reductions in heart rate, blood pressure and central sympathetic outflow. In patients with heart failure, the central chemoreflex response to hypercapnia is markedly and selectively enhanced. This increased central chemoreflex sensitivity may contribute to the development of central sleep apnoea in heart failure patients. Administration of 100% oxygen does not lower sympathetic activity in patients with heart failure, providing further evidence against any peripheral chemoreflex potentiation. The peripheral and central chemoreflexes have powerful effects on sympathetic activity in both health and disease and may contribute importantly to disease pathophysiology, particularly in conditions such as hypertension, obstructive sleep apnoea and heart failure.

Differential strain patterns of the human gastrocnemius aponeurosis and free tendon, in vivo.

Abstract: AIM: The mechanical characteristics of the human free tendon and aponeurosis, in vivo, remains largely unknown. The present study evaluated the longitudinal displacement of the separate free Achilles tendon and distal (deep) aponeurosis of the medial gastrocnemius muscle during voluntary isometric contraction. METHODS: Ultrasonography-obtained displacement of the free tendon and tendon-aponeurosis complex, electromyography of the gastrocnemius, soleus, and dorsiflexor muscles, and joint angular rotation were recorded during isometric plantarflexion (n = 5). Tendon cross-sectional area, moment arm and segment lengths (L(o)) were measured using magnetic resonance imaging. Tendon force was calculated from joint moments and tendon moment arm, and stress was obtained by dividing force by cross-sectional area. The difference between the free tendon and tendon-aponeurosis complex deformation yielded separate distal aponeurosis deformation. Longitudinal aponeurosis and tendon strain were obtained from the deformations normalized to segment lengths. RESULTS: At a common tendon force of 2641 +/- 306 N, the respective deformation and Lo were 5.85 +/- 0.85 and 74 +/- 0.8 mm for the free tendon and 2.12 +/- 0.64 and 145 +/- 1.3 mm for the distal aponeurosis, P < 0.05. Longitudinal strain was 8.0 +/- 1.2% for the tendon and 1.4 +/- 0.4% for the aponeurosis, P < 0.01. Stiffness and stored energy was 759 +/- 132 N mm(-1) and 6.14 +/- 1.89 J, respectively, for the free tendon. Cross-sectional area of the Achilles tendon was 73 +/- 4 mm2, yielding a stress of 36.5 +/- 4.6 MPa and Young's modulus of 788 +/- 181 MPa. CONCLUSION: The free Achilles tendon demonstrates greater strain compared with that of the distal (deep) aponeurosis during voluntary isometric contraction, which suggests that separate functional roles may exist during in vivo force transmission.

Medullary and supramedullary mechanisms regulating sympathetic vasomotor tone.

Abstract: Aim: Neurons in the rostral ventrolateral medulla (RVLM) that project directly to sympathetic preganglionic neurons in the spinal cord play a critical role in maintaining tonic activity in sympathetic vasomotor nerves. Intracellular recordings in vivo from putative RVLM presympathetic neurons have demonstrated that under resting conditions these neurons display an irregular tonic firing rate, and also receive both excitatory and inhibitory synaptic inputs. This paper will briefly review some recent findings on the role of glutamate, GABA and angiotensin II (Ang II) receptors in maintaining the tonic activity of RVLM presympathetic neurons. Results: Based on these findings, the following hypotheses will be discussed: (1) RVLM neurons receive tonic glutamatergic excitatory inputs, which originate from both medullary and supramedullary sources; (2) at least some neurons that project to and tonically inhibit RVLM presympathetic neurons are themselves tonically inhibited by GABAergic inputs originating from neurons in the caudalmost part of the ventrolateral medulla (caudal pressor area); (3) under normal conditions, Ang II receptors in the RVLM do not contribute significantly to the tonic activity of RVLM presympathetic neurons, but may do so in abnormal conditions such as heart failure or neurogenic hypertension; (4) RVLM presympathetic neurons maintain a significant level of tonic resting activity even when glutamate, GABA and Ang II receptors on the neurons are completely blocked. Under these conditions, the tonic activity is a consequence either of the intrinsic membrane properties of the neurons (autoactivity) or of synaptic inputs mediated by receptors other than glutamate, GABA or Ang II receptors. Conclusion: The current evidence indicates that the resting activity of RVLM presympathetic neurons is determined by the balance of powerful tonic excitatory and inhibitory synaptic inputs. Ang II receptors also contribute to the raised resting activity of these neurons in some pathological conditions.

The role of intramuscular lipid in insulin resistance

Abstract: There is interest in how altered lipid metabolism could contribute to muscle insulin resistance. Many animal and human states of insulin resistance have increased muscle triglyceride content, and there are now plausible mechanistic links between muscle lipid accumulation and insulin resistance, which go beyond the classic glucose-fatty acid cycle. We postulate that muscle cytosolic accumulation of the metabolically active long-chain fatty acyl CoAs (LCACoA) is involved, leading to insulin resistance and impaired insulin signalling or impaired enzyme activity (e.g. glycogen synthase or hexokinase) either directly or via chronic translocation/activation of mediators such as a protein kinase C (particularly PKC theta and epsilon ). Ceramides and diacylglycerols (DAGs) have also been implicated in forms of lipid-induced muscle insulin resistance. Dietary lipid-induced muscle insulin resistance in rodents is relatively easily reversed by manipulations that lessen cytosolic lipid accumulation (e.g. diet change, exercise or fasting). PPAR agonists (both gamma and alpha) also lower muscle LCACoA and enhance insulin sensitivity. Activation of AMP-activated protein kinase (AMPK) by AICAR leads to muscle enhancement (especially glycolytic muscle) of insulin sensitivity, but involvement of altered lipid metabolism is less clear cut. In rodents there are similarities in the pattern of muscle lipid accumulation/PKC translocation/altered insulin signalling/insulin resistance inducible by 3-5-h acute free fatty acid elevation, 1-4 days intravenous glucose infusion or several weeks of high-fat feeding. Recent studies extend findings and show relevance to humans. Muscle cytosolic lipids may accumulate either by increased fatty acid flux into muscle, or by reduced fatty acid oxidation. In some circumstances muscle insulin resistance may be an adaptation to optimize use of fatty acids when they are the predominant available energy fuel. The interactions described here are fundamental to optimizing therapy of insulin resistance based on alterations in muscle lipid metabolism.

Interaction of fibre type, potentiation and fatigue in human knee extensor muscles.

Abstract: Aim: To examine the effect of fibre type on potentiation and fatigue. Methods: Young men (n = 4 per group) with a predominance of type I [61.4 ± 6.9% (SD), group I (GI)] or type II [71.8 ± 9.2%, group II (GII)] fibres in vastus lateralis, performed a fatigue protocol of sixteen 5-s maximal voluntary isometric contractions (MVCs) of the right knee extensors. Maximal twitches and corresponding muscle action potentials (M-waves) were evoked before the first MVC, during the 3-s rest period after each MVC and at intervals during the 5-min recovery period after the last MVC. Results: Group II [49.3 ± 2.6% (SE)] had a greater decrease in MVC force than GI (22.8 ± 6.2%) during the fatigue protocol. Group II (126.4 ± 13.6%) showed greater twitch force potentiation early in the fatigue protocol than GI (38.2 ± 2.3%), but greater depression at the end (33.7 ± 13.7% vs.17.4 ± 3.4%). Twitch time-to-peak torque (TPT) and half relaxation time (HRT) initially decreased but then increased as the fatigue protocol progressed; GII had a greater increase in HRT. During a 5-min recovery period twitch force increased above the prefatigue level and remained so until the end of the recovery period; the pattern was similar in GI and GII. Twitch TPT and HRT remained elevated during recovery. M-wave area increased throughout the fatigue protocol and the first part of recovery before returning to baseline values in GII, whereas there were no significant changes in GI. The interaction between potentiation and fatigue was amplified in GII early in the fatigue protocol with concurrently greater twitch and M-wave potentiation, and greater MVC force decrease and HRT increase. Late in the protocol, GII had a greater decrease in twitch and MVC force combined with greater M-wave potentiation. Conclusion: It is concluded that fibre type distribution influences potentiation and fatigue of the twitch, and potentiation of the M-wave during fatiguing exercise.

AMPK as a metabolic switch in rat muscle, liver and adipose tissue after exercise

Abstract: An increasing body of evidence has revealed that activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK)-activated protein kinase increases fatty acid oxidation by lowering the concentration of malonyl coenzyme A (CoA), an inhibitor of carnitine palmitoyl transferase 1. Studies carried out primarily in skeletal muscle suggest that AMPK modulates the concentration of malonyl CoA by concurrently phosphorylating and inhibiting acetyl CoA carboxylase (ACC), the rate limiting enzyme in malonyl CoA synthesis, and phosphorylating and activating malonyl CoA decarboxylase (MCD), an enzyme involved in its degradation. We have recently observed that AMPK and MCD activities are increased and ACC activity diminished in skeletal muscle, liver and, surprisingly, in adipose tissue 30 min following exercise (treadmill run) in normal rats. In liver and adipose tissue these changes were associated with a decrease in the activity of glycerol-3-phosphate acyltransferase (GPAT), which catalyses the first committed reaction in glycerolipid synthesis and, which like ACC, is phosphorylated and inhibited by AMPK. Similar changes in ACC, MCD and GPAT were observed following the administration of 5-aminoimidazole 4-carboxamide-riboside (AICAR), further indicating that the exercise-induced alterations in these enzymes were AMPK-mediated. Conclusions: (1) AMPK plays a major role in regulating lipid metabolism in multiple tissues following exercise. (2) The net effect of its activation is to increase fatty acid oxidation and diminish glycerolipid synthesis. (3) The relevance of these findings to the regulation of muscle glycogen repletion in the post-exercise state and to the demonstrated ability of AMPK activation to decrease adiposity and increase insulin sensitivity in rodents remains to be determined.

Molecular regulation of individual skeletal muscle fibre types.

Abstract: The purpose of this review is to present current understanding of cellular and molecular regulation of fibre type expression in skeletal muscle. Published literature seems to conclusively suggest that muscle fibre type expression is regulated by multiple signalling pathways and transcription factors rather than a single 'master' switch or signalling pathway. While the current nomenclature for fibre types is convenient for communication, based upon the evolution of this nomenclature, the prediction that fibre type classifications may change in the future to incorporate post-genomic information is made. It is predicted that future fibre type classifications could be based upon the contractile-activity-induced changes in a common regulatory factor(s) within a subpopulation of genes whose expressions are altered to modify and maintain the new muscle fibre phenotype.

Non-invasive assessment of cardiac output during exercise in healthy young humans: comparison between Modelflow method and Doppler echocardiography method.

Abstract: Aims: The Modelflow method can estimate cardiac output from arterial blood pressure waveforms using a three-element model of aortic input impedance (aortic characteristic impedance, arterial compliance, and systemic vascular resistance). We tested the reliability of a non-invasive cardiac output estimation during submaximal exercise using the Modelflow method from finger arterial pressure waveforms collected by Portapres in healthy young humans. Methods: The Doppler echocardiography method was used as a reference method. Sixteen healthy young subjects (nine males and seven females) performed a multi-stage cycle ergometer exercise at an intensity corresponding to 70, 90, 110 and 130% of their individual ventilatory threshold for 2 min each. The simultaneous estimation of cardiac output (15 s averaged data) using the Modelflow and Doppler echocardiography methods was performed at rest and during exercise. Results and Conclusion: The Modelflow-estimated cardiac output correlated significantly with the simultaneous estimates by the Doppler method in all subjects (r = 0.87, P < 0.0001) and the SE of estimation was 1.93 L min−1. Correlation coefficients in each subject ranged from 0.91 to 0.98. Although the Modelflow method overestimated cardiac output, the errors between two estimates were not significantly different among the exercise levels. These results suggest that the Modelflow method using Portapres could provide a reliable estimation of the relative change in cardiac output non-invasively and continuously during submaximal exercise in healthy young humans, at least in terms of the relative changes in cardiac output.

Sympathetic nerve activity and neurotransmitter release in humans: translation from pathophysiology into clinical practice.

Abstract: Aim: There has been a revolution in cardiovascular neuroscience in recent years with, in some cases, translation into clinical practice of the knowledge of pathophysiology gained through application of sympathetic nerve recording and catecholamine isotope dilution methodology. Obesity-related hypertension: An earlier hypothesis, based on findings in most models, was that weight gain in obesity is due in part to sympathetic nervous underactivity reducing thermogenesis. Microneurography and regional noradrenaline spillover measurements in human obesity have disproven this hypothesis, weakening the case for the use of b3-adrenergic agonists to stimulate thermogenesis. Sympathetic nerve firing rates in postganglionic fibres directed to the skeletal muscle vasculature are increased, as is renal sympathetic tone, with a doubling of the spillover rate of noradrenaline from the kidneys. Given these findings, antiadrenergic antihypertensive drugs may be the preferred agents for obesity-related hypertension, but this has not been adequately tested. Essential hypertension: Whether stress causes high blood pressure, previously hotly debated, has been under recent review by an Australian Government body, the Specialist Medical Review Council. Despite medicolegal implications, the ruling was that stress is one proven cause of hypertension. The judgment was reached after consideration of the epidemiological evidence, but in particular the described neural pathophysiology of essential hypertension: (a) persistent sympathetic nervous stimulation is commonly present, (b) suprabulbar projections of noradrenergic brainstem neurones are activated and (c) adrenaline is released as a cotransmitter in sympathetic nerves. These were taken to be biological markers of stress. Cardiac failure: At one time, the failing heart was thought to be sympathetically denervated. Longterm administration of inotropic adrenergic agonists, to provide the cardiac catecholamine stimulation thought to be lacking, increased mortality. Noradrenaline isotope dilution methodology subsequently demonstrated that the sympathetic outflow to the heart was preferentially activated, cardiac noradrenaline spillover being increased as much as 50-fold. The level of stimulation of the cardiac sympathetic nerves was the most powerful predictor of death. These observations provide the theoretical foundation for the very successful introduction of b-adrenergic blockers for treatment of heart failure.

From molecules to mammals: what's NOS got to do with it?

Abstract: Nitric oxide synthases (NOSs) generate nitric oxide (NO) and the by-product l-citrulline, via the catalytic combination of l-arginine and molecular oxygen. In mammals, there are three NOS genes: nNOS (NOS1), iNOS (NOS2) and eNOS (NOS3). The molecular structure, enzymology and pharmacology of these enzymes have been well defined, and reveal critical roles for the NOS system in a variety of important processes. The studies of NOS enzymes using knockout and transgenic mouse models have provided an invaluable contribution, highlighting critical roles in neuronal, renal, pulmonary, gastro-intestinal, skeletal muscle, reproductive and cardiovascular biology. This review will outline the data gleaned from complementary knockout and transgenic over-expression models in mice, and focus on the interactions between NOS enzymes and pathophysiology of the vascular system. These studies are a paradigm for the near future, which will involve the translation of an enormous amount of genomic data into physiological insights that penetrate the realms of both health care and biology.

Peroxisome proliferator-activated receptors (PPARS): regulators of gene expression in heart and skeletal muscle.

Abstract: The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily. The three isoforms (PPARα, β/δ and γ) have been implicated in the regulation of the expression of genes involved in lipid metabolism. Although their prominent role in lipid homeostasis is well established, the way in which the activity of each of the PPAR isoforms is regulated under physiological and pathological conditions is still subject of intensive research. In skeletal as well as cardiac muscle cells it has been demonstrated that the expression of a large panel of proteins involved in the transport and metabolic conversion of fatty acids is under control of PPARs. The pivotal role of the PPARα isoform in cardiac fatty acid metabolism has been confirmed in PPARα-null mice. The exact role of PPARβ/δ in the regulation of muscle metabolism is still a matter of debate. Whereas several studies provided evidence to support the notion that PPARα and PPARβ/δ have redundant roles, other studies suggest that PPARα activity is counteracted by PPARβ/δ. Marked effects of bona fide PPARγ ligands (the antidiabetic thiazolidinediones) on skeletal and cardiac muscle function and phenotype, have also been reported. However, next to activating PPARy, the thiazolidinediones do affect other cellular processes as well. To date it is being realized that the control of the trans-activating capacity of each of the PPAR isoforms is multi-factorial and, in addition to ligand availability, depends on such factors as isoform-specific phosphorylation and selective interaction with various proteins acting either as co-activator or co-repressor.

Exercise-induced HSP27, HSP70 and MAPK responses in human skeletal muscle

Abstract: Aim: The present work examined protein and messenger RNA (mRNA) expression of intramuscular heat shock protein 27 (HSP27), heat shock cognate (HSC70) and HSP70 in human biceps brachii (BB) and vastus lateralis (VL) subsequent to two different exercises. Methods: Untrained subjects performed 50 high-force eccentric contractions with their non-dominant BB and ran downhill (−10°) for 30 min. The 48-h PX stress response was evaluated with immunoblotting and reverse transcriptase-polymerase chain reaction (RT-PCR). Muscle damage was indicated indirectly at 48 h post-exercise (PX) [loss of mobility, muscle soreness and serum creatine kinase (CK) activity]. Results: On the protein level, HSP27 and HSP70 increased significantly PX in the BB (384 and 227%, respectively; P < 0.01), but there were no significant HSP changes in the VL or in HSC70 in either muscle. The RT-PCR data complemented these findings: BB HSP27 and HSP70C mRNA levels increased (135 and 128%, respectively; P < 0.05); in the VL only HSP70B increased (206%; P < 0.05). Phosphorylation of e-jun NH2-terminal kinase (JNK) and extracellular regulated kinase (ERK) increased significantly in the BB (226 and 200%, respectively; P < 0.05) but not in the VL, indicating activation of these pathways only after the resistance exercise. Conclusion: These data indicate that the PX HSP and mitogen-activated protein kinase responses are exercise-specific and local, not systemic. Further, only the resistance exercise induced HSP expression (protein and mRNA) and JNK/ERK activation at 48 h PX, suggesting that these molecules may be important to long-term skeletal muscle adaptations such as hypertrophy.

AMP-activated protein kinase and muscle glucose uptake.

Abstract: The AMP-activated protein kinase (AMPK) is an enzyme that is activated in situations where there are changes in the cellular energy status such as muscle contraction and hypoxia. AMPK can also be pharmacologically activated by the compound 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and the antidiabetic agent metformin. Several studies support the hypothesis that AMPK plays an important role in the stimulation of muscle glucose uptake by these physiological and pharmacological stimuli. In isolated rat muscles, activation of AMPK is associated with increases in glucose uptake through an insulin-independent mechanism. Studies done in rodents have shown that the activation of AMPK by AICAR is accompanied by decreases in blood glucose concentrations, in part due to enhanced muscle glucose uptake. Similar to exercise, AICAR not only directly stimulates glucose uptake into the skeletal muscle, but also enhances insulin sensitivity. The activation of AMPK and associated increases in muscle glucose uptake are affected by factors such as glycogen content, exercise training and fibre type. The effects of AMPK on muscle glucose uptake makes this protein a promising pharmacological target for the treatment of type 2 diabetes.

Comparison of force-velocity relationships of vastus lateralis muscle in isokinetic and in stretch-shortening cycle exercises.

Abstract: Aim: This study investigated the force–velocity characteristics of the vastus lateralis (VL) muscle fascicle and muscle–tendon unit (MTU) in isolated lengthening and shortening actions, and during natural movement. Methods: Four subjects performed maximal eccentric and concentric knee extensions (60, 120 and 180° s−1). Unilateral counter movement jumps and drop jumps in the sledge apparatus served as natural movements. Vastus lateralis fascicle lengths were determined from ultrasonography. In vivo patellar tendon forces (PTF) were measured with an optic fibre technique. Patellar tendon force was derived to VL force according to the cross-sectional area of the muscle. Force in the direction of fascicle was calculated by dividing the VL force value by cosine of the fascicle angle. Force–velocity curves were constructed using angle specific values from isokinetic knee extensions (classical curve) and using instantaneous values from jumping exercises. Results: In the fascicle level, we did not find an enhanced muscle force in the jumping performances as compared with the classical force–velocity curve. In the muscle–tendon level, the instantaneous force at high muscle–tendon shortening speeds exceeded that extrapolated according to Hill's equation. Conclusion: This difference between fascicle and muscle–tendon behaviour suggests that the neural input in fast stretch-shortening cycle exercises minimizes the length changes in muscle fascicle and enables storage and recoil of energy from elastic components that contributes to the enhanced mechanical output of the MTU during the push-off phase.

Central osmotic regulation of sympathetic nerve activity

Abstract: Aim: In this review, we will focus on the central neural mechanisms that couple osmotic perturbations to changes in sympathetic nerve discharge, and the possible impact these actions have in cardiovascular diseases such as arterial hypertension and congestive heart failure. Results: Changes in extracellular fluid osmolality lead to specific regulatory responses in defence of body fluid and cardiovascular homeostasis. Systemic hyperosmolality is well known to stimulate thirst and the release of anti-diuretic hormone. These responses are largely due to osmosensing neurones in the forebrain lamina terminalis and hypothalamus and are critical elements in a control system that operates to restore body fluid osmolality. An equally important, but less characterized, target of central osmoregulatory processes is the sympathetic nervous system. Conclusion: Understanding the neurobiology of sympathetic responses to changes in osmolality has important implications for body fluid and cardiovascular physiology. By stabilizing osmolality, vascular volume is preserved and thereby relatively normal levels of cardiac output and arterial pressure are maintained.

Paraventricular nucleus of the hypothalamus and elevated sympathetic activity in heart failure: the altered inhibitory mechanisms

Abstract: Aim: There is a characteristic neurohumoral activation in heart failure (HF) However, few studies have been performed to examine the role of the central nervous system in the activation of sympathetic outflow during HF In this paper we review some of our studies, with particular emphasis on examining the role of the paraventricular nucleus (PVN) in the exaggerated sympathetic outflow commonly observed in HF Results: Our studies have revealed that the inhibitory mechanisms regulating sympathetic outflow are mediated by nitric oxide (NO) and γ-aminobutyric acid (GABA) within the PVN and are attenuated in HF These alterations are associated with elevated sympathetic activity Furthermore, these studies have indicated that the interactions among excitatory (angiotensin II and glutamate) and inhibitory (NO and GABA) neurotransmitters/ mediators within the PVN significantly influence sympathetic outflow Conclusion: Reduced inhibitory actions of NO and/or GABA within the PVN may exaggerate an increase in the actions of excitatory neurotransmitters such as glutamate and angiotensin II within the PVN and this may contribute to the overall sympatho-excitation commonly observed in HF

Gap junction hemichannels in astrocytes of the CNS.

Abstract: Connexins are protein subunits that oligomerize into hexamers called connexons, gap junction hemichannels or just hemichannels. Because some gap junction channels are permeable to negatively and/or positively charged molecules up to approximately 1kDa in size, it was thought that hemichannels should not open to the extracellular space. A growing amount of evidence indicates that opening of hemichannels does occur under both physiological and pathological conditions in astrocytes and other cell types. Electrophysiological studies indicate that hemichannels have a low open probability under physiological conditions but may have a much higher open probability under certain pathological conditions. Some of the physiological behaviours of astrocytes that have been attributed to gap junctions may, in fact, be mediated by hemichannels. Hemichannels constituted of Cx43, the main connexin expressed by astrocytes, are permeable to small physiologically significant molecules, such as ATP, NAD+ and glutamate, and may mediate paracrine as well as autocrine signalling. Hemichannels tend to be closed by negative membrane potentials, high concentrations of extracellular Ca2+ and intracellular H+ ions, gap junction blockers and protein phosphorylation. Hemichannels tend to be opened by positive membrane potentials and low extracellular Ca2+, and possibly by as yet unidentified cytoplasmic signalling molecules. Exacerbated hemichannel opening occurs in metabolically inhibited cells, including cortical astrocytes, which contributes to the loss of chemical gradients across the plasma membrane and speeds cell death.

Renal and vascular oxidative stress and salt-sensitivity of arterial pressure.

Abstract: Oxidative stress occurs in a tissue or in the whole body when the total oxidant production exceeds the antioxidant capacity. Recent studies in human essential hypertension indicate that free radical production is increased and antioxidant levels are decreased, and more than one-half of these hypertensives have a salt-sensitive type of hypertension with progressive renal damage. Increased oxidative stress may also play a critical role in animal models of salt-sensitive hypertension. The stroke-prone spontaneously hypertensive rats (SHRSP) exhibits salt-sensitivity, vascular release of superoxide is increased, and total plasma antioxidant capacity is decreased. The superoxide release in the SHRSP rats inactivates nitric oxide, and superoxide dismutase (SOD) administration returns the bioactive nitric oxide levels to normal. The deoxycorticosterone acetate (DOCA)-salt hypertensive rat is salt-sensitive, aortic superoxide production is increased, and renal inflammation is significant. Treatment of the DOCA-salt rats with apocynin, an NADPH oxidase inhibitor, decreased aortic superoxide production and decreased arterial pressure. The Dahl salt-sensitive (S) rat has increased mesenteric microvascular and renal superoxide production and increased plasma levels of H2O2. The renal protein expression of SOD is decreased in the kidney of Dahl S rats, and long-term administration of Tempol, a superoxide mimetic, significantly decreased arterial pressure and renal damage. In conclusion, both human hypertension and experimental models of salt-sensitive hypertension have increased superoxide release, decreased antioxidant capacity and elevated renal damage.

Cytoplasmic fatty acid-binding protein facilitates fatty acid utilization by skeletal muscle

Abstract: The intracellular transport of long-chain fatty acids in muscle cells is facilitated to a great extent by heart-type cytoplasmic fatty acid-binding protein (H-FABP). By virtue of the marked affinity of this 14.5-kDa protein for fatty acids, H-FABP dramatically increases their concentration in the aqueous cytoplasm by non-covalent binding, thereby facilitating both the transition of fatty acids from membranes to the aqueous space and their diffusional transport from membranes (e.g. sarcolemma) to other cellular compartments (e.g. mitochondria). Striking features are the relative abundance of H-FABP in muscle, especially in oxidative muscle fibres, and the modulation of the muscular H-FABP content in concert with the modulation of other proteins and enzymes involved in fatty acid handling and utilization. Newer studies with mice carrying a homozygous or heterozygous deletion of the H-FABP gene show that, in comparison with wild-type mice, hindlimb muscles from heterozygous animals have a markedly lowered (-66%) H-FABP content but unaltered palmitate uptake rate, while in hindlimb muscles from homozygous animals (no H-FABP present) palmitate uptake was reduced by 45%. These findings indicate that H-FABP is present in relative excess and plays a substantial, but merely permissive role in fatty acid uptake by skeletal muscles.

Oestrogen receptor beta is expressed in adult human skeletal muscle both at the mRNA and protein level.

Abstract: Aim There are two known oestrogen receptors (ER), oestrogen receptor alpha (ERalpha) and the recently cloned oestrogen receptor beta (ERbeta). ERalpha mRNA has been detected in mouse, rat, bovine and human skeletal muscle. ERbeta mRNA has been detected in bovine skeletal muscle. To our knowledge, no study has investigated the expression of oestrogen receptor beta in human skeletal muscle. Therefore, the primary aim of the present investigation was to study ERbeta mRNA and protein expression in human skeletal muscle. In addition the ERalpha expression was also studied. Methods Muscle biopsies were taken from vastus lateralis in six healthy adults (three women and three men). mRNA expression was detected with real-time PCR (TaqMan) and protein localization by immunohistochemistry. Results A clear expression of ERalpha and ERbeta mRNA was seen in skeletal muscle in all subjects. The ERalpha mRNA expression was 180 fold higher compared with that of ERbeta mRNA. Immunohistochemistry demonstrated positive staining for ERbeta, but not for ERalpha, with localization to the nuclei of skeletal muscle fibres. On average, 70% of all nuclei were ERbeta-positive. Conclusion The present study shows for the first time ERbeta mRNA and protein expression in human skeletal muscle tissue in both males and females.

Neurophysiological analysis of target-related sympathetic pathways--from animal to human: similarities and differences.

Abstract: The sympathetic nervous system regulates many different target tissues in the somatic and visceral domains of the body in a differentiated manner, indicating that there exist separate sympathetic pathways that are functionally defined by their target cells. Signals generated by central integration and channelled through the preganglionic neurons into the final sympathetic pathways are precisely transmitted through the para- and prevertebral ganglia and at the neuroeffector junctions to the effector cells. Neurophysiological recordings of activity in postganglionic neurons in skin and muscle nerves using microneurography in human subjects and in skin, muscle and visceral nerves, using conventional recording techniques in anaesthetized animals, clearly show that each type of sympathetic neuron exhibits a discharge pattern that is characteristic for its target cells and, therefore, its function. These findings justify labelling the neurons as muscle vasoconstrictor, cutaneous vasoconstrictor, sudomotor, lipomotor, cardiomotor, secretomotor neurons, etc. The discharge patterns monitor aspects of the central organization of the respective sympathetic system in the neuraxis and forebrain. They can be dissected into several distinct reflexes (initiated by peripheral and central afferent inputs) and reactions connected to central signals (related to respiration, circadian and other rhythms, command signals generated in the forebrain, etc). They are functional markers for the sympathetic final pathways. These neurophysiological recordings of the discharge patterns from functionally identified neurons of sympathetic pathways in the human and in animals are the ultimate reference for all experimental investigations that aim to unravel the central organization of the sympathetic systems. The similarities of the results obtained in the in vivo studies in the human and in animals justify concluding that the principles of the central organization of sympathetic systems are similar, if not identical, at least in the neuraxis, in both species. Future progress in the analysis of the central neuronal circuits that are associated with the different final sympathetic pathways will very much depend on whether we are able to align the human models and the animal models. Human models using microneurography have the advantage to work under awake conditions. The activity in the postganglionic neurons can be correlated with various other (afferent, centrally generated) signals, effector responses, perceptions, central changes monitored by imaging methods, etc. However, human models have considerable limitations. Animal models can be divided into in vivo models and various types of reduced in vitro models. Animal models allow using various methodological approaches (e.g., neurophysiological, pharmacological, modern anatomical tracing methods; behavioural animal models; transgenic animals), which cannot be used in the human. Interaction of the research performed in the human and animals will allow to design animal models that are relevant for diseases in which the sympathetic nervous systems is involved and to trace down the underlying pathophysiological mechanisms. The scientific questions to be asked are formulated on the basis of clinical observations resulting in testable hypotheses that are investigated in the in vivo human and animal models.

The role of uncoupling proteins in the regulation of metabolism

Abstract: Investigations of variations in metabolic efficiency and thermogenesis have a short and turbulent history. In small animals, non-shivering thermogenesis and diet-induced thermogenesis have a great impact on overall body weight, and the question is whether mechanisms to waste energy have evolved also in human energy metabolism. The candidate molecules for this adaptive thermogenesis are the uncoupling proteins. This is a newly discovered family of proteins, consisting of at least five proteins, namely UCP1, UCP2, UCP3, UCP4 and UCP5. Although a role for UCP1 in thermogenesis is unequivocal, the physiological function of the newer uncoupling proteins is as yet unclear. UCP1 is present in brown adipose tissue and has a well-documented role in cold-induced thermogenesis. The targeted disruption of the UCP1-gene rendered animals that were cold sensitive, but not obese. UCP2 mRNA has a ubiquitous distribution in tissue, namely, in skeletal muscle, white and brown adipose tissue, the gastro-intestinal tract, the lung and the spleen. By targeting the UCP2-gene there was no effect on whole body energy metabolism, but instead, a reduced ability to protect against free-radical oxygen species. UCP2 has also been shown to act as a negative regulator for insulin secretion. UCP3 is present in skeletal muscle. Targeted disruption of the UCP3-gene gave no effect on whole body energy metabolism, but showed the mitochondria in muscle to be more coupled. In conclusion, the uncoupling proteins may be important in various specific ways, as protectors of free radical oxygen species and as regulators of ATP-dependent processes.

Effect of low-load resistance training on the tendon properties in middle-aged and elderly women

Abstract: Aim: The purposes of this study were to determine the age-related changes in the tendon-aponeurosis structures and to investigate the effects of lowload resistance training on the tendon-aponeurosis structures in middle-aged and elderly women Methods: Fifty-one women (558 � 137 years, range: 21–77 years) volunteered to take part in the present study Furthermore, 11 middle-aged and elderly women (497 � 92 years) performed the low-load resistance training, ie squat using body weight, for 6 months The elongation of the tendon and aponeurosis of the vastus lateralis muscle was directly measured by ultrasonography, while the subjects performed ramp isometric knee extension up to the voluntary maximum, followed by a ramp relaxation The relationship between the estimated muscle force (Fm) and tendon elongation (L) during the ascending phase was fitted to a linear regression, the slope of which was defined as stiffness The percentage of the area within the Fm-L loop to the area beneath the curve during the ascending phase was calculated as hysteresis Results: Maximal strain (L/initial tendon length) and stiffness of the tendon-aponeurosis structures decreased significantly with ageing In contrast, the hysteresis increased significantly with ageing In addition, low-load resistance training produced no significant change in stiffness and hysteresis, but significantly increased the maximal elongation of tendon-aponeurosis structures from 233 � 21 mm to 248 � 22 mm (P ¼ 0045) Conclusion: These results suggest that increasing age results in a decrease in the elasticity of tendon-aponeurosis structures and an increase in their viscosity Furthermore, the low-load resistance training made the elasticity of tendon-aponeurosis structures increase

The paraventricular nucleus: an important component of the central neurocircuitry regulating sympathetic nerve outflow

Abstract: Aim: The sympathetic nervous system plays an important role in the regulation of physiological homeostasis under basal conditions and in response to acute and chronic stressors. It is known that multiple levels of the neuroaxis, including the paraventricular nucleus (PVN) of the hypothalamus, are involved in regulation of efferent sympathetic nerve discharge (SND). This review focuses on the role of the PVN in regulation of functional characteristics of efferent SND. Results: The available experimental evidence indicates that the level of efferent sympathetic nerve activity is altered after microinjection of numerous substances into the PVN, including excitatory amino acids, -aminobutyric acid (GABA A ) receptor agonists and antagonists, and PVN nitric oxide synthase inhibitors. In addition, antagonism of PVN GABA A receptors changes the pattern of synchronized discharge bursts in efferent sympathetic nerves and enhances the frequency-domain coupling between low-frequency bursts in sympathetic nerve pairs. Finally, PVN microinjections of excitatory amino acids (L-glutamate, D,L-homocysteic acid) have been shown to produce non-uniform changes in the level of efferent sympathetic nerve activity. Conclusion: These findings support the concept that the PVN is an important component of the central neurocircuitry regulating functional characteristics (basal level of activity, bursting pattern, and relationships between discharges in nerves innervating different targets) of efferent sympathetic nerve outflow.

Exercise training improves ageing‐induced decrease in eNOS expression of the aorta

Abstract: Aims: Ageing impairs endothelial function such as the regulation of vascular tone. The release of nitric oxide (NO), which has a potent vasodilator effect and antiatherosclerotic property, is decreased in the aorta of aged rats. Exercise training, however, has been reported to increase the expression of endothelial NO synthase (eNOS) in the aorta of young rats. In aged rats, it is not known whether the expression of eNOS is altered by exercise training. We hypothesized that exercise training would improve the ageing-induced decrease in eNOS expression in vessels, and examined the messenger RNA (mRNA) and protein expression of eNOS in the aorta of sedentary-young rats (sedentary-young group; 4 months old), sedentary-aged rats (sedentary-aged group; 23 months old), and swim-trained aged rats (training-aged group; 23 months old, swimming training for 8 weeks, 5 days week−1, 90 min day−1). Results: Body weight was significantly lower, and citrate synthase activity in the epitrochlearis muscle was significantly higher in the training-aged group compared with the sedentary-aged group. The mRNA expression of eNOS in the aorta was significantly higher in the training-aged group than in the sedentary-aged group, while it was significantly lower in both the sedentary-aged and training-aged groups than in the sedentary-young group. The expression of eNOS protein in the aorta was also significantly higher in the training-aged group than in the sedentary-aged group, while it was also significantly lower in the sedentary-aged group, but not in the training-aged group, than in the sedentary-young group. Conclusion: The present results revealed that the production of eNOS in the aorta decreases with ageing, and that the decreased production is increased by exercise training in aged rats, which may produce beneficial effects on the impaired cardiovascular system caused by ageing.

Redox regulation of the afferent arteriole and tubuloglomerular feedback.

Abstract: Oxidative stress implies an increased production of reactive oxygen species (ROS) or a decreased capacity to metabolize them. Superoxide anion (O) can bioinactivate nitric oxide (NO). Therefore, many effects of ROS are manifest as NO deficiency. The afferent arteriole and macula densa cell both contain a full complement of components of nicotine adenine dinucleotide phosphate (NADPH) oxidase that generates O. Nitric oxide synthase (NOS) type 1 or neuronal NOS (nNOS) is expressed in the macula densa and NOS type II or endothelial NOS (eNOS) in the afferent arteriole. Whole animal studies in models of hypertension and oxidative stress demonstrate that metabolism of O by a superoxide dismutase (SOD) mimetic can reduce renal vascular resistance. In vivo studies of single nephron function and in vitro studies with the double-perfused juxtaglomerular apparatus preparation have shown extensive interaction between O and NO in macula densa to regulate afferent arteriolar tone mediated by the tubuloglomerular feedback response. In vitro studies of rabbits isolated, perfused afferent arterioles have shown a similar interaction in this vessel. These data indicate important roles for O in the macula densa and afferent arterioles to enhance preglomerular resistance in animal models of oxidative stress. As an increase in afferent arteriolar resistance can precede hypertension, oxidative stress could be important in determining the long-term blood pressure and thereby contribute to hypertension.