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The optimal exercise intensity to improve endothelial function remains unclear, as well as whether the addition of hypoxia could potentiate this function. Therefore, the aim of this study was to compare the effects of different exercise intensities in normoxia and hypoxia on vascular reactivity and nitric oxide (NO) bioavailability in mice. C57BL/6 mice underwent treadmill running three times per week, for 4 weeks at either low, maximal or supramaximal intensity in normoxia or hypoxia (inspire oxygen fraction = 0.13). Vascular reactivity and expression of genes and proteins involved in NO production/bioavailability were assessed in aorta using isolated vessel tension experiments, RT-qPCR and western blot, respectively. Circulating NO metabolites and pro-/antioxidant markers were measured. Hypoxic exercise improved both acetylcholine-induced vasorelaxation and phenylephrine-induced vasoconstriction compared to normoxic exercise, independently of intensity. In hypoxia, a higher acetylcholine-induced vasorelaxation was observed with high intensities (supramaximal and maximal) compared to low intensity. Exercise protocols modulated endothelial nitric oxide synthase (eNOS) and α1-adrenergic receptor (α 1 -AR) mRNA level, but not superoxide dismutase 3 (SOD3) and p47phox. No significant differences were observed for protein expression of α 1 -AR, total eNOS, phosphorylated eNOS, SOD isoforms and p47phox. However, plasma SOD and catalase activities were significantly increased in hypoxic supramaximal compared to hypoxic low intensity, while concentration of nitrotyrosine significantly decreased. The latter was also observed in hypoxic maximal and supramaximal compared to the same intensities in normoxia. Hypoxic high-intensity exercise increases NO bioavailability and improves vascular function, opening promising clinical perspectives for cardiovascular disease prevention.

Aim The tools that have been used to assess the function of the vagus nerve lack specificity. This could explain discrepancies about the role of vagal gut‐brain signalling in long‐term control of energy balance. Here we use a validated approach to selectively ablate sensory vagal neurones that innervate the gut to determine the role of vagal gut‐brain signalling in the control of food intake, energy expenditure and glucose homoeostasis in response to different diets. Methods Rat nodose ganglia were injected bilaterally with either the neurotoxin saporin conjugated to the gastrointestinal hormone cholecystokinin (CCK), or unconjugated saporin as a control. Food intake, body weight, glucose tolerance and energy expenditure were measured in both groups in response to chow or high‐fat high‐sugar (HFHS) diet. Willingness to work for fat or sugar was assessed by progressive ratio for orally administered solutions, while post‐ingestive feedback was tested by measuring food intake after an isocaloric lipid or sucrose pre‐load. Results Vagal deafferentation of the gut increases meal number in lean chow‐fed rats. Switching to a HFHS diet exacerbates overeating and body weight gain. The breakpoint for sugar or fat solution did not differ between groups, suggesting that increased palatability may not drive HFHS‐induced hyperphagia. Instead, decreased satiation in response to intra‐gastric infusion of fat, but not sugar, promotes hyperphagia in CCK‐Saporin‐treated rats fed with HFHS diet. Conclusions We conclude that intact sensory vagal neurones prevent hyperphagia and exacerbation of weight gain in response to a HFHS diet by promoting lipid‐mediated satiation.

Aim NG2 cells in the brain are comprised of pericytes and NG2 glia and play an important role in the execution of cerebral hypoxia responses, including the induction of erythropoietin (EPO) in pericytes. Oxygen‐dependent angiogenic responses are regulated by hypoxia‐inducible factor (HIF), the activity of which is controlled by prolyl 4‐hydroxylase domain (PHD) dioxygenases and the von Hippel‐Lindau (VHL) tumour suppressor. However, the role of NG2 cells in HIF‐regulated cerebral vascular homeostasis is incompletely understood. Methods To examine the HIF/PHD/VHL axis in neurovascular homeostasis, we used a Cre‐loxP‐based genetic approach in mice and targeted Vhl, Epo, Phd1, Phd2, Phd3 and Hif2a in NG2 cells. Cerebral vasculature was assessed by immunofluorescence, RNA in situ hybridization, gene and protein expression analysis, gel zymography and in situ zymography. Results Vhl inactivation led to a significant increase in angiogenic gene and Epo expression. This was associated with EPO‐independent expansion of capillary networks in cortex, striatum and hypothalamus, as well as pericyte proliferation. A comparable phenotype resulted from the combined inactivation of Phd2 and Phd3, but not from Phd2 inactivation alone. Concomitant PHD1 function loss led to further expansion of the neurovasculature. Genetic inactivation of Hif2a in Phd1/Phd2/Phd3 triple mutant mice resulted in normal cerebral vasculature. Conclusion Our studies establish (a) that HIF2 activation in NG2 cells promotes neurovascular expansion and remodelling independently of EPO, (b) that HIF2 activity in NG2 cells is co‐controlled by PHD2 and PHD3 and (c) that PHD1 modulates HIF2 transcriptional responses when PHD2 and PHD3 are inactive.

Aim Skeletal muscles are distributed throughout the body, presenting a variety of sizes, shapes and functions. Here, we examined whether muscle regeneration and atrophy occurred homogeneously throughout the body in mouse models. Methods Acute muscle regeneration was induced by a single intramuscular injection of cardiotoxin in adult mice. Chronic muscle regeneration was assessed in mdx mice. Muscle atrophy in different muscles was evaluated by cancer cachexia, ageing and castration mouse models. Results We found that, in the cardiotoxin‐injected acute muscle injury model, head muscles slowly regenerated, while limb muscles exhibited a rapid regeneration and even overgrowth. This overgrowth was also observed in limb muscles alone (but not in head muscles) in mdx mice as chronic injury models. We described the body region–specific decline in the muscle mass in muscle atrophy models: cancer cachexia‐induced, aged and castrated mice. The positional identities, including gene expression profiles and hormone sensitivity, were robustly preserved in the ectopically engrafted satellite cell‐derived muscles in the castrated model. Conclusion Our results indicate that positional identities in muscles should be considered for the development of efficient regenerative therapies for muscle weakness, such as muscular dystrophy and age‐related sarcopenia.

Aim High‐altitude pulmonary oedema (HAPE) is a non‐cardiogenic pulmonary oedema that can occur during rapid ascent to a high‐altitude environment. Classically, HAPE has been described as a condition resulting from a combination of pulmonary vasoconstriction and hypertension. Inflammation has been described as important in HAPE, although as a side effect of pulmonary oedema rather than as a causative factor. In this study, we aim to understand the role of hypoxic response in myeloid cells and its involvement in pathogenesis of HAPE. Methods We have generated a conditional deletion in mice of the von Hippel‐Lindau factor (VHL) in myeloid cells to determine the effect of a deregulated hypoxic response in pulmonary oedema. Results The deletion of VHL in pulmonary myeloid cells gave rise to pulmonary oedema, increased pulmonary vascular permeability and reduced performance during exertion. These changes were accompanied by reduced stroke volume in the left ventricle. Conclusion In this model, we show that a deregulated myeloid cell hypoxic response can trigger some of the most important symptoms of HAPE, and thus mice with a deletion of VHL in the myeloid lineage can function as a model of HAPE.

Aim To identify microRNAs (miRs) involved in the regulation of skeletal muscle mass. For that purpose, we have initially utilized an in silico analysis, resulting in the identification of miR‐29c as a positive regulator of muscle mass. Methods miR‐29c was electrotransferred to the tibialis anterior to address its morphometric and functional properties and to determine the level of satellite cell proliferation and differentiation. qPCR was used to investigate the effect of miR‐29c overexpression on trophicity‐related genes. C2C12 cells were used to determine the impact of miR‐29c on myogenesis and a luciferase reporter assay was used to evaluate the ability of miR‐29c to bind to the MuRF1 3′UTR. Results The overexpression of miR‐29c in the tibialis anterior increased muscle mass by 40%, with a corresponding increase in fibre cross‐sectional area and force and a 30% increase in length. In addition, satellite cell proliferation and differentiation were increased. In C2C12 cells, miR‐29c oligonucleotides caused increased levels of differentiation, as evidenced by an increase in eMHC immunostaining and the myotube fusion index. Accordingly, the mRNA levels of myogenic markers were also increased. Mechanistically, the overexpression of miR‐29c inhibited the expression of the muscle atrophic factors MuRF1, Atrogin‐1 and HDAC4. For the key atrogene MuRF1, we found that miR‐29c can bind to its 3′UTR to mediate repression. Conclusions The results herein suggest that miR‐29c can improve skeletal muscle size and function by stimulating satellite cell proliferation and repressing atrophy‐related genes. Taken together, our results indicate that miR‐29c might be useful as a future therapeutic device in diseases involving decreased skeletal muscle mass.

Aim This study aimed to explore the molecular mechanisms for the parietal cell loss and fundic hyperplasia observed in gastric mucosa of mice lacking the carbonic anhydrase 9 (CAIX). Methods We assessed the ability of CAIX‐knockout and WT gastric surface epithelial cells to withstand a luminal acid load by measuring the pH i of exteriorized gastric mucosa in vivo using two‐photon confocal laser scanning microscopy. Cytokines and claudin‐18A2 expression was analysed by RT‐PCR. Results CAIX‐knockout gastric surface epithelial cells showed significantly faster pH i decline after luminal acid load compared to WT. Increased gastric mucosal IL‐1β and iNOS, but decreased claudin‐18A2 expression (which confer acid resistance) was observed shortly after weaning, prior to the loss of parietal and chief cells. At birth, neither inflammatory cytokines nor claudin‐18 expression were altered between CAIX and WT gastric mucosa. The gradual loss of acid secretory capacity was paralleled by an increase in serum gastrin, IL‐11 and foveolar hyperplasia. Mild chronic proton pump inhibition from the time of weaning did not prevent the claudin‐18 decrease nor the increase in inflammatory markers at 1 month of age, except for IL‐1β. However, the treatment reduced the parietal cell loss in CAIX‐KO mice in the subsequent months. Conclusions We propose that CAIX converts protons that either backflux or are extruded from the cells rapidly to CO 2 and H2O, contributing to tight junction protection and gastric epithelial pH i regulation. Lack of CAIX results in persistent acid backflux via claudin‐18 downregulation, causing loss of parietal cells, hypergastrinaemia and foveolar hyperplasia.

In recent years, most of the manuscripts published as a regular paper in ACTA Physiologica are based on animal experiments. As the mouse is the workhorse of physiologic research, most of the papers would not exist without animal experiments. All authors willing to submit manuscripts to ACTA Physiologica are expected to follow the 3R principle to reduce, refine, and replace animal experiments. 1 Most of the major research funding bodies in Europe require that the projects funded by them take full account of the 3Rs principle. It is a major issue for much of the public who are concerned about animal welfare and the need to avoid causing unnecessary or unjustified suffering. Applying the 3R strategy in physiologic research is not without difficulties but it is possible.

The response to exercise training (trainability) has been shown to have a strong heritable component. There is growing evidence suggesting that traits such as trainability do not only depend on the genetic code, but also on epigenetic signals. Epigenetic signals play an important role in the modulation of gene expression, through mechanisms such as DNA methylation and histone modifications. There is an emerging evidence to show that physical activity influences DNA methylation in humans. The present review aims to summarize current knowledge on the link between DNA methylation and physical activity in humans. We have critically reviewed the literature and only papers focused on physical activity and its influence on DNA methylation status were included; a total of 25 papers were selected. We concluded that both acute and chronic exercises significantly impact DNA methylation, in a highly tissue- and gene-specific manner. This review also provides insights into the molecular mechanisms of exercise-induced DNA methylation changes, and recommendations for future research.

The present paper deals with a fundamental issue in neuroscience: the inter-neuronal communication. The paper gives a brief account of our previous and more recent theoretical contributions to the subject and also reports new recent data that support some aspects of our proposal on two major modes of communication in the central nervous system: the wiring and the volume transmission. There exist two competing theories on inter-neuronal communication: the neuron doctrine and the theory of the diffuse nerve network, supported by Cajal and Golgi, respectively (see their respective Nobel Lectures). The present paper gives a brief account of a view on inter-neuronal communication in the brain, the volume and wiring transmission concept that to a great extent reconcile these two theories. Thus, the theory of volume and wiring transmission are summarized and its recent developments that allow to extend these two modes of communication from the cellular network to the molecular network level is also briefly illustrated. The explanatory value of this broadened view is further enhanced by our recent proposal on the existence of a Global Molecular Network enmeshing the entire central nervous system. It may be interesting to note that also the Global Molecular Network theory is reminiscent of the old reticular theory of Apathy. Finally, the so-called 'tide hypothesis' for diffusion of signals in the brain is briefly discussed and its possible extension to the molecular level is for the first time introduced. Early indirect evidence supporting volume transmission in the brain was the discovery of transmitter-receptor mismatches. Thus, as an experimental part of the present paper a new approach to evaluate transmitter-receptor mismatches is given and evidence for inter-relationships between temperature micro-gradients and mismatches is provided.

Chemically skinned fibres allow the study of human muscle contractile function in vitro. A particularly important parameter is specific force (SF), that is, maximal isometric force divided by cross-sectional area, representing contractile quality. Although SF varies substantially between studies, the magnitude and cause of this variability remains puzzling. Here, we aimed to summarize and explore the cause of variability in SF between studies. A systematic search was conducted in Medline, Embase and Web of Science databases in June 2020, yielding 137 data sets from 61 publications which studied healthy, young adults. Five-fold differences in mean SF data were observed. Adjustments to the reported data for key methodological differences allowed between-study comparisons to be made. However, adjustment for fibre shape, swelling and sarcomere length failed to significantly reduce SF variance (I2 = 96%). Interestingly, grouping papers based on shared authorship did reveal consistency within research groups. In addition, lower SF was found to be associated with higher phosphocreatine concentrations in the fibre activating solution and with Triton X-100 being used as a skinning agent. Although the analysis showed variance across the literature, the ratio of SF in single fibres containing myosin heavy chain isoforms IIA or I was found to be consistent across research groups. In conclusion, whilst the skinned fibre technique is reliable for studying in vitro force generation of single fibres, the composition of the solution used to activate fibres, which differs between research groups, is likely to heavily influence SF values.

Dr DeGroat and Wickens has reviewed the central neural mechanisms controlling the lower urinary tract with a major focus on the brain stem circuitry that mediates the switch-like characteristics of micturition, in particular the periaqueductal grey and the pontine micturition centre (de 2012). The review culminates in a computer model of how the brainstem switch operates in animals in which forebrain influences on micturition have been removed by decerebration. In this complementary paper, we review the mechanisms of forebrain involvement in the voluntary control of human micturition and the maintenance of continence with evidence based heavily on the results of functional brain imaging experiments.

The comorbidity of cardiovascular disease (CVD) and depression/anxiety disorders is well established, but the mechanisms are not well understood. This paper will review the epidemiological and biological evidence for the role of depression in CVD, as well as the pathophysiological process underlying both depression and CVD. The focus will be on the roles of serotonin, platelets, and the immune system, with an emphasis on the relevance of sex differences in both depression/anxiety and CVD as they pertain to women.

Aim: To identify the mechanisms of P2X3 receptor inhibition by extracellular cyclic adenosine monophosphate (cAMP) in rat dorsal root ganglion (DRG) neurones. Methods: Whole-cell currents were measured in cultured DRG neurones using the combination of voltage and concentration clamp. Results: We have found that extracellular cAMP inhibits P2X3-mediated currents in a concentration- and use-dependent manner. The P2X3 currents, activated by ATP applied every 4 min, were inhibited by 55% in the presence of 10 lm cAMP and by 81% in the presence of 30 lm cAMP. At 8 min interval between ATP applications the same concentration of cAMP did not alter the currents. Addition of 0.5 mm of guanosine 5¢-O-(2-thiodiphosphate) to intracellular solution blocked the inhibitory action of cAMP. The inhibitory effects of cAMP were not mimicked by extracellular application of 30 lm adenosine. Conclusions: In this paper, we demonstrate, for the first time, that extracellular application of cAMP to rat sensory neurones inhibits P2X3 receptors via a G protein-coupled mechanism in a use-dependent manner, thus indicating the neuronal expression of specific plasmalemmal cAMP receptor.

Long chain fatty acids (LCFAs) are an important substrate for ATP production within the skeletal muscle. The process of LCFA delivery from adipose tissue to muscle mitochondria involves many regulatory steps. Recently, it has been recognized that LCFA oxidation is not only dependent on LCFA delivery to the muscle, but also on regulatory steps within the muscle. Increasing selected fatty acid binding proteins/transporters on the plasma membrane facilitates a very rapid LCFA increase into the muscle, independent of any changes in LCFA delivery to the muscle. Such a mechanism of LCFA transporter translocation is activated by muscle contraction. Intramuscular triacylglycerols may also be hydrolysed to provide fatty acids for mitochondrial oxidation, particularly during exercise, when hormone-sensitive lipase and other enzymes are activated. Mitochondrial LCFA entry is also highly regulated. This however does not involve only the malonyl CoA carnitine palmitoyltransferase-I (CPTI) axis. Exercise-induced fatty acid entry into mitochondria is also regulated by at least one of the proteins (FAT/CD36) that also regulates plasma membrane fatty acid transport. Among individuals, differences in mitochondrial fatty acid oxidation appear to be correlated with the content of mitochondrial CPTI and FAT/CD36. This paper provides a brief overview of mechanisms that regulate LCFA uptake and oxidation in skeletal muscle during exercise and in obesity. We focus largely on our own work on FAT/CD36, which contributes to regulating, in a coordinated fashion, LCFA uptake across the plasma membrane and the mitochondrial membrane. Very little is known about the roles of FATP1-6 on fatty acid transport in skeletal muscle.

Voltage-gated Ca 2+ channels (Cav) are highly expressed in the adrenal chromaffin cells of mammalian species. Besides shaping action potential waveforms, they are directly involved in the excitation-secretion coupling underlying catecholamine release and, possibly, control other Ca 2+ -dependent events that originate near the membrane. These functions are shared by a number of Cav channel types (L, N, P/Q, R and T) which have different structure-function characteristics and whose degree of expression changes remarkably among mammalian species. Understanding precisely the functioning of each voltage-gated Ca 2+ channels is a crucial task that helps clarifying the Ca 2+ -dependent mechanisms controlling exocytosis during physiological and pathological conditions.In this paper, we focus on classical and new roles that L- and T-type channels play in the control of chromaffin cell excitability and neurotransmitter release. Interestingly, L-type channels are shown to be implicated in the spontaneous autorhythmicity of chromaffin cells, while T-type channels, which are absent in adult chromaffin cells, are coupled with secretion and can be recruited following long-term ;8-adrenergic stimulation or chronic hypoxia. This suggests that like other cells, adrenal chromaffin cells undergo effective remodelling of membrane ion channels and cell functioning during prolonged stress conditions.

The paper reviews findings in humans regarding interindividual differences in sympathetic nerve activity. Data come predominantly from microneurographic multi- or single fibre recordings of sympathetic nerve activity in healthy subjects. Findings relate to interindividual differences in muscle sympathetic nerve activity (MSNA) during resting conditions and in response to surprising sensory stimuli. At rest there are marked interindividual differences in the number of multiunit MSNA bursts. At the single fibre level the differences are because of more vasoconstrictor fibres being active in subjects with high than in subjects with low number of bursts. There are inverse relationships between (i) sympathetic burst incidence and cardiac output (CO) and (ii) between sympathetic burst incidence and vascular responsiveness to noradrenaline. Both findings contribute to explaining the absence of correlation between resting levels of MSNA and blood pressure. Surprising visual, somatosensory or auditory stimuli of sufficient strength cause a short lasting inhibition of MSNA in approx. 50-60% of healthy subjects. In subjects who display significant inhibition, the stimulus-induced blood pressure increase is smaller than in subjects without inhibition. The underlying mechanism may be related to fear of blood/injury. It is concluded that analysis of interindividual differences in sympathetic activity improves the understanding of central nervous control of the circulation.