Conduit artery structure and function in lowlanders and native highlanders: relationships with oxidative stress and role of sympathoexcitation
Research detailing the normal vascular adaptions to high altitude is minimal and often confounded by pathology (e.g. chronic mountain sickness) and methodological issues. We examined vascular function and structure in: (1) healthy lowlanders during acute hypoxia and prolonged (~2 weeks) exposure to high altitude, and (2) high-altitude natives at 5050 m (highlanders). In 12 healthy lowlanders (aged 32 ± 7 years) and 12 highlanders (Sherpa; 33 ± 14 years) we assessed brachial endothelium-dependent flow-mediated dilatation (FMD), endothelium-independent dilatation (via glyceryl trinitrate; GTN), common carotid intima–media thickness (CIMT) and diameter (ultrasound), and arterial stiffness via pulse wave velocity (PWV; applanation tonometry). Cephalic venous biomarkers of free radical-mediated lipid peroxidation (lipid hydroperoxides, LOOH), nitrite (NO2–) and lipid soluble antioxidants were also obtained at rest. In lowlanders, measurements were performed at sea level (334 m) and between days 3–4 (acute high altitude) and 12–14 (chronic high altitude) following arrival to 5050 m. Highlanders were assessed once at 5050 m. Compared with sea level, acute high altitude reduced lowlanders’ FMD (7.9 ± 0.4 vs. 6.8 ± 0.4%; P = 0.004) and GTN-induced dilatation (16.6 ± 0.9 vs. 14.5 ± 0.8%; P = 0.006), and raised central PWV (6.0 ± 0.2 vs. 6.6 ± 0.3 m s–1; P = 0.001). These changes persisted at days 12–14, and after allometrically scaling FMD to adjust for altered baseline diameter. Compared to lowlanders at sea level and high altitude, highlanders had a lower carotid wall:lumen ratio (~19%, P ≤ 0.04), attributable to a narrower CIMT and wider lumen. Although both LOOH and NO2– increased with high altitude in lowlanders, only LOOH correlated with the reduction in GTN-induced dilatation evident during acute (n = 11, r = –0.53) and chronic (n = 7, r = –0.69; P ≤ 0.01) exposure to 5050 m. In a follow-up, placebo-controlled experiment (n = 11 healthy lowlanders) conducted in a normobaric hypoxic chamber (inspired O2 fraction (F IO 2) = 0.11; 6 h), a sustained reduction in FMD was evident within 1 h of hypoxic exposure when compared to normoxic baseline (5.7 ± 1.6 vs. 8.0 ±1.3%; P < 0.01); this decline in FMD was largely reversed following α1-adrenoreceptor blockade. In conclusion, high-altitude exposure in lowlanders caused persistent impairment in vascular function, which was mediated partially via oxidative stress and sympathoexcitation. Although a lifetime of high-altitude exposure neither intensifies nor attenuates the impairments seen with short-term exposure, chronic high-altitude exposure appears to be associated with arterial remodelling.
Effects of natriuretic peptides on electrical conduction in the sinoatrial node and atrial myocardium of the heart
Natriuretic peptides, including B-type and C-type natriuretic peptide (BNP and CNP), are powerful regulators of the cardiovascular system; however, their electrophysiological effects in the heart, particularly in the sinoatrial node (SAN), are incompletely understood. We have used high-resolution optical mapping to measure the effects of BNP and CNP, and the roles of natriuretic peptide receptors (NPR-A, NPR-B and NPR-C), on electrical conduction within the SAN and atrial myocardium. In basal conditions BNP and CNP (50–500 nm) increased conduction velocity (CV) within the SAN by ~30% at the high dose and shifted the initial exit site superiorly. These effects sped conduction from the SAN to the surrounding atrial myocardium and were mediated by the NPR-A and NPR-B receptors. In the presence of isoproterenol (1 μm) the NPR-C receptor made a major contribution to the effects of BNP and CNP in the heart. In these conditions BNP, CNP and the NPR-C agonist cANF each decreased SAN CV and shifted the initial exit site inferiorly. The effects of cANF (30% reduction) were larger than BNP or CNP (~15% reduction), indicating that BNP and CNP activate multiple natriuretic peptide receptors. In support of this, the inhibitory effects of BNP were absent in NPR-C knockout mice, where BNP instead elicited a further increase (~25%) in CV. Measurements in externally paced atrial preparations demonstrate that the effects of natriuretic peptides on CV are partially independent of changes in cycle length. These data provide detailed novel insight into the complex effects of natriuretic peptides and their receptors on electrical conduction in the heart.
Innate collateral segments are predominantly present in the subendocardium without preferential connectivity within the left ventricular wall
Functional collateral vessels often stem from outward remodelling of pre-existing connections between perfusion territories. Knowledge of the distribution and morphology of innate collateral connections may help in identifying myocardial areas with protection against risk for ischaemia. The coronary network of six healthy canine hearts was investigated with an imaging cryomicrotome. Innate collateral connections ranged from 286 to 1015 μm in diameter. Left ventricular collateral density (number per gram of tissue) was about five in the subendocardium vs. 2.5 in the mid-myocardium (P < 0.01) and 1.3 in the epicardium (P < 0.01). Subendocardial collateral connections were oriented parallel to the long axis of the heart. For the major coronary arteries, five times more intracoronary than intercoronary connections were found, while their median diameter and interquartile range were not significantly different, at 96.1 (16.9) vs. 94.7 (18.9) μm. Collateral vessels connecting crowns from sister branches from a stem are denoted intercrown connections and those within crowns intracrown connections. The number of intercrown connections was related to the mean tissue weight of the crowns (y = 0.73x – 0.33, r2 = 0.85, P < 0.0001). This relation was likewise found to describe intercoronary connections. The median collateral diameter and length were independent of the tissue volumes bridged. We conclude that connectivity and morphology of the innate collateral network are distributed with no preference for intra- or intercrown connections, independent of stem diameter, including epicardial arteries. This renders all sites of the myocardium equally protected in case of coronary artery disease. The orientation of subendocardial collateral vessels indicates the longitudinal direction of subendocardial collateral flow.
Short-term hypoxic vasodilation in vivo is mediated by bioactive nitric oxide metabolites, rather than free nitric oxide derived from haemoglobin-mediated nitrite reduction
Local increases in blood flow – ‘hypoxic vasodilation’ – confer cellular protection in the face of reduced oxygen delivery. The physiological relevance of this response is well established, yet ongoing controversy surrounds its underlying mechanisms. We sought to confirm that early hypoxic vasodilation is a nitric oxide (NO)-mediated phenomenon and to study putative pathways for increased levels of NO, namely production from NO synthases, intravascular nitrite reduction, release from preformed stores and reduced deactivation by cytochrome c oxidase. Experiments were performed on spontaneously breathing, anaesthetized, male Wistar rats undergoing short-term systemic hypoxaemia, who received pharmacological inhibitors and activators of the various NO pathways. Arterial blood pressure, cardiac output, tissue oxygen tension and the circulating pool of NO metabolites (oxidation, nitrosation and nitrosylation products) were measured in plasma and erythrocytes. Hypoxaemia caused a rapid and sustained vasodilation, which was only partially reversed by non-selective NO synthase inhibition. This was associated with significantly lower plasma nitrite, and marginally elevated nitrate levels, suggestive of nitrite bioinactivation. Administration of sodium nitrite had little effect in normoxia, but produced significant vasodilation and increased nitrosylation during hypoxaemia that could not be reversed by NO scavenging. Methodological issues prevented assessment of the contribution, if any, of reduced deactivation of NO by cytochrome c oxidase. In conclusion, acute hypoxic vasodilation is an adaptive NO-mediated response conferred through bioactive metabolites rather than free NO from haemoglobin-mediated reduction of nitrite.
Effect of endogenous hydrogen sulfide on the transwall gradient of the mouse colon circular smooth muscle
A transwall gradient in resting membrane potential (RMP) exists across the circular muscle layer in the mouse colon. This gradient is dependent on endogenous generation of CO. H2S is also generated in muscle layers of the mouse colon. The effect of endogenously generated H2S on the transwall gradient is not known. The aim was to investigate the role of endogenous H2S. Our results showed that the CSE inhibitor dl-propargylglycine (PAG, 500 μm) had no effect on the transwall gradient. However, in preparations pretreated with the nitric oxide synthase inhibitor N-nitro-l-arginine (l-NNA, 200 μm) and in nNOS-knockout (KO) mouse preparations, PAG shifted the transwall gradient in the depolarizing direction. In CSE-KO–nNOS-KO mice, the gradient was shifted in the depolarizing direction. Endogenous generation of NO was significantly higher in muscle preparations of CSE-KO mice compared to wild-type (WT) mice. The amplitude of NO-mediated slow inhibitory junction potentials (S-IJPs) evoked by electric field stimulation was significantly higher in CSE-KO mouse preparations compared to the amplitude of S-IJPs in wild-type mouse preparations. CSE was present in all submucosal ganglion neurons and in almost all myenteric ganglion neurons. Eleven per cent of CSE positive neurons in the submucosal plexus and 50% of CSE positive neurons in the myenteric plexus also contained nNOS. Our results suggest that endogenously generated H2S acts as a stealth hyperpolarizing factor on smooth muscle cells to maintain the CO-dependent transwall gradient and inhibits NO production from nNOS.
Effects of obesity, diabetes and exercise on Fndc5 gene expression and irisin release in human skeletal muscle and adipose tissue: in vivo and in vitro studies
Irisin was identified as a myokine secreted by contracting skeletal muscle, possibly mediating some exercise health benefits via ‘browning’ of white adipose tissue. However, a controversy exists concerning irisin origin, regulation and function in humans. Thus, we have explored Fndc5 gene and irisin protein in two clinical studies: (i) a cross-sectional study (effects of type 2 diabetes (T2D) in drug-naive men) and (ii) an intervention study (exercise effects in sedentary, overweight/obese individuals). Glucose tolerance and insulin sensitivity were assessed. Maximal aerobic capacity and muscle strength were measured before and after training. Body composition (magnetic resonance imaging), muscle and liver fat content (1H-magnetic resonance spectroscopy (MRS)) and in vivo muscle metabolism (32P-MRS) were determined. Skeletal muscle and subcutaneous abdominal adipose tissue samples were taken in the fasted state and during euglycaemic hyperinsulinaemia (adipose tissue) and before/after exercise training (muscle). We found that muscle Fndc5 mRNA was increased in prediabetes but not T2D. Fndc5 in adipose tissue and irisin in plasma were reduced in T2D by 40% and 50%, respectively. In contrast, T2D-derived myotubes expressed/secreted the highest levels of Fndc5/irisin. Neither hyperinsulinaemia (adipose tissue/plasma) nor exercise (muscle/plasma) affected Fndc5/irisin in vivo. Circulating irisin was positively associated with muscle mass, strength and metabolism and negatively with fasting glycaemia. Glucose and palmitate decreased Fndc5 mRNA in myotubes in vitro. We conclude that distinct patterns of Fndc5/irisin in muscle, adipose tissue and circulation, and concordant in vivo down-regulation in T2D, indicate that irisin might distinguish metabolic health and disease. Moreover, Fndc5/irisin was discordantly regulated in diabetic muscle and myotubes in vitro, suggesting that whole body factors, such as glucose and fatty acids, might be important for irisin regulation. Exercise did not affect Fndc5/irisin. However, irisin was positively linked to muscle mass, strength and metabolism, pointing to common regulatory factors and/or the potential for irisin to modify muscle phenotype.
Force in striated muscle is due to attachment of the heads of the myosin, the molecular motors extending from the myosin filament, to the actin filament in each half-sarcomere, the functional unit where myosin motors act in parallel. Mechanical and X-ray structural evidence indicates that at the plateau of isometric contraction (force T0), less than half of the elastic strain of the half-sarcomere is due to the strain in the array of myosin motors (s), with the remainder being accounted for by the compliance of filaments acting as linear elastic elements in series with the motor array. Early during the development of isometric force, however, the half-sarcomere compliance has been found to be less than that expected from the linear elastic model assumed above, and this non-linearity may affect the estimate of s. This question is investigated here by applying nanometre–microsecond-resolution mechanics to single intact fibres from frog skeletal muscle at 4°C, to record the mechanical properties of the half-sarcomere throughout the development of force in isometric contraction. The results are interpreted with mechanical models to estimate the compliance of the myosin motors. Our conclusions are as follows: (i) early during the development of an isometric tetanus, an elastic element is present in parallel with the myosin motors, with a compliance of ~200 nm MPa–1 (~20 times larger than the compliance of the motor array at T0); and (ii) during isometric contraction, s is 1.66 ± 0.05 nm, which is not significantly different from the value estimated with the linear elastic model.
X-ray diffraction patterns were recorded at beamline ID02 of the European Synchrotron Radiation Facility from small bundles of skeletal muscle fibres from Rana esculenta at sarcomere lengths between 2.1 and 3.5 μm at 4°C. The intensities of the X-ray reflections from resting fibres associated with the quasi-helical order of the myosin heads and myosin binding protein C (MyBP-C) decreased in the sarcomere length range 2.6–3.0 μm but were constant outside it, suggesting that an OFF conformation of the thick filament is maintained by an interaction between MyBP-C and the thin filaments. During active isometric contraction the intensity of the M3 reflection from the regular repeat of the myosin heads along the filaments decreased in proportion to the overlap between thick and thin filaments, with no change in its interference fine structure. Thus, myosin heads in the regions of the thick filaments that do not overlap with thin filaments are highly disordered during isometric contraction, in contrast to their quasi-helical order at rest. Heads in the overlap region that belong to two-headed myosin molecules that are fully detached from actin are also highly disordered, in contrast to the detached partners of actin-attached heads. These results provide strong support for the concept of a regulatory structural transition in the thick filament involving changes in both the organisation of the myosin heads on its surface and the axial periodicity of the myosin tails in its backbone, mediated by an interaction between MyBP-C and the thin filaments.
A network of kinases, including WNKs, SPAK and Sgk1, is critical for the independent regulation of K+ and Na+ transport in the distal nephron. Angiotensin II is thought to act as a key hormone in orchestrating these kinases to switch from K+ secretion during hyperkalaemia to Na+ reabsorption during intravascular volume depletion, thus keeping disturbances in electrolyte and blood pressure homeostasis at a minimum. It remains unclear, however, how K+ and Na+ transport are regulated during a high Na+ intake, which is associated with suppressed angiotensin II levels and a high distal tubular Na+ load. We therefore investigated the integrated blood pressure, renal, hormonal and gene and protein expression responses to large changes of K+ intake in Na+ replete mice. Both low and high K+ intake increased blood pressure and caused Na+ retention. Low K+ intake was accompanied by an upregulation of the sodium-chloride cotransporter (NCC) and its activating kinase SPAK, and inhibition of NCC normalized blood pressure. Renal responses were unaffected by angiotensin AT1 receptor antagonism, indicating that low K+ intake activates the distal nephron by an angiotensin-independent mode of action. High K+ intake was associated with elevated plasma aldosterone concentrations and an upregulation of the epithelial sodium channel (ENaC) and its activating kinase Sgk1. Surprisingly, high K+ intake increased blood pressure even during ENaC or mineralocorticoid receptor antagonism, suggesting the contribution of aldosterone-independent mechanisms. These findings show that in a Na+ replete state, changes in K+ intake induce specific molecular and functional adaptations in the distal nephron that cause a functional coupling of renal K+ and Na+ handling, resulting in Na+ retention and high blood pressure when K+ intake is either restricted or excessively increased.
The mitochondrial calcium uniporter complex: molecular components, structure and physiopathological implications
Although it has long been known that mitochondria take up Ca2+, the molecular identities of the channels and transporters involved in this process were revealed only recently. Here, we discuss the recent work that has led to the characterization of the mitochondrial calcium uniporter complex, which includes the channel-forming subunit MCU (mitochondrial calcium uniporter) and its regulators MICU1, MICU2, MCUb, EMRE, MCUR1 and miR-25. We review not only the biochemical identities and structures of the proteins required for mitochondrial Ca2+ uptake but also their implications in different physiopathological contexts.
Herein, we review mechanisms regulating cerebral blood flow (CBF), with specific focus on humans. We revisit important concepts from the older literature and describe the interaction of various mechanisms of cerebrovascular control. We amalgamate this broad scope of information into a brief review, rather than detailing any one mechanism or area of research. The relationship between regulatory mechanisms is emphasized, but the following three broad categories of control are explicated: (1) the effect of blood gases and neuronal metabolism on CBF; (2) buffering of CBF with changes in blood pressure, termed cerebral autoregulation; and (3) the role of the autonomic nervous system in CBF regulation. With respect to these control mechanisms, we provide evidence against several canonized paradigms of CBF control. Specifically, we corroborate the following four key theses: (1) that cerebral autoregulation does not maintain constant perfusion through a mean arterial pressure range of 60–150 mmHg; (2) that there is important stimulatory synergism and regulatory interdependence of arterial blood gases and blood pressure on CBF regulation; (3) that cerebral autoregulation and cerebrovascular sensitivity to changes in arterial blood gases are not modulated solely at the pial arterioles; and (4) that neurogenic control of the cerebral vasculature is an important player in autoregulatory function and, crucially, acts to buffer surges in perfusion pressure. Finally, we summarize the state of our knowledge with respect to these areas, outline important gaps in the literature and suggest avenues for future research.
Neurotransmitter transporters are ion-coupled symporters that drive the uptake of neurotransmitters from neural synapses. In the past decade, the structure of a bacterial amino acid transporter, leucine transporter (LeuT), has given valuable insights into the understanding of architecture and mechanism of mammalian neurotransmitter transporters. Different conformations of LeuT, including a substrate-free state, inward-open state, and competitive and non-competitive inhibitor-bound states, have revealed a mechanistic framework for the transport and transport inhibition of neurotransmitters. The current review integrates our understanding of the mechanistic and pharmacological properties of eukaryotic neurotransmitter transporters obtained through structural snapshots of LeuT.
Dietary proteins are cleaved within the intestinal lumen to oligopeptides which are further processed to small peptides (di- and tripeptides) and free amino acids. Although the transport of amino acids is mediated by several specific amino acid transporters, the proton-coupled uptake of the more than 8000 different di- and tripeptides is performed by the high-capacity/low-affinity peptide transporter isoform PEPT1 (SLC15A1). Its wide substrate tolerance also allows the transport of a repertoire of structurally closely related compounds and drugs, which explains their high oral bioavailability and brings PEPT1 into focus for medical and pharmaceutical approaches. Although the first evidence for the interplay of nutrient supply and PEPT1 expression and function was described over 20 years ago, many aspects of the molecular processes controlling its transcription and translation and modifying its transporter properties are still awaiting discovery. The present review summarizes the recent knowledge on the factors modulating PEPT1 expression and function in Caenorhabditis elegans, Danio rerio, Mus musculus and Homo sapiens, with focus on dietary ingredients, transcription factors and functional modulators, such as the sodium–proton exchanger NHE3 and selected scaffold proteins.
Teleost fish models in membrane transport research: the PEPT1(SLC15A1) H+-oligopeptide transporter as a case study
Human genes for passive, ion-coupled transporters and exchangers are included in the so-called solute carrier (SLC) gene series, to date consisting of 52 families and 398 genes. Teleost fish genes for SLC proteins have also been described in the last two decades, and catalogued in preliminary SLC-like form in 50 families and at least 338 genes after systematic GenBank database mining (December 2010–March 2011). When the kinetic properties of the expressed proteins are studied in detail, teleost fish SLC transporters always reveal extraordinary ‘molecular diversity’ with respect to the mammalian counterparts, which reflects peculiar adaptation of the protein to the physiology of the species and/or to the environment where the species lives. In the case of the H+–oligopeptide transporter PEPT1(SLC15A1), comparative analysis of diverse teleost fish orthologs has shown that the protein may exhibit very eccentric properties in terms of pH dependence (e.g. the adaptation of zebrafish PEPT1 to alkaline pH), temperature dependence (e.g. the adaptation of icefish PEPT1 to sub-zero temperatures) and/or substrate specificity (e.g. the species-specificity of PEPT1 for the uptake of l-lysine-containing peptides). The revelation of such peculiarities is providing new contributions to the discussion on PEPT1 in both basic (e.g. molecular structure–function analyses) and applied research (e.g. optimizing diets to enhance growth of commercially valuable fish).
Phosphatase activity of the voltage-sensing phosphatase, VSP, shows graded dependence on the extent of activation of the voltage sensor
The voltage-sensing phosphatase (VSP) consists of a voltage sensor and a cytoplasmic phosphatase region, and the movement of the voltage sensor is coupled to the phosphatase activity. However, its coupling mechanisms still remain unclear. One possible scenario is that the phosphatase is activated only when the voltage sensor is in a fully activated state. Alternatively, the enzymatic activity of single VSP proteins could be graded in distinct activated states of the voltage sensor, and partial activation of the voltage sensor could lead to partial activation of the phosphatase. To distinguish between these two possibilities, we studied a voltage sensor mutant of zebrafish VSP, where the voltage sensor moves in two steps as evidenced by analyses of charge movements of the voltage sensor and voltage clamp fluorometry. Measurements of the phosphatase activity toward phosphatidylinositol 4,5-bisphosphate revealed that both steps of voltage sensor activation are coupled to the tuning of phosphatase activities, consistent with the idea that the phosphatase activity is graded by the magnitude of the movement of the voltage sensor.