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Cerebrovascular reactivity is the change in cerebral blood flow in response to a vasodilatory or vasoconstrictive stimulus. Measuring variations of cerebrovascular reactivity between different regions of the brain has the potential to not only advance understanding of how the cerebral vasculature controls the distribution of blood flow but also to detect cerebrovascular pathophysiology. While there are standardized and repeatable methods for estimating the changes in cerebral blood flow in response to a vasoactive stimulus, the same cannot be said for the stimulus itself. Indeed, the wide variety of vasoactive challenges currently employed in these studies impedes comparisons between them. This review therefore critically examines the vasoactive stimuli in current use for their ability to provide a standard repeatable challenge and for the practicality of their implementation. Such challenges include induced reductions in systemic blood pressure, and the administration of vasoactive substances such as acetazolamide and carbon dioxide. We conclude that many of the stimuli in current use do not provide a standard stimulus comparable between individuals and in the same individual over time. We suggest that carbon dioxide is the most suitable vasoactive stimulus. We describe recently developed computer-controlled MRI compatible gas delivery systems which are capable of administering reliable and repeatable vasoactive CO2 stimuli.
Important considerations for protein analyses using antibody based techniques: down-sizing Western blotting up-sizes outcomes
Western blotting has been used for protein analyses in a wide range of tissue samples for >30 years. Fundamental to Western blotting success are a number of important considerations, which unfortunately are often overlooked or not appreciated. Firstly, lowly expressed proteins may often be better detected by dramatically reducing the amount of sample loaded. Single cell (fibre) Western blotting demonstrates the ability to detect proteins in small sample sizes, 5–10 g total mass (1–3 g total protein). That is an order of magnitude less than often used. Using heterogeneous skeletal muscle as the tissue of representation, the need to undertake Western blotting in sample sizes equivalent to single fibre segments is demonstrated. Secondly, incorrect results can be obtained if samples are fractionated and a proportion of the protein of interest inadvertently discarded during sample preparation. Thirdly, quantitative analyses demand that a calibration curve be used. This is regardless of using a loading control, which must be proven to not change with the intervention and also be appropriately calibrated. Fourthly, antibody specificity must be proven using whole tissue analyses, and for immunofluorescence analyses it is vital that only a single protein is detected. If appropriately undertaken, Western blotting is reliable, quantitative, both in relative and absolute terms, and extremely valuable.
Exosomes are vesicles that are released from the kidney into urine. They contain protein and RNA from the glomerulus and all sections of the nephron and represent a reservoir for biomarker discovery. Current methods for the identification and quantification of urinary exosomes are time consuming and only semi-quantitative. Nanoparticle tracking analysis (NTA) counts and sizes particles by measuring their Brownian motion in solution. In this study, we applied NTA to human urine and identified particles with a range of sizes. Using antibodies against the exosomal proteins CD24 and aquaporin 2 (AQP2), conjugated to a fluorophore, we could identify a subpopulation of CD24- and AQP2-positive particles of characteristic exosomal size. Extensive pre-NTA processing of urine was not necessary. However, the intra-assay variability in the measurement of exosome concentration was significantly reduced when an ultracentrifugation step preceded NTA. Without any sample processing, NTA tracked exosomal AQP2 upregulation induced by desmopressin stimulation of kidney collecting duct cells. Nanoparticle tracking analysis was also able to track changes in exosomal AQP2 concentration that followed desmopressin treatment of mice and a patient with central diabetes insipidus. When urine was stored at room temperature, 4°C or frozen, nanoparticle concentration was reduced; freezing at –80°C with the addition of protease inhibitors produced the least reduction. In conclusion, with appropriate sample storage, NTA has potential as a tool for the characterization and quantification of extracellular vesicles in human urine.
Intracellular water plays a critical role in apoptotic and necrotic cell death. We describe a method for quantifying cell water by application of two previously described variants of transmission microscopy. By taking two axially displaced brightfield images, the phase shift of the transmitted wave was computed using the transport-of-intensity equation. At the same time, cell thickness was determined by transmission through an externally applied dye (‘transmission-through-dye' microscopy); switching between these two imaging modalities was accomplished by simply changing the illumination wavelength. The sets of data thus obtained allow computation of the refractive index and cell water content within individual cells. The method was illustrated using cells treated with apoptotic agents staurosporine and actinomycin D and with necrosis inducer ionomycin. Water imaging allows discrimination between apoptotic volume decrease due to dehydration from that due to detachment of apoptotic bodies and can be used on samples where cell volume determination alone would be difficult or insufficient.
Increases in intracellular pH facilitate endocytosis and decrease availability of voltage-gated proton channels in osteoclasts and microglia
Voltage-gated proton channels (H+ channels) are highly proton-selective transmembrane pathways. Although the primary determinants for activation are the pH and voltage gradients across the membrane, the current amplitudes fluctuate often when these gradients are constant. The aim of this study was to investigate the role of the intracellular pH (pHi) in regulating the availability of H+ channels in osteoclasts and microglia. In whole-cell clamp recordings, the pHi was elevated after exposure to NH4Cl and returned to the control level after washout. However, the H+ channel conductance did not recover fully when the exposure was prolonged (>5 min). Similar results were observed in osteoclasts and microglia, but not in COS7 cells expressing a murine H+ channel gene (mVSOP). As other electrophysiological properties, like the gating kinetics and voltage dependence for activation, were unchanged, the decreases in the H+ channel conductance were probably due to the decreases in H+ channels available at the plasma membrane. The decreases in the H+ channel conductances were accompanied by reductions in the cell capacitance. Exposure to NH4Cl increased the uptake of the endocytosis marker FM1-43, substantiating the idea that pHi increases facilitated endocytosis. In osteoclasts, whose plasma membrane expresses V-ATPases and H+ channels, pHi increases by these H+-transferring molecules in part facilitated endocytosis. The endocytosis and decreases in the H+ channel conductance were reduced by dynasore, a dynamin blocker. These results suggest that pHi increases in osteoclasts and microglia decrease the numbers of H+ channels available at the plasma membrane through facilitation of dynamin-dependent endocytosis.
Myosin molecules from smooth muscle and non-muscle cells are known to self-assemble into side-polar filaments in vitro. However, the in situ mechanism of filament assembly is not clear and the question of whether there is a unique length for myosin filaments in smooth muscle is still under debate. In this study we measured the lengths of 16,587 myosin filaments in three types of smooth muscle cells using serial electron microscopy (EM). Sheep airway and pulmonary arterial smooth muscle as well as rabbit carotid arterial smooth muscle were fixed for EM and serial ultra-thin (50–60 nm) sections were obtained. Myosin filaments were traced in consecutive sections to determine their lengths. The results indicate that there is not a single length for the myosin filaments; instead there is a wide variation in lengths. The plots of observation frequency versus myosin filament length follow an exponential decay pattern. Analysis suggests that in situ assembly of myosin filaments in smooth muscle is governed by random processes of linear polymerization and de-polymerization, and that the dynamic equilibrium of these processes determines the observed length distribution.
ClC-5 is a 2Cl–/1H+ antiporter highly expressed in endosomes of proximal tubule cells. It is essential for endocytosis and mutations in ClC-5 cause Dent's disease, potentially leading to renal failure. However, the physiological role of ClC-5 is still unclear. One of the main issues is whether the strong rectification of ClC-5 currents observed in heterologous systems, with currents elicited only at positive voltages, is preserved in vivo and what is the origin of this rectification. In this work we identified a ClC-5 mutation, D76H, which, besides the typical outward currents of the wild-type (WT), shows inward tail currents at negative potentials that allow the estimation of the reversal of ClC-5 currents for the first time. A detailed analysis of the dependence of these inward tail currents on internal and external pH and [Cl–] shows that they are generated by a coupled transport of Cl– and H+ with a 2 : 1 stoichiometry. From this result we conclude that the inward tail currents are caused by a gating mechanism that regulates ClC-5 transport activity and not by a major alteration of the transport mechanism itself. This implies that the strong rectification of the currents of WT ClC-5 is at least in part caused by a gating mechanism that activates the transporter at positive potentials. These results elucidate the biophysical properties of ClC-5 and contribute to the understanding of its physiological role.
Redox modification of ryanodine receptors by mitochondria-derived reactive oxygen species contributes to aberrant Ca2+ handling in ageing rabbit hearts
Ageing is associated with a blunted response to sympathetic stimulation and an increased risk of arrhythmia and sudden cardiac death. Aberrant calcium (Ca2+) handling is an important contributor to the electrical and contractile dysfunction associated with ageing. Yet, the specific molecular mechanisms underlying abnormal Ca2+ handling in ageing heart remain poorly understood. In this study, we used ventricular myocytes isolated from young (5–9 months) and old (4–6 years) rabbit hearts to test the hypothesis that changes in Ca2+ homeostasis are caused by post-translational modification of ryanodine receptors (RyRs) by mitochondria-derived reactive oxygen species (ROS) generated in the ageing heart. Changes in parameters of Ca2+ handling were determined by measuring cytosolic and intra-sarcoplasmic reticulum (SR) Ca2+ dynamics in intact and permeabilized ventricular myocytes using confocal microscopy. We also measured age-related changes in ROS production and mitochondria membrane potential using a ROS-sensitive dye and a mitochondrial voltage-sensitive fluorescent indicator, respectively. In permeablized myocytes, ageing did not change SERCA activity and spark frequency but decreased spark amplitude and SR Ca2+ load suggesting increased RyR activity. Treatment with the antioxidant dithiothreitol reduced RyR-mediated SR Ca2+ leak in permeabilized myocytes from old rabbit hearts to the level comparable to young. Moreover, myocytes from old rabbits had more depolarized mitochondria membrane potential and increased rate of ROS production. Under -adrenergic stimulation, Ca2+ transient amplitude, SR Ca2+ load, and latency of pro-arrhythmic spontaneous Ca2+ waves (SCWs) were decreased while RyR-mediated SR Ca2+ leak was increased in cardiomyocytes from old rabbits. Additionally, with -adrenergic stimulation, scavenging of mitochondrial ROS in myocytes from old rabbit hearts restored redox status of RyRs, which reduced SR Ca2+ leak, ablated most SCWs, and increased latency to levels comparable to young. These data indicate that an age-associated increase of ROS production by mitochondria leads to the thiol-oxidation of RyRs, which underlies the hyperactivity of RyRs and thereby shortened refractoriness of Ca2+ release in cardiomyocytes from the ageing heart. This mechanism probably plays an important role in the increased incidence of arrhythmia and sudden death in the ageing population.
Chronic renin inhibition lowers blood pressure and reduces upright muscle sympathetic nerve activity in hypertensive seniors
Cardiovascular risk remains high in patients with hypertension even with adequate blood pressure (BP) control. One possible mechanism may be sympathetic activation via the baroreflex. We tested the hypothesis that chronic inhibition of renin reduces BP without sympathetic activation, but diuresis augments sympathetic activity in elderly hypertensives. Fourteen patients with stage-I hypertension (66 ± 5 (SD) years) were treated with a direct renin inhibitor, aliskiren (n = 7), or a diuretic, hydrochlorothiazide (n = 7), for 6 months. Muscle sympathetic nerve activity (MSNA), BP, direct renin and aldosterone were measured during supine and a graded head-up tilt (HUT; 5 min 30° and 20 min 60°), before and after treatment. Sympathetic baroreflex sensitivity (BRS) was assessed. Both groups had similar BP reductions after treatment (all P < 0.01), while MSNA responses were different between hydrochlorothiazide and aliskiren (P = 0.006 pre/post x drug). Both supine and upright MSNA became greater after hydrochlorothiazide treatment (supine, 72 ± 18 post vs. 64 ± 15 bursts (100 beats)–1 pre; 60° HUT, 83 ± 10 vs. 78 ± 13 bursts (100 beats)–1; P = 0.002). After aliskiren treatment, supine MSNA remained unchanged (69 ± 13 vs. 64 ± 8 bursts (100 beats)–1), but upright MSNA was lower (74 ± 15 vs. 85 ± 10 bursts (100 beats)–1; P = 0.012 for pre/post x posture). Direct renin was greater after both treatments (both P < 0.05), while upright aldosterone was greater after hydrochlorothiazide only (P = 0.002). The change in upright MSNA by the treatment was correlated with the change of aldosterone (r = 0.74, P = 0.002). Upright sympathetic BRS remained unchanged after either treatment. Thus, chronic renin inhibition may reduce upright MSNA through suppressed renin activity, while diuresis may evoke sympathetic activation via the upregulated renin–angiotensin–aldosterone system, without changing intrinsic sympathetic baroreflex function in elderly hypertensive patients.
Abnormal ventricular repolarization in ion channelopathies and heart disease is a major cause of ventricular arrhythmias and sudden cardiac death. K+ channel-interacting protein 2 (KChIP2) expression is significantly reduced in human heart failure (HF), contributing to a loss of the transient outward K+ current (Ito). We aim to investigate the possible significance of a changed KChIP2 expression on the development of HF and proarrhythmia. Transverse aortic constrictions (TAC) and sham operations were performed in wild-type (WT) and KChIP2–/– mice. Echocardiography was performed before and every 2 weeks after the operation. Ten weeks post-surgery, surface ECG was recorded and we paced the heart in vivo to induce arrhythmias. Afterwards, tissue from the left ventricle was used for immunoblotting. Time courses of HF development were comparable in TAC-operated WT and KChIP2–/– mice. Ventricular protein expression of KChIP2 was reduced by 70% after 10 weeks TAC in WT mice. The amplitudes of the J and T waves were enlarged in KChIP2–/– control mice. Ventricular effective refractory period, RR, QRS and QT intervals were longer in mice with HF compared to sham-operated mice of either genotype. Pacing-induced ventricular tachycardia (VT) was observed in 5/10 sham-operated WT mice compared with 2/10 HF WT mice with HF. Interestingly, and contrary to previously published data, sham-operated KChIP2–/– mice were resistant to pacing-induced VT resulting in only 1/10 inducible mice. KChIP2–/– with HF mice had similar low vulnerability to inducible VT (1/9). Our results suggest that although KChIP2 is downregulated in HF, it is not orchestrating the development of HF. Moreover, KChIP2 affects ventricular repolarization and lowers arrhythmia susceptibility. Hence, downregulation of KChIP2 expression in HF may be antiarrhythmic in mice via reduction of the fast transient outward K+ current.
Important role of mucosal serotonin in colonic propulsion and peristaltic reflexes: in vitro analyses in mice lacking tryptophan hydroxylase 1
Although there is general agreement that mucosal 5-hydroxytryptamine (5-HT) can initiate peristaltic reflexes in the colon, recent studies have differed as to whether or not the role of mucosal 5-HT is critical. We therefore tested the hypothesis that the secretion of 5-HT from mucosal enterochromaffin (EC) cells is essential for the manifestation of murine colonic peristaltic reflexes. To do so, we analysed the mechanisms underlying faecal pellet propulsion in isolated colons of mice lacking tryptophan hydroxylase 1 (Tph1–/– mice), which is the rate-limiting enzyme in the biosynthesis of mucosal but not neuronal 5-HT. We used video analysis of faecal pellet propulsion, tension transducers to record colonic migrating motor complexes (CMMCs) and intracellular microelectrodes to record circular muscle activity occurring spontaneously or following intraluminal distension. When compared with control (Tph1+/+) mice, Tph1–/– animals exhibited: (1) an elongated colon; (2) larger faecal pellets; (3) orthograde propulsion followed by retropulsion (not observed in Tph1+/+ colon); (4) slower in vitro propulsion of larger faecal pellets (28% of Tph1+/+); (5) CMMCs that infrequently propagated in an oral to anal direction because of impaired descending inhibition; (6) reduced CMMCs and inhibitory responses to intraluminal balloon distension; (7) an absence of reflex activity in response to mucosal stimulation. In addition, (8) thin pellets that propagated along the control colon failed to do so in Tph1–/– colon; and (9) the 5-HT3 receptor antagonist ondansetron, which reduced CMMCs and blocked their propagation in Tph1+/+ mice, failed to alter CMMCs in Tph1–/– animals. Our observations suggest that mucosal 5-HT is essential for reflexes driven by mucosal stimulation and is also important for normal propagation of CMMCs and propulsion of pellets in the isolated colon.
Identification of unique release kinetics of serotonin from guinea-pig and human enterochromaffin cells
The major source of serotonin (5-HT) in the body is the enterochromaffin (EC) cells lining the intestinal mucosa of the gastrointestinal tract. Despite the fact that EC cells synthesise ~95% of total body 5-HT, and that this 5-HT has important paracrine and endocrine roles, no studies have investigated the mechanisms of 5-HT release from single primary EC cells. We have developed a rapid primary culture of guinea-pig and human EC cells, allowing analysis of single EC cell function using electrophysiology, electrochemistry, Ca2+ imaging, immunocytochemistry and 3D modelling. Ca2+ enters EC cells upon stimulation and triggers quantal 5-HT release via L-type Ca2+ channels. Real time amperometric techniques reveal that EC cells release 5-HT at rest and this release increases upon stimulation. Surprisingly for an endocrine cell storing 5-HT in large dense core vesicles (LDCVs), EC cells release 70 times less 5-HT per fusion event than catecholamine released from similarly sized LDCVs in endocrine chromaffin cells, and the vesicle release kinetics instead resembles that observed in mammalian synapses. Furthermore, we measured EC cell density along the gastrointestinal tract to create three-dimensional (3D) simulations of 5-HT diffusion using the minimal number of variables required to understand the physiological relevance of single cell 5-HT release in the whole-tissue milieu. These models indicate that local 5-HT levels are likely to be maintained around the activation threshold for mucosal 5-HT receptors and that this is dependent upon stimulation and location within the gastrointestinal tract. This is the first study demonstrating single cell 5-HT release in primary EC cells. The mode of 5-HT release may represent a unique mode of exocytosis amongst endocrine cells and is functionally relevant to gastrointestinal sensory and motor function.
Oxygen and mitochondrial inhibitors modulate both monomeric and heteromeric TASK-1 and TASK-3 channels in mouse carotid body type-1 cells
In rat arterial chemoreceptors, background potassium channels play an important role in maintaining resting membrane potential and promoting depolarization and excitation in response to hypoxia or acidosis. It has been suggested that these channels are a heterodimer of TASK-1 and TASK-3 based on their similarity to heterologously expressed TASK-1/3 fusion proteins. In this study, we sought to confirm the identity of these channels through germline ablation of Task-1 (Kcnk3) and Task-3 (Kcnk9) in mice. Background K-channels were abundant in carotid body type-1 cells from wild-type mice and comparable to those previously described in rat type-1 cells with a main conductance state of 33 pS. This channel was absent from both Task-1–/– and Task-3–/– cells. In its place we observed a larger (38 pS) K+-channel in Task-1–/– cells and a smaller (18 pS) K+-channel in Task-3–/– cells. None of these channels were observed in Task-1–/–/Task-3–/– double knock-out mice. We therefore conclude that the predominant background K-channel in wild-type mice is a TASK-1/TASK-3 heterodimer, whereas that in Task-1–/– mice is TASK-3 and, conversely, that in Task-3–/– mice is TASK-1. All three forms of TASK channel in type-1 cells were inhibited by hypoxia, cyanide and the uncoupler FCCP, but the greatest sensitivity was seen in TASK-1 and TASK-1/TASK-3 channels. In summary, the background K-channel in type-1 cells is predominantly a TASK-1/TASK-3 heterodimer. Although both TASK-1 and TASK-3 are able to couple to the oxygen and metabolism sensing pathways present in type-1 cells, channels containing TASK-1 appear to be more sensitive.