Hypoxia and metabolic inhibitors alter the intracellular ATP:ADP ratio and membrane potential in human coronary artery smooth muscle cells.

ATP-sensitive potassium (KATP) channels couple cellular metabolism to excitability, making them ideal candidate sensors for hypoxic vasodilation. However, it is still unknown whether cellular nucleotide levels are affected sufficiently to activate vascular KATP channels during hypoxia. To address this fundamental issue, we measured changes in the intracellular ATP:ADP ratio using the biosensors Perceval/PercevalHR, and membrane potential using the fluorescent probe DiBAC4(3) in human coronary artery smooth muscle cells (HCASMCs). ATP:ADP ratio was significantly reduced by exposure to hypoxia. Application of metabolic inhibitors for oxidative phosphorylation also reduced ATP:ADP ratio. Hyperpolarization caused by inhibiting oxidative phosphorylation was blocked by either 10 µM glibenclamide or 60 mM K+. Hyperpolarization caused by hypoxia was abolished by 60 mM K+ but not by individual K+ channel inhibitors. Taken together, these results suggest hypoxia causes hyperpolarization in part by modulating K+ channels in SMCs

Bioenergetic profile of human coronary artery smooth muscle cells and effect of metabolic intervention

Bioenergetics of artery smooth muscle cells is critical in cardiovascular health and disease. An acute rise in metabolic demand causes vasodilation in systemic circulation while a chronic shift in bioenergetic profile may lead to vascular diseases. A decrease in intracellular ATP level may trigger physiological responses while dedifferentiation of contractile smooth muscle cells to a proliferative and migratory phenotype is often observed during pathological processes. Although it is now possible to dissect multiple building blocks of bioenergetic components quantitatively, detailed cellular bioenergetics of artery smooth muscle cells is still largely unknown. Thus, we profiled cellular bioenergetics of human coronary artery smooth muscle cells and effects of metabolic intervention. Mitochondria and glycolysis stress tests utilizing Seahorse technology revealed that mitochondrial oxidative phosphorylation accounted for 54.5% of ATP production at rest with the remaining 45.5% due to glycolysis. Stress tests also showed that oxidative phosphorylation and glycolysis can increase to a maximum of 3.5 fold and 1.25 fold, respectively, indicating that the former has a high reserve capacity. Analysis of bioenergetic profile indicated that aging cells have lower resting oxidative phosphorylation and reduced reserve capacity. Intracellular ATP level of a single cell was estimated to be over 1.1 mM. Application of metabolic modulators caused significant changes in mitochondria membrane potential, intracellular ATP level and ATP:ADP ratio. The detailed breakdown of cellular bioenergetics showed that proliferating human coronary artery smooth muscle cells rely more or less equally on oxidative phosphorylation and glycolysis at rest. These cells have high respiratory reserve capacity and low glycolysis reserve capacity. Metabolic intervention influences both intracellular ATP concentration and ATP:ADP ratio, where subtler changes may be detected by the latter

An orthogonal C-H borylation - cross-coupling strategy for the preparation of tetrasubstituted "A(2)B(2)''-chrysene derivatives with tuneable photophysical properties

Cl-substituents serve as a functionalisable regiocontrol element for the orthogonal functionalisation of chrysene.</p

Human Alpha Defensin 5 Expression in the Human Kidney and Urinary Tract

The mechanisms that maintain sterility in the urinary tract are incompletely understood. Recent studies have implicated the importance of antimicrobial peptides (AMP) in protecting the urinary tract from infection. Here, we characterize the expression and relevance of the AMP human alpha-defensin 5 (HD5) in the human kidney and urinary tract in normal and infected subjects.Using RNA isolated from human kidney, ureter, and bladder tissue, we performed quantitative real-time PCR to show that DEFA5, the gene encoding HD5, is constitutively expressed throughout the urinary tract. With pyelonephritis, DEFA5 expression significantly increased in the kidney. Using immunoblot analysis, HD5 production also increased with pyelonephritis. Immunostaining localized HD5 to the urothelium of the bladder and ureter. In the kidney, HD5 was primarily produced in the distal nephron and collecting tubules. Using immunoblot and ELISA assays, HD5 was not routinely detected in non-infected human urine samples while mean urinary HD5 production increased with E.coli urinary tract infection.DEFA5 is expressed throughout the urinary tract in non-infected subjects. Specifically, HD5 is expressed throughout the urothelium of the lower urinary tract and in the collecting tubules of the kidney. With infection, HD5 expression increases in the kidney and levels become detectable in the urine. To our knowledge, our findings represent the first to quantitate HD5 expression and production in the human kidney. Moreover, this is the first report to detect the presence of HD5 in infected urine samples. Our results suggest that HD5 may have an important role in maintaining urinary tract sterility

Elevations of intracellular calcium reflect normal voltage-dependent behavior, and not constitutive activity, of voltage-dependent calcium channels in gastrointestinal and vascular smooth muscle

In smooth muscle, the gating of dihydropyridine-sensitive Ca2+ channels may either be stochastic and voltage dependent or coordinated among channels and constitutively active. Each form of gating has been proposed to be largely responsible for Ca2+ influx and determining the bulk average cytoplasmic Ca2+ concentration. Here, the contribution of voltage-dependent and constitutively active channel behavior to Ca2+ signaling has been studied in voltage-clamped single vascular and gastrointestinal smooth muscle cells using wide-field epifluorescence with near simultaneous total internal reflection fluorescence microscopy. Depolarization (−70 to +10 mV) activated a dihydropyridine-sensitive voltage-dependent Ca2+ current (ICa) and evoked a rise in [Ca2+] in each of the subplasma membrane space and bulk cytoplasm. In various regions of the bulk cytoplasm the [Ca2+] increase ([Ca2+]c) was approximately uniform, whereas that of the subplasma membrane space ([Ca2+]PM) had a wide range of amplitudes and time courses. The variations that occurred in the subplasma membrane space presumably reflected an uneven distribution of active Ca2+ channels (clusters) across the sarcolemma, and their activation appeared consistent with normal voltage-dependent behavior. Indeed, in the present study, dihydropyridine-sensitive Ca2+ channels were not normally constitutively active. The repetitive localized [Ca2+]PM rises (“persistent Ca2+ sparklets”) that characterize constitutively active channels were observed rarely (2 of 306 cells). Neither did dihydropyridine-sensitive constitutively active Ca2+ channels regulate the bulk average [Ca2+]c. A dihydropyridine blocker of Ca2+ channels, nimodipine, which blocked ICa and accompanying [Ca2+]c rise, reduced neither the resting bulk average [Ca2+]c (at −70 mV) nor the rise in [Ca2+]c, which accompanied an increased electrochemical driving force on the ion by hyperpolarization (−130 mV). Activation of protein kinase C with indolactam-V did not induce constitutive channel activity. Thus, although voltage-dependent Ca2+ channels appear clustered in certain regions of the plasma membrane, constitutive activity is unlikely to play a major role in [Ca2+]c regulation. The stochastic, voltage-dependent activity of the channel provides the major mechanism to generate rises in [Ca2+]

Steady-state modulation of voltage-gated K+ channels in rat arterial smooth muscle by cyclic AMP-dependent protein kinase and protein phosphatase 2B

Voltage-gated potassium channels (Kv) are important regulators of membrane potential in vascular smooth muscle cells, which is integral to controlling intracellular Ca2+ concentration and regulating vascular tone. Previous work indicates that Kv channels can be modulated by receptor-driven alterations of cyclic AMP-dependent protein kinase (PKA) activity. Here, we demonstrate that Kv channel activity is maintained by tonic activity of PKA. Whole-cell recording was used to assess the effect of manipulating PKA signalling on Kv and ATP-dependent K+ channels of rat mesenteric artery smooth muscle cells. Application of PKA inhibitors, KT5720 or H89, caused a significant inhibition of Kv currents. Tonic PKA-mediated activation of Kv appears maximal as application of isoprenaline (a β-adrenoceptor agonist) or dibutyryl-cAMP failed to enhance Kv currents. We also show that this modulation of Kv by PKA can be reversed by protein phosphatase 2B/calcineurin (PP2B). PKA-dependent inhibition of Kv by KT5720 can be abrogated by pre-treatment with the PP2B inhibitor cyclosporin A, or inclusion of a PP2B auto-inhibitory peptide in the pipette solution. Finally, we demonstrate that tonic PKA-mediated modulation of Kv requires intact caveolae. Pre-treatment of the cells with methyl-β-cyclodextrin to deplete cellular cholesterol, or adding caveolin-scaffolding domain peptide to the pipette solution to disrupt caveolae-dependent signalling each attenuated PKA-mediated modulation of the Kv current. These findings highlight a novel, caveolae-dependent, tonic modulatory role of PKA on Kv channels providing new insight into mechanisms and the potential for pharmacological manipulation of vascular tone

Challenges Enrolling Children Into Traumatic Brain Injury Trials: An Observational Study

ObjectivesIn preparation for a clinical trial of therapeutic agents for children with moderate‐to‐severe blunt traumatic brain injuries (TBIs) in emergency departments (EDs), we conducted this feasibility study to (1) determine the number and clinical characteristics of eligible children, (2) determine the timing of patient and guardian arrival to the ED, and (3) describe the heterogeneity of TBIs on computed tomography (CT) scans.MethodsWe conducted a prospective observational study at 16 EDs of children ≤ 18 years of age presenting with blunt head trauma and Glasgow Coma Scale scores of 3–12. We documented the number of potentially eligible patients, timing of patient and guardian arrival, patient demographics and clinical characteristics, severity of injuries, and cranial CT findings.ResultsWe enrolled 295 eligible children at the 16 sites over 6 consecutive months. Cardiac arrest and nonsurvivable injuries were the most common characteristics that would exclude patients from a future trial. Most children arrived within 2 hours of injury, but most guardians did not arrive until 2–3 hours after the injury. There was a substantial range in types of TBIs, with subdural hemorrhages being the most common.ConclusionEnrolling children with moderate‐to‐severe TBI into time‐sensitive clinical trials will require large numbers of sites and meticulous preparation and coordination and will prove challenging to obtain informed consent given the timing of patient and guardian arrival. The Federal Exception from Informed Consent for Emergency Research will be an important consideration for enrolling these children.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135996/1/acem13085_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135996/2/acem13085.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135996/3/acem13085-sup-0001-DataSupplementS1.pd

Principal role of adenylyl cyclase 6 in K+ channel regulation and vasodilator signalling in vascular smooth muscle cells

AIMS: Membrane potential is a key determinant of vascular tone and many vasodilators act through the modulation of ion channel currents [e.g. the ATP-sensitive potassium channel (K(ATP))] involved in setting the membrane potential. Adenylyl cyclase (AC) isoenzymes are potentially important intermediaries in such vasodilator signalling pathways. Vascular smooth muscle cells (VSMCs) express multiple AC isoenzymes, but the reason for such redundancy is unknown. We investigated which of these isoenzymes are involved in vasodilator signalling and regulation of vascular ion channels important in modulating membrane potential. METHODS AND RESULTS: AC isoenzymes were selectively depleted (by >75%) by transfection of cultured VSMCs with selective short interfering RNA sequences. AC6 was the predominant isoenzyme involved in vasodilator-mediated cAMP accumulation in VSMCs, accounting for ∼60% of the total response to β-adrenoceptor (β-AR) stimulation. AC3 played a minor role in β-AR signalling, whereas AC5 made no significant contribution. AC6 was also the principal isoenzyme involved in β-AR-mediated protein kinase A (PKA) signalling (determined using the fluorescent biosensor for PKA activity, AKAR3) and the substantial β-AR/PKA-dependent enhancement of K(ATP) current. K(ATP) current was shown to play a vital role in setting the resting membrane potential and in mediating the hyperpolarization observed upon β-AR stimulation. CONCLUSION: AC6, but not the closely related AC5, plays a principal role in vasodilator signalling and regulation of the membrane potential in VSMCs. These findings identify AC6 as a vital component in the vasodilatory apparatus central to the control of blood pressure

Creative destruction in science

Drawing on the concept of a gale of creative destruction in a capitalistic economy, we argue that initiatives to assess the robustness of findings in the organizational literature should aim to simultaneously test competing ideas operating in the same theoretical space. In other words, replication efforts should seek not just to support or question the original findings, but also to replace them with revised, stronger theories with greater explanatory power. Achieving this will typically require adding new measures, conditions, and subject populations to research designs, in order to carry out conceptual tests of multiple theories in addition to directly replicating the original findings. To illustrate the value of the creative destruction approach for theory pruning in organizational scholarship, we describe recent replication initiatives re-examining culture and work morality, working parents\u2019 reasoning about day care options, and gender discrimination in hiring decisions. Significance statement It is becoming increasingly clear that many, if not most, published research findings across scientific fields are not readily replicable when the same method is repeated. Although extremely valuable, failed replications risk leaving a theoretical void\u2014 reducing confidence the original theoretical prediction is true, but not replacing it with positive evidence in favor of an alternative theory. We introduce the creative destruction approach to replication, which combines theory pruning methods from the field of management with emerging best practices from the open science movement, with the aim of making replications as generative as possible. In effect, we advocate for a Replication 2.0 movement in which the goal shifts from checking on the reliability of past findings to actively engaging in competitive theory testing and theory building. Scientific transparency statement The materials, code, and data for this article are posted publicly on the Open Science Framework, with links provided in the article