Autocrine activation of P2Y1 receptors couples Ca (2+) influx to Ca (2+) release in human pancreatic beta cells.

AIMS/HYPOTHESIS: There is evidence that ATP acts as an autocrine signal in beta cells but the receptors and pathways involved are incompletely understood. Here we investigate the receptor subtype(s) and mechanism(s) mediating the effects of ATP on human beta cells. METHODS: We examined the effects of purinergic agonists and antagonists on membrane potential, membrane currents, intracellular Ca(2+) ([Ca(2+)]i) and insulin secretion in human beta cells. RESULTS: Extracellular application of ATP evoked small inward currents (3.4 ± 0.7 pA) accompanied by depolarisation of the membrane potential (by 14.4 ± 2.4 mV) and stimulation of electrical activity at 6 mmol/l glucose. ATP increased [Ca(2+)]i by stimulating Ca(2+) influx and evoking Ca(2+) release via InsP3-receptors in the endoplasmic reticulum (ER). ATP-evoked Ca(2+) release was sufficient to trigger exocytosis in cells voltage-clamped at -70 mV. All effects of ATP were mimicked by the P2Y(1/12/13) agonist ADP and the P2Y1 agonist MRS-2365, whereas the P2X(1/3) agonist α,ß-methyleneadenosine-5-triphosphate only had a small effect. The P2Y1 antagonists MRS-2279 and MRS-2500 hyperpolarised glucose-stimulated beta cells and lowered [Ca(2+)]i in the absence of exogenously added ATP and inhibited glucose-induced insulin secretion by 35%. In voltage-clamped cells subjected to action potential-like stimulation, MRS-2279 decreased [Ca(2+)]i and exocytosis without affecting Ca(2+) influx. CONCLUSIONS/INTERPRETATION: These data demonstrate that ATP acts as a positive autocrine signal in human beta cells by activating P2Y1 receptors, stimulating electrical activity and coupling Ca(2+) influx to Ca(2+) release from ER stores.

Choice of Oral Anticoagulant: Outcomes in Atrial Fibrillation Patients Post-Stroke Despite Direct Oral Anticoagulant Use.

Background: For patients with atrial fibrillation who have an ischemic stroke or transient ischemic attack (TIA) despite taking direct oral anticoagulants (DOACs), the optimal strategy for ongoing anticoagulation is unknown. Methods: Using provincial administrative databases in Alberta, Canada, we compared anticoagulant use before/after the breakthrough stroke/TIA and assessed recurrence of stroke/TIA or bleeding, with consideration of medication adherence. Adherence was defined as the proportion of days covered (PDC) being ≥ 80%. Results: Among 985 patients, the median age was 80 years (interquartile range 13), with a mean CHADS2 score of 1.7± 1 prior to the index event. Patients were followed for a median of 643 days (interquartile range 836). Following the index stroke/TIA event, 623 patients (63%) filled a prescription for the same DOAC regimen, 83 (8%) filled a prescription for a different dose, 155 (16%) switched DOAC agents, 51 (5%) switched to warfarin, and 73 (7%) filled no oral anticoagulant prescription. Patients who kept the same regimen more commonly had TIA index events (59%); patients who changed dose or drug more often had stroke index events (55%-78%). During follow-up, 135 (14%) had stroke/TIA recurrence, and 46 (5%) had bleeding; rates of each did not differ between prescribing patterns. Post-index event, the proportion of patients with a proportion of days covered ≥ 80% improved from 55% to 80%. Conclusions: Although most maintained the same DOAC regimen after stroke/TIA, rates of recurrent stroke/TIA and bleeding were similar across prescribing patterns. Stroke/TIA severity may have influenced prescribing practices. DOAC prescription adherence improved poststroke/TIA and signals an opportunity for optimization in patients with atrial fibrillation.

Contexte: Chez les patients atteints de fibrillation auriculaire qui subissent un accident vasculaire cérébral (AVC) ischémique ou un accident ischémique transitoire (AIT) malgré la prise d'anticoagulants oraux directe (AOD), la stratégie optimale pour la poursuite de l'anticoagulation est inconnue. Méthodologie: À partir des bases de données administratives provinciales en Alberta, au Canada, nous avons comparé l'utilisation d'anticoagulants avant/après l'AVC/AIT survenu pendant l'anticoagulothérapie et avons évalué la récurrence d'un AVC/AIT ou d'un saignement, en tenant compte de l'adhésion au traitement médicamenteux. L'adhésion a été définie comme une proportion de jours couverts (PJC) de 80 % ou plus. Résultats: Chez 985 patients, l'âge médian était de 80 ans (écart interquartile de 13) et le score CHADS2 moyen, de 1,7 ± 1 avant l'événement de référence. Les patients ont été suivis pendant une médiane de 643 jours (écart interquartile de 836). Après l'AVC/AIT de référence, 623 patients (63 %) ont fait exécuter une ordonnance du même schéma d'AOD, 83 (8 %) ont fait exécuter une ordonnance d'une dose différente, 155 (16 %) sont passés à d'autres AOD, 51 (5 %) sont passés à la warfarine et 73 (7 %) n'ont fait exécuter aucune ordonnance d'anticoagulant oral. Chez les patients qui ont continué à recevoir le même schéma, la plupart (59 %) avaient eu un AIT comme événement de référence; chez les patients qui ont changé de dose ou de médicament, la plupart (55 à 78 %) avaient eu un AVC comme événement de référence. Durant le suivi, 135 (14 %) ont connu un AVC/AIT récurrent et 46 (5 %) ont présenté un saignement; les taux de chaque manifestation ont été similaires pour les différents schémas de prescription. Après l'événement de référence, le pourcentage de patients ayant une PJC ≥ 80 % a augmenté, passant de 55 à 80 %. Conclusions: Malgré le maintien du même schéma d'AOD chez la plupart des patients après l'AVC/AIT, les taux d'AVC/AIT récurrent et de saignement ont été similaires avec tous les schémas de prescription. La gravité d'un AVC/AIT pourrait avoir influencé les pratiques de prescription. L'adhésion aux AOD prescrits s'est améliorée après un AVC/AIT et témoigne d'une possibilité d'optimisation chez les patients atteints de fibrillation auriculaire.

Chronoprints: Identifying Samples by Visualizing How They Change over Space and Time.

The modern tools of chemistry excel at identifying a sample, but the cost, size, complexity, and power consumption of these instruments often preclude their use in resource-limited settings. In this work, we demonstrate a simple and low-cost method for identifying a sample based on visualizing how the sample changes over space and time in response to a perturbation. Different types of perturbations could be used, and in this proof-of-concept we use a dynamic temperature gradient that rapidly cools different parts of the sample at different rates. We accomplish this by first loading several samples into long parallel channels on a "microfluidic thermometer chip." We then immerse one end of the chip in liquid nitrogen to create a dynamic temperature gradient along the channels, and we use an inexpensive USB microscope to record a video of how the samples respond to the changing temperature gradient. The video is then converted into several bitmap images (one per sample) that capture each sample's response to the perturbation in both space (the y-axis; the distance along the dynamic temperature gradient) and time (the x-axis); we call these images "chronological fingerprints" or "chronoprints" of each sample. If two samples' chronoprints are similar, this suggests that the samples are the same chemical substance or mixture, but if two samples' chronoprints are significantly different, this proves that the samples are chemically different. Since chronoprints are just bitmap images, they can be compared using a variety of techniques from computer science, and in this work we use three different image comparison algorithms to quantify chronoprint similarity. As a demonstration of the versatility of chronoprints, we use them in three different applications: distinguishing authentic olive oil from adulterated oil (an example of the over $10 billion global problem of food fraud), identifying adulterated or counterfeit medication (which represents around 10% of all medication in low- and middle-income countries), and distinguishing the occasionally confused pharmaceutical ingredients glycerol and diethylene glycol (whose accidental or intentional substitution has led to hundreds of deaths). The simplicity and versatility of chronoprints should make them valuable analytical tools in a variety of different fields.

Cause of death analysis and temporal trends in survival after liver transplantation for transthyretin familial amyloid polyneuropathy.

BACKGROUND: Hereditary transthyretin amyloidosis (ATTR) is a multisystemic disease involving mainly the peripheral nervous system and the heart. Liver transplantation (LT) is the reference treatment for ATTR neuropathy and preoperative detection of high risk patients is crucial. We aimed to document the causes of death of ATTR patients after LT, their temporal trends, and to evaluate whether the available preoperative tools that predict the risk of death after LT for hereditary ATTR amyloidosis matched with these trends. METHODS: A retrospective longitudinal cohort study was performed on 215 consecutive ATTR patients who underwent LT between January 1993 and January 2011. Each patient's death cause and timing were classified. RESULTS: Over a median follow up of 5.9 years, 84 patients died. The rate of death was higher in the first year following LT than thereafter (13.0 vs. 4.3 ± 1.8%/year; p = .004). Cardiac events ranked as the leading cause of death (C: 38%), followed by infections (I: 24%), graft complications (G: 17%), end stage amyloidosis, stroke and others (ASO: 7% each). Deaths due to graft complications and infections (GI) occurred earlier than those due to end stage amyloidosis and stroke. Death prediction was less accurate for GI-related mortality than for other causes, which blunted the accuracy of the early-term risk prediction scores. Conclusions In ATTR amyloidosis, cardiac events were the leading cause of death after liver transplantation. Close preoperative evaluation allowed for accurate mid-term prediction of mortality, but the high rate of graft complications and infections blunted the early-term risk prediction.

MicroRNA-135a regulates sodium-calcium exchanger gene expression and cardiac electrical activity.

BACKGROUND: Complete atrioventricular block (CAVB) causes arrhythmogenic remodeling and increases the risk of torsades de pointes arrhythmias. MicroRNAs (miRNAs) are key regulators of gene expression that contribute to cardiac remodeling. OBJECTIVE: The purpose of this study was to assess miRNA changes after CAVB and identify novel candidates potentially involved in arrhythmogenic cardiac remodeling. METHODS: CAVB was induced in mice via His-bundle ablation. Expression of miRNAs was evaluated by pan-miRNA microarray with quantitative polymerase chain reaction (qPCR) confirmation, on samples obtained 24 hours and 4 weeks post-CAVB. MiRNA target prediction algorithms were used to identify potential target genes. Targets confirmed by luciferase assays in HEK293 cells were followed up with overexpression studies in neonatal rat ventricular myocytes to evaluate regulation using real time- quantitative polymerase chain reaction (RT-qPCR), western blots, cell shortening measurements, and fura-2 Ca2+ fluorescence imaging. RESULTS: Of >400 miRNAs assayed, only miRNA-135a (miR-135a) was altered at 24 hours, down-regulated 78% (P <.001). Algorithms predicted miR-135a regulation of the sodium-calcium exchanger type 1 (NCX1). miR-135a transfection suppressed NCX1 3'UTR reporter activity by 42% (P <.001), mRNA expression by 34% (P <.001), and protein levels by 45% (P <.001) vs noncoding miRNA control. miR-135a overexpression reduced spontaneous beating frequency of neonatal rat ventricular myocytes by 63% (P <.001) while slowing decay (by 56%, P <.05) of caffeine-induced Ca2+ transients. miR-135a also suppressed the Ca2+ loading effects of ouabain and ouabain-induced spontaneous Ca2+ release events. CONCLUSION: NCX1 is negatively regulated by miR-135a, a microRNA that is down-regulated in the heart after CAVB in mice. By controlling NCX1 expression, miR-135a modulates cardiomyocyte automaticity, Ca2+ extrusion, and arrhythmogenic Ca2+ loading/spontaneous Ca2+ release events. Therefore, miR-135a may contribute to proarrhythmic remodeling after CAVB.

A Glycine-Insulin Autocrine Feedback Loop Enhances Insulin Secretion From Human ß-Cells and Is Impaired in Type 2 Diabetes.

The secretion of insulin from pancreatic islet ß-cells is critical for glucose homeostasis. Disrupted insulin secretion underlies almost all forms of diabetes, including the most common form, type 2 diabetes (T2D). The control of insulin secretion is complex and affected by circulating nutrients, neuronal inputs, and local signaling. In the current study, we examined the contribution of glycine, an amino acid and neurotransmitter that activates ligand-gated Cl(-) currents, to insulin secretion from islets of human donors with and without T2D. We find that human islet ß-cells express glycine receptors (GlyR), notably the GlyRα1 subunit, and the glycine transporter (GlyT) isoforms GlyT1 and GlyT2. ß-Cells exhibit significant glycine-induced Cl(-) currents that promote membrane depolarization, Ca(2+) entry, and insulin secretion from ß-cells from donors without T2D. However, GlyRα1 expression and glycine-induced currents are reduced in ß-cells from donors with T2D. Glycine is actively cleared by the GlyT expressed within ß-cells, which store and release glycine that acts in an autocrine manner. Finally, a significant positive relationship exists between insulin and GlyR, because insulin enhances the glycine-activated current in a phosphoinositide 3-kinase-dependent manner, a positive feedback loop that we find is completely lost in ß-cells from donors with T2D.