scholarly journals Simulation of P2X-mediated calcium signaling in microglia

2018 ◽  
Author(s):  
Ben Chun ◽  
Bradley D. Stewart ◽  
Darin Vaughan ◽  
Adam D. Bachstetter ◽  
Peter M. Kekenes-Huskey
Keyword(s):  
Computational Model ◽  
Purinergic Receptor ◽  
Receptor Activation ◽  
Quantitative Model ◽  
Published Data ◽  
Transcriptional Responses ◽  
Extracellular Adenosine ◽  
Wide Range ◽  
Intracellular Ca

AbstractMicroglia function is orchestrated through highly-coupled signaling pathways that depend on calcium (Ca2+). In response to extracellular adenosine triphosphate (ATP), transient increases in intracellular Ca2+ driven through the activation of purinergic receptors, P2X and P2Y, are sufficient to promote cytokine synthesis and potentially their release. While steps comprising the pathways bridging purinergic receptor activation with transcriptional responses have been probed in great detail, a quantitative model for how these steps collectively control cytokine production has not been established. Here we developed a minimal computational model that quantitatively links extracellular stimulation of two prominent ionotropic puriner-gic receptors, P2X4 and P2X7, with the graded production of a gene product, namely the tumor necrosis factor α (TNFα) cytokine. In addition to Ca2+ handling mechanisms common to eukaryotic cells, our model includes microglia-specific processes including ATP-dependent P2X4 and P2X7 activation, activation of NFAT transcription factors, and TNFα production. Parameters for this model were optimized to reproduce published data for these processes, where available. With this model, we determined the propensity for TNFα production in microglia, subject to a wide range of ATP exposure amplitudes, frequencies and durations that the cells could encounter in vivo. Furthermore, we have investigated the extent to which modulation of the signal transduction pathways influence TNFα production. Our key findings are that TNFα production via P2X4 is maximized at low ATP when subject to high frequency ATP stimulation, whereas P2X7 contributes most significantly at millimolar ATPranges. Given that Ca2+ homeostasis in microglia is profoundly important to its function, this computational model provides a quantitative framework to explore hypotheses pertaining to microglial physiology.

scholarly journals TRPM4 Channels Couple Purinergic Receptor Mechanoactivation and Myogenic Tone Development in Cerebral Parenchymal Arterioles

2014 ◽  
Vol 34 (10) ◽  
pp. 1706-1714 ◽  
Author(s):  
Yao Li ◽  
Rachael L Baylie ◽  
Matthew J Tavares ◽  
Joseph E Brayden
Keyword(s):  
Transient Receptor Potential ◽  
Purinergic Receptor ◽  
Critical Role ◽  
Receptor Potential ◽  
Transient Receptor Potential Channel ◽  
Receptor Activation ◽  
Myogenic Tone ◽  
Pial Arteries ◽  
Myogenic Regulation

Cerebral parenchymal arterioles (PAs) have a critical role in assuring appropriate blood flow and perfusion pressure within the brain. They are unique in contrast to upstream pial arteries, as defined by their critical roles in neurovascular coupling, distinct sensitivities to chemical stimulants, and enhanced myogenic tone development. The objective of the present study was to reveal some of the unique mechanisms of myogenic tone regulation in the cerebral microcirculation. Here, we report that in vivo suppression of TRPM4 (transient receptor potential) channel expression, or inhibition of TRPM4 channels with 9-phenanthrol substantially reduced myogenic tone of isolated PAs, supporting a key role of TRPM4 channels in PA myogenic tone development. Further, downregulation of TRPM4 channels inhibited vasoconstriction induced by the specific P2Y4 and P2Y6 receptor ligands (UTP γS and UDP) by 37% and 42%, respectively. In addition, 9-phenanthrol substantially attenuated purinergic ligand-induced membrane depolarization and constriction of PAs, and inhibited ligand-evoked TRPM4 channel activation in isolated PA myocytes. In concert with our previous work showing the essential contributions of P2Y4 and P2Y6 receptors to myogenic regulation of PAs, the current results point to TRPM4 channels as an important link between mechanosensitive P2Y receptor activation and myogenic constriction of cerebral PAs.

Relationship between flow rate and NO production in postnatal mesenteric arteries

2001 ◽  
Vol 280 (1) ◽  
pp. G43-G50 ◽  
Author(s):  
Kristina M. Reber ◽  
Gennifer M. Mager ◽  
Charles E. Miller ◽  
Philip T. Nowicki
Keyword(s):  
Flow Rate ◽  
Kinase Inhibitor ◽  
Age Groups ◽  
Mesenteric Arteries ◽  
No Production ◽  
Herbimycin A ◽  
Wide Range ◽  
Intracellular Ca ◽  
Controlled Flow

We studied mesenteric arterial arcades from 3- and 35-day-old swine to determine the relationship between perfusate flow rate and release of nitric oxide (NO) into mesenteric effluent. Mesenteric arterial arcades were perfused under controlled-flow conditions with a peristaltic pump using warm oxygenated Krebs buffer. Basal rates of NO production were 43.6 ± 4.2 vs. 12.1 ± 2.5 nmol/min in 3- vs. 35-day-old mesentery during perfusion at in vivo flow rates (9 vs. 20 ml/min, respectively). Rate of NO production was directly related to flow rate over a wide range of flows (5–40 ml/min) in 3- but not 35-day-old mesentery. Both age groups demonstrated a brisk, albeit brief, increase in NO production in response to infusion of NO-dependent vasodilator substance P (10−8 M/min). Tyrosine kinase inhibitor herbimycin A andl-arginine analog l-NMMA significantly attenuated flow-induced increase in NO production, and phosphatase inhibitor phenylarsine oxide increased magnitude of flow-induced increase in NO production in 3-day-olds. Removal of extracellular Ca2+ and depletion of intracellular Ca2+ stores (Ca2+-free Krebs with EGTA plus thapsigargin) had no effect on NO production in either group. Thus, basal rate of NO production is greater in mesenteric arterial arcades from 3- than from 35-day old swine, a direct relationship between flow rate and NO production rate is present in mesentery from 3- but not 35-day-olds, and phosphorylation events are necessary for this interaction to occur.

scholarly journals Integrated experimental-computational analysis of a liver-islet microphysiological system for human-centric diabetes research

2021 ◽  
Author(s):  
Belén Casas ◽  
Liisa Vilén ◽  
Sophie Bauer ◽  
Kajsa Kanebratt ◽  
Charlotte Wennberg Huldt ◽  
...  
Keyword(s):  
Glucose Metabolism ◽  
Computational Model ◽  
In Silico ◽  
Experimental Results ◽  
Published Data ◽  
Diabetes Research ◽  
Glucose Response ◽  
High Blood Glucose

Microphysiological systems (MPS) are powerful tools for emulating human physiology and replicating disease progression in vitro. MPS could be better predictors of human outcome than current animal models, but mechanistic interpretation and in vivo extrapolation of the experimental results remain significant challenges. Here, we address these challenges using an integrated experimental-computational approach. This approach allows for in silico representation and predictions of glucose metabolism in a previously reported MPS with two organ compartments (liver and pancreas) connected in a closed loop with circulating medium. We developed a computational model describing glucose metabolism over 15 days of culture in the MPS. The model was calibrated on an experiment-specific basis using data from seven experiments, where single-liver or liver-islet cultures were exposed to both normal and hyperglycemic conditions resembling high blood glucose levels in diabetes. The calibrated models reproduced the fast (i.e. hourly) variations in glucose and insulin observed in the MPS experiments, as well as the long-term (i.e. over weeks) decline in both glucose tolerance and insulin secretion. We also investigated the behavior of the system under hypoglycemia by simulating this condition in silico, and the model could correctly predict the glucose and insulin responses measured in new MPS experiments. Last, we used the computational model to translate the experimental results to humans, showing good agreement with published data of the glucose response to a meal in healthy subjects. The integrated experimental-computational framework opens new avenues for future investigations toward disease mechanisms and the development of new therapies for metabolic disorders.

scholarly journals Temperature effects on morphological integrity and Ca2+ signaling in freshly isolated murine feed artery endothelial cell tubes

2011 ◽  
Vol 301 (3) ◽  
pp. H773-H783 ◽  
Author(s):  
Matthew J. Socha ◽  
Chady H. Hakim ◽  
William F. Jackson ◽  
Steven S. Segal
Keyword(s):  
Endothelial Cell ◽  
Enzymatic Digestion ◽  
Receptor Activation ◽  
Abdominal Muscle ◽  
Fura 2 ◽  
Intracellular Ca ◽  
Feed Arteries ◽  
In Vitro Stability

To study Ca2+ signaling in the endothelium of murine feed arteries, we determined the in vitro stability of endothelial cell (EC) tubes freshly isolated from abdominal muscle feed arteries of male and female C57BL/6 mice (5–9 mo, 25–35 g). We tested the hypothesis that intracellular Ca2+ concentration ([Ca2+]i) responses to muscarinic receptor activation would increase with temperature. Intact EC tubes (length: 1–2 mm, width: 65–80 μm) were isolated using gentle enzymatic digestion with trituration to remove smooth muscle cells. A freshly isolated EC tube was secured in a chamber and superfused at 24 (room temperature), 32, or 37°C. Using fura-2 dye, [Ca2+]i was monitored (ratio of fluorescence at 340- to 380-nm wavelength) at rest and in response to bolus doses of ACh (20 nmol to 200 μmol). The morphological integrity of EC tubes was preserved at 24 and 32°C. Based on the Ca2+ Kd values we determined for fura-2 (174 nM at 24°C and 146 nM at 32°C), resting [Ca2+]i remained stable for 180 min at both 24 and 32°C (27 ± 4 and 34 ± 2 nM, respectively), with peak responses to ACh (20 μmol) increasing from ∼220 nM at 24°C to ∼500 nM at 32°C ( P < 0.05). There was no difference in responses to ACh between EC tubes from male versus female mice. When EC tubes were maintained at 37°C (typical in vivo temperature), resting [Ca2+]i increased by ∼30% within 15 min, and gaps formed between individual ECs as they retracted and extruded dye, precluding further study. We conclude that EC tubes enable Ca2+ signaling to be evaluated in the freshly isolated endothelium of murine feed arteries. While Ca2+ responses are enhanced by approximately twofold at 32 versus 24°C, the instability of EC tubes at 37°C precludes their study at typical body temperature.

Mechanical strain-induced Ca2+waves are propagated via ATP release and purinergic receptor activation

2000 ◽  
Vol 279 (2) ◽  
pp. C295-C307 ◽  
Author(s):  
H. Sauer ◽  
J. Hescheler ◽  
M. Wartenberg
Keyword(s):  
Gap Junctions ◽  
Purinergic Receptor ◽  
Mechanical Strain ◽  
Atp Release ◽  
Anion Channel ◽  
Receptor Activation ◽  
Disulfonic Acid ◽  
Channel Blockers ◽  
Junctional Coupling ◽  
Intracellular Ca

Mechanical strain applied to prostate cancer cells induced an intracellular Ca2+ (Cai 2+) wave spreading with a velocity of 15 μm/s. Cai 2+ waves were not dependent on extracellular Ca2+ and membrane potential because propagation was unaffected in high-K+ and Ca2+-free solution. Waves did not depend on the cytoskeleton or gap junctions because cytochalasin B and nocodazole, which disrupt microfilaments and microtubules, respectively, and 1-heptanol, which uncouples gap junctions, were without effects. Fluorescence recovery after photobleaching experiments revealed an absence of gap junctional coupling. Cai 2+ waves were inhibited by the purinergic receptor antagonists basilen blue and suramin; by pretreatment with ATP, UTP, ADP, UDP, 2-methylthio-ATP, and benzoylbenzoyl-ATP; after depletion of ATP by 2-deoxyglucose; and after ATP scavenging by apyrase. Waves were abolished by the anion channel inhibitors 5-nitro-2-(3-phenylpropylamino)benzoic acid, tamoxifen, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, niflumic acid, and gadolinium. ATP release following strain was significantly inhibited by anion channel blockers. Hence, ATP is secreted via mechanosensitive anion channels and activates purinergic receptors on the same cell or neighboring cells in an autocrine and paracrine manner, thus leading to Cai 2+ wave propagation.

scholarly journals Network analysis reveals a distinct axis of macrophage activation in response to conflicting inflammatory cues

2019 ◽  
Author(s):  
Xiaji Liu ◽  
Jingyuan Zhang ◽  
Angela C. Zeigler ◽  
Anders R. Nelson ◽  
Merry L. Lindsey ◽  
...  
Keyword(s):  
Computational Model ◽  
Macrophage Activation ◽  
Expression Profiles ◽  
Interleukin 4 ◽  
Cytokine Signaling ◽  
Arginase 1 ◽  
M2 Polarization ◽  
Wide Range ◽  
Polarization Axis

AbstractMacrophages are subject to a wide range of cytokine and pathogen signals in vivo, which contribute to differential activation and modulation of inflammation. Understanding the response to multiple, often conflicting, cues that macrophages experience requires a network perspective. Here, we integrate data from literature curation and mRNA expression profiles to develop a large-scale computational model of the macrophage signaling network. In response to stimulation across all pairs of 9 cytokine inputs, the model predicted activation along the classic M1-M2 polarization axis but also a second axis of macrophage activation that distinguishes unstimulated macrophages from a mixed phenotype induced by conflicting cues. Along this second axis, combinations of conflicting stimuli, interleukin 4 (IL4) with lipopolysaccharide (LPS), interferon-γ (IFNγ), IFNβ, or tumor necrosis factor-α (TNFα), produced mutual inhibition of several signaling pathways, e.g. nuclear factor κB (NFκB) and signal transducer and activator of transcription 6 (STAT6), but also mutual activation of the phosphoinositide 3-kinases (PI3K) signaling module. In response to combined IFNγ and IL4, the model predicted genes whose expression was mutually inhibited, e.g. inducible nitric oxide synthase (iNOS) and arginase 1 (Arg1), or mutually enhanced, e.g. IL4 receptor-α (IL4Rα) and suppressor of cytokine signaling 1 (SOCS1), which was validated by independent experimental data. Knockdown simulations further predicted network mechanisms underlying functional crosstalk, such as mutual STAT3/STAT6-mediated enhancement of IL4Rα expression. In summary, the computational model predicts that network crosstalk mediates a broadened spectrum of macrophage activation in response to mixed pro- and anti-inflammatory cytokine cues, making it useful for modeling in vivo scenarios.Summary sentenceNetwork modeling of macrophage activation predicts responses to combinations of cytokines along both the M1-M2 polarization axis and a second axis associated with a mixed macrophage activation phenotype.

In vivo foregut digestion of starch-based concentrate feeds in horses using the mobile bag technique

2006 ◽  
Vol 35 ◽  
pp. 171-174
Author(s):  
M.J.S. Moore-Colyer ◽  
T. Clercq ◽  
V. Julliand ◽  
C. Drogoul
Keyword(s):  
Crude Protein ◽  
Metabolic Disorders ◽  
Published Data ◽  
Wide Range ◽  
Pelleted Feed ◽  
Ad Libitum ◽  
Horse Owners

Metabolic disorders from poor foregut digestion of starch-based concentrate feeds are one of the most serious challenges that face today’s horse owners. Although there are many different cereal and legume-based concentrates used to formulate compound horse feeds in Europe relatively little is known about their in vivo foregut digestibility. While Meyer et al (1993) has examined the effects of processing on digestibility, there is no published data comparing the in vivo foregut digestibility of a wide range of concentrates. Therefore this study sought to determine the digestibility of 20 different starch rich feeds in the foregut of horses using the mobile bag technique.Four mature geldings (BW ranging from 395 to 478 kg) fitted with caecal and colonic cannulae, were maintained in individual stalls, where water was available ad libitum and fed a diet of pellets and straw. The pelleted feed, fed at 0.8% BW/d, contained 14% fibre, 13% crude protein, 3.2% fat and 8.7% ash. This feed was equally divided and offered at 8am and 5pm.

scholarly journals P2Y1R-Initiated, IP3R-Dependent Stimulation of Astrocyte Mitochondrial Metabolism Reduces and Partially Reverses Ischemic Neuronal Damage in Mouse

2013 ◽  
Vol 33 (4) ◽  
pp. 600-611 ◽  
Author(s):  
Wei Zheng ◽  
Lora Talley Watts ◽  
Deborah M Holstein ◽  
Jimmy Wewer ◽  
James D Lechleiter
Keyword(s):  
Fluorescent Protein ◽  
Neuronal Damage ◽  
Purinergic Receptor ◽  
Yellow Fluorescent Protein ◽  
Neuronal Morphology ◽  
Mitochondrial Metabolism ◽  
Astrocyte Swelling ◽  
Intracellular Ca

Glia-based neuroprotection strategies are emerging as promising new avenues to treat brain damage. We previously reported that activation of the glial-specific purinergic receptor, P2Y1R, reduces both astrocyte swelling and brain infarcts in a photothrombotic mouse model of stroke. These restorative effects were dependent on astrocyte mitochondrial metabolism. Here, we extend these findings and report that P2Y1R stimulation with the purinergic ligand 2-methylthioladenosine 5′ diphosphate (2MeSADP) reduces and partially reverses neuronal damage induced by photothrombosis. In vivo neuronal morphology was confocally imaged in transgenic mice expressing yellow fluorescent protein under the control of the Thy1 promoter. Astrocyte mitochondrial membrane potentials, monitored with the potential sensitive dye tetra-methyl rhodamine methyl ester, were depolarized after photothrombosis and subsequently repolarized when P2Y1Rs were stimulated. Mice deficient in the astrocyte-specific type 2 inositol 1,4,5 trisphosphate (IP3) receptor exhibited aggravated ischemic dendritic damage after photothrombosis. Treatment of these mice with 2MeSADP did not invoke an intracellular Ca2+ response, did not repolarize astrocyte mitochondria, and did not reduce or partially reverse neuronal lesions induced by photothrombotic stroke. These results demonstrate that IP3-Ca2+ signaling in astrocytes is not only critical for P2Y1R-enhanced protection, but suggest that IP3-Ca2+ signaling is also a key component of endogenous neuroprotection.

scholarly journals Venular endothelium-derived NO can affect paired arteriole: a computational model

2006 ◽  
Vol 290 (2) ◽  
pp. H716-H723 ◽  
Author(s):  
Mahendra Kavdia ◽  
Aleksander S. Popel
Keyword(s):  
Smooth Muscle ◽  
Computational Model ◽  
Reaction Rate ◽  
Soluble Guanylate Cyclase ◽  
Experimental Studies ◽  
Vascular Wall ◽  
No Production ◽  
Wide Range ◽  
Venular Endothelium

Venular endothelial cells can release nitric oxide (NO) in response to intraluminal flow both in isolated venules and in vivo. Experimental studies suggest that venular endothelium-released NO causes dilation of the adjacent paired arteriole. In the vascular wall, NO stimulates its target hemoprotein, soluble guanylate cyclase (sGC), which relaxes smooth muscle cells. In this study, a computational model of NO transport for an arteriole and venule pair was developed to determine the importance of the venular endothelium-released NO and its transport to the adjacent arteriole in the tissue. The model predicts that the tissue NO levels are affected within a wide range of parameters, including NO-red blood cell reaction rate and NO production rate in the arteriole and venule. The results predict that changes in the venular NO production affected not only venular endothelial and smooth muscle NO concentration but also endothelial and smooth muscle NO concentration in the adjacent arteriole. This suggests that the anatomy of microvascular tissue can permit the transport of NO from arteriolar to venular side, and vice versa, and may provide a mechanism for dilation of proximal arterioles by venules. These results will have significant implications for our understanding of tissue NO levels in both physiological and pathophysiological conditions.

scholarly journals Mixed-Species Genomic Microarray Analysis of Fecal Samples Reveals Differential Transcriptional Responses of Bifidobacteria in Breast- and Formula-Fed Infants

2009 ◽  
Vol 75 (9) ◽  
pp. 2668-2676 ◽  
Author(s):  
Eline S. Klaassens ◽  
Rolf J. Boesten ◽  
Monique Haarman ◽  
Jan Knol ◽  
Frank H. Schuren ◽  
...  
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Rrna Genes ◽  
Transcriptional Responses ◽  
Fecal Samples ◽  
Young Infants ◽  
Exact Function ◽  
Wide Range ◽  
Protein Encoding ◽  
Encoding Genes

ABSTRACT Although their exact function remains enigmatic, bifidobacteria are among the first colonizers of the newborn infant gut and further develop into abundant communities, notably in response to diet. Therefore, the transcriptional responses of bifidobacteria in rapidly processed fecal samples from young infants that were fed either breast milk or a formula containing a mixture of galacto- and fructo-oligosaccharides were studied. The presence and diversity of the bifidobacterial fecal communities were determined using PCR-denaturing gradient gel electrophoresis and quantitative real-time PCR for specific species. Changes in the total number of bifidobacteria as well as in species diversity were observed, indicating the metabolic activities of the bifidobacteria within the infant gut. In addition, total RNAs isolated from infant feces were labeled and hybridized to a bifidobacterium-specific microarray comprising approximately 6,000 clones of the major bifidobacterial species of the human gut. Approximately 270 clones that showed the most prominent hybridization with the samples were sequenced. Fewer than 10% of the hybridizing clones contained rRNA genes, whereas the vast majority of the inserts showed matches with protein-encoding genes predicted to originate from bifidobacteria. Although a wide range of functional groups was covered by the obtained sequences, the largest fraction (14%) of the transcribed genes assigned to a functional category were predicted to be involved in carbohydrate metabolism, while some were also implicated in exopolysaccharide production or folate production. A total of three of the above-described protein-encoding genes were selected for quantitative PCR and sequence analyses, which confirmed the expression of the corresponding genes and the expected nucleotide sequences. In conclusion, the results of this study show the feasibility of obtaining insight into the transcriptional responses of intestinal bifidobacteria by analyzing fecal RNA and highlight the in vivo expression of bifidobacterial genes implicated in host-related functions.

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