Alteration of microtubule polymerization modulates arteriolar vasomotor tone

1999 ◽  
Vol 277 (1) ◽  
pp. H100-H106 ◽  
Author(s):  
Steven H. Platts ◽  
Jeff C. Falcone ◽  
William T. Holton ◽  
Michael A. Hill ◽  
Gerald A. Meininger
Keyword(s):  
Time Course ◽  
Contractile Function ◽  
Vasomotor Tone ◽  
Microtubule Depolymerization ◽  
Similar Time ◽  
Cellular Processes ◽  
Endothelial Denudation ◽  
Wide Range ◽  
Intracellular Ca ◽  
Cytoskeletal Elements

Microtubules are important cytoskeletal elements that have been shown to play a major role in many cellular processes because of their mechanical properties and/or their participation in various cell signaling pathways. We tested the hypothesis that depolymerization of microtubules would alter vascular smooth muscle (VSM) tone and hence contractile function. In our studies, isolated cremaster arterioles exhibited significant vasoconstriction that developed over a 20- to 40-min period when they were treated with microtubule depolymerizing drugs colchicine (10 μM), nocodazole (10 μM), or demecolcine (10 μM). Immunofluorescent labeling of microtubules in cultured rat VSM revealed that both colchicine and nocodazole caused microtubule depolymerization over a similar time course. The vasoconstriction was maintained over a wide range of intraluminal pressures (30–170 cmH2O). The increased tone was not affected by endothelial denudation, suggesting that it was due to an effect on VSM. Microtubule depolymerization with demecolcine or colchicine had no effect on VSM intracellular Ca2+ concentration ([Ca2+]i). These data indicate that microtubules significantly interact with processes leading to the expression of vasomotor tone. The mechanism responsible for the effect of microtubules on vasomotor tone appears to be independent of both the endothelium and an increase in VSM [Ca2+]i.

Intraluminal pressure stimulates MAPK phosphorylation in arterioles: temporal dissociation from myogenic contractile response

2003 ◽  
Vol 285 (4) ◽  
pp. H1764-H1773 ◽  
Author(s):  
Brian E. Spurrell ◽  
Timothy V. Murphy ◽  
Michael A. Hill
Keyword(s):  
Smooth Muscle ◽  
Time Course ◽  
Contractile Function ◽  
Intraluminal Pressure ◽  
Growth Responses ◽  
P38 Inhibitor ◽  
Pressure Step ◽  
Late Event ◽  
Intracellular Ca ◽  
Sb 203580

Members of the MAPK family of enzymes, p42/44 and p38, have been implicated in both the regulation of contractile function and growth responses in vascular smooth muscle. We determined whether such kinases are activated during the arteriolar myogenic response after increases in intraluminal pressure. Particular emphasis was placed on temporal aspects of activation to determine whether such phosphorylation events parallel the known time course for myogenic contraction. Experiments used single cannulated arterioles isolated from the cremaster muscle of rats with some vessels loaded with the fluorescent Ca2+-sensitive dye fura 2 (2 μM). The p42/44 inhibitor PD-98059 (50 μM) caused vasodilation but did not prevent pressure-induced myogenic constriction. The vasodilator response was accompanied by decreased smooth muscle intracellular Ca2+. Western blotting revealed a significant increase in the level of phosphorylation of p42/44 15 min after the application of a 30- to 100-mmHg pressure step. Phosphorylation of p42/44 was a late event that appeared to be temporally dissociated from contraction, which was complete within 1–5 min. EGF (80 nM) caused marked phosphorylation of p42/44 but only acted as a weak vasoconstrictor. The p38 inhibitor SB-203580 (10 μM) did not alter baseline diameter, nor did it prevent myogenic vasoconstriction. Consistent with these observations, SB-203580 did not cause a measurable change in intracellular Ca2+. The results demonstrate activation of the p42/44 class of MAPK resulting from increased transmural pressure. Such activation is, however, dissociated from the acute pressure-induced vasoconstrictor response in terms of time course and may represent the activation of compensatory, but parallel, pathways, including those related to growth and remodeling.

scholarly journals Analysis of neuronal Ca2+ handling properties by combining perforated patch clamp recordings and the added buffer approach

2020 ◽  
Author(s):  
Simon Hess ◽  
Christophe Pouzat ◽  
Lars Paeger ◽  
Andreas Pippow ◽  
Peter Kloppenburg
Keyword(s):  
Patch Clamp ◽  
Intracellular Signaling ◽  
Whole Cell ◽  
Perforated Patch ◽  
Cellular Processes ◽  
Wide Range ◽  
Intracellular Ca ◽  
Precise Quantification ◽  
Perforated Patch Clamp ◽  
Whole Cell Recordings

AbstractCa2+ functions as an important intracellular signal for a wide range of cellular processes. These processes are selectively activated by controlled spatiotemporal dynamics of the free cytosolic Ca2+. Intracellular Ca2+ dynamics are regulated by numerous cellular parameters. Here, we established a new way to determine neuronal Ca2+ handling properties by combining the ‘added buffer’ approach (Neher and Augustine, 1992) with perforated patch-clamp recordings (Horn and Marty, 1988). Since the added buffer approach typically employs the standard whole-cell configuration for concentration-controlled Ca2+ indicator loading, it only allows for the reliable estimation of the immobile fraction of intracellular Ca2+ buffers. Furthermore, crucial components of intracellular signaling pathways are being washed out during prolonged whole-cell recordings, leading to cellular deterioration. By combining the added buffer approach with perforated patch-clamp recordings, these issues are circumvented, allowing the precise quantification of the cellular Ca2+ handling properties, including immobile as well as mobile Ca2+ buffers.

scholarly journals Single cell characterization of a synthetic bacterial clock with a hybrid feedback loop containing dCas9-sgRNA

2020 ◽  
Author(s):  
John Henningsen ◽  
Matthaeus Schwarz-Schilling ◽  
Andreas Leibl ◽  
Joaquin A. M. Guttierez ◽  
Sandra Sagredo ◽  
...  
Keyword(s):  
Single Cell ◽  
Transcriptional Repression ◽  
Time Course ◽  
Cellular Growth ◽  
Gene Circuits ◽  
Rna Molecules ◽  
Guide Rna ◽  
Oscillatory Dynamics ◽  
Cellular Processes ◽  
Wide Range

AbstractGenetic networks that generate oscillations in gene expression activity are found in a wide range of organisms throughout all kingdoms of life. Oscillatory dynamics facilitates the temporal orchestration of metabolic and growth processes inside cells and organisms, as well as the synchronization of such processes with periodically occurring changes in the environment. Synthetic oscillator gene circuits such as the ‘repressilator’ can perform similar functions in bacteria. Until recently, such circuits were mainly based on a relatively small set of well-characterized transcriptional repressors and activators. A promising, sequence-programmable alternative for gene regulation is given by CRISPR interference (CRISPRi), which enables transcriptional repression of nearly arbitrary gene targets directed by short guide RNA molecules. In order to demonstrate the use of CRISPRi in the context of dynamic gene circuits, we here replaced one of the nodes of a repressilator circuit by the RNA-guided dCas9 protein. Using single cell experiments in microfluidic reactors we show that this system displays robust relaxation oscillations over multiple periods and over the time course of several days. Through statistical analysis of the single cell data, the potential for the circuit to act as a synthetic pacemaker for cellular processes is evaluated. The use of CRISPRi in the context of an oscillator circuit is found to have profound effects on its dynamics. Specifically, irreversible binding of dCas9-sgRNA appears to prolong the period of the oscillator. Further, we demonstrate that the oscillator affects cellular growth, leading to variations in growth rate with the oscillator’s frequency.

scholarly journals Role of Apamin-Sensitive KCa Channels for Reticulospinal Synaptic Transmission to Motoneuron and for the Afterhyperpolarization

2002 ◽  
Vol 88 (1) ◽  
pp. 289-299 ◽  
Author(s):  
Lorenzo Cangiano ◽  
Peter Wallén ◽  
Sten Grillner
Keyword(s):  
Synaptic Transmission ◽  
Nmda Receptors ◽  
Time Course ◽  
Frequency Regulation ◽  
Similar Time ◽  
Calcium Dependent ◽  
Before And After ◽  
Voltage Dependent ◽  
Intracellular Ca

Single motoneurons and pairs of a presynaptic reticulospinal axon and a postsynaptic motoneuron were recorded in the isolated lamprey spinal cord, to investigate the role of calcium-dependent K+ channels (KCa) during the afterhyperpolarization following the action potential (AHP), and glutamatergic synaptic transmission on the dendritic level. The AHP consists of a fast phase due to transient K+ channels (fAHP) and a slower phase lasting 100–200 ms (sAHP), being the main determinant of spike frequency regulation. We now present evidence that the sAHP has two components. The larger part, around 80%, is abolished by superfusion of Cd2+ (blocker of voltage-dependent Ca2+ channels), by intracellular injection of 1,2-bis-( 2-aminophenoxy)-ethane- N,N,N′,N′-tetraacetic acid (BAPTA; fast Ca2+ chelator), and by apamin (selective toxin for KCa channels of the SK subtype). While 80% of the sAHP is thus due to KCa channels, the remaining 20% is not mediated by Ca2+, either entering through voltage-dependent Ca2+ channels or released from intracellular Ca2+ stores. This Ca2+-independent sAHP component has a similar time course as the KCa portion and is not due to a Cl− conductance. It may be caused by Na+-activated K+ channels. Glutamatergic excitatory postsynaptic potentials (EPSPs) evoked by single reticulospinal axons give rise to a local Ca2+ increase in the postsynaptic dendrite, mediated in part by N-methyl-d-aspartate (NMDA) receptors. The Ca2+ levels remain elevated for several hundred milliseconds and could be expected to activate KCa channels. If so, this activation should cause a local conductance increase in the dendrite that would shunt EPSPs following the first EPSP in a spike train. We have tested this in reticulospinal/motoneuronal pairs, by stimulating the presynaptic axon with spike trains at different frequencies. We compared the first EPSP and the following EPSPs in the control and after blockade with apamin. No difference was observed in EPSP amplitude or shape before and after apamin, either in normal Ringer or in Mg2+-free Ringer removing the voltage-dependent block of NMDA receptors. In conclusion, the local Ca2+ entry during reticulospinal EPSPs does not cause an activation of KCa channels sufficient to affect the efficacy of synaptic transmission. Thus the integration of synaptic signals at the dendritic level in motoneurons appears simpler than would otherwise have been the case.

Abstract 144: Erythropoietin Induces Positive Inotropy And Lusitropy Mediated By Phosphatidylinositol 3-kinase And Novel PKC Isoforms In Murine Cardiomyocytes

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
David Hefer ◽  
David E Fishbaugher ◽  
Sarah M Tremble ◽  
Martin LeWinter ◽  
Bradley M Palmer ◽  
...  
Keyword(s):  
Time Course ◽  
Contractile Function ◽  
Ischemia Reperfusion ◽  
Dose Finding ◽  
Phosphatidylinositol 3 Kinase ◽  
Shortening Velocity ◽  
Buffer Solution ◽  
Pkc Isoforms ◽  
Intracellular Ca ◽  
Phosphatidylinositol 3

Introduction Clinical studies indicate a potentially beneficial effect of erythropoietin (EPO) in patients with anemia and heart failure. Moreover, EPO is known to be cardioprotective in ischemia-reperfusion models, an effect mediated through a cardiac EPO receptor. However it is not known if EPO has a direct effect on cardiac contractility. Methods Isolated cardiomyocytes were obtained from C57BL/6 mice and exposed to either 50 U/mL of EPO or to a buffer solution. Cells were paced at 8 Hz. Sarcomere shortening dynamics were measured using a Fourier video transformation method. Global intracellular Ca 2+ dynamics were determined after loading cells with fura 2-AM. EPO concentrations from 0.1 – 50 U/mL and exposure times from 0 – 90 min were used for dose finding and time course experiments. The phosphatidylinositol 3-kinase (PI3K) blocker Wortmannin, the non-isozyme specific PKC blocker Chelerythrine or the PKC α and β blocker Gö6976 were administered concurrently with EPO to determine the signaling pathways involved. Results In control cells peak sarcomere shortening was 2.3 ± 0.3 %, while in EPO treated cells it was 4.4 ± 0.5 % (P=0.007, N=40 cells in each group from 5 hearts). Baseline sarcomere length was 1.55 ± 0.04 μm in controls compared with 1.72 ± 0.04 μm in EPO cells (P=0.008). Both shortening velocity [2.2 ± 0.3 μm/s versus 4.2 ± 0.5 μm/s (P=0.01)] and relengthening velocity [1.3 ± 0.2 μm/s versus 3.5 ± 0.5 μm/s (P=0.002)] were higher with EPO treatment. Ca 2+ dynamics studied in 25 cells in each group showed no differences between EPO treated and control cells. Positive inotropic and lusitropic effects occurred at EPO concentrations above 1 U/mL and after an incubation period of at least 30 min. The EPO mediated increase in peak sarcomere shortening was abrogated by concurrent PI3K blockade or by non-isozyme specific PKC blockade. However, it was preserved if only the classical PKC isozymes αand β were blocked. Conclusions In addition to its antiapoptotic and cytoprotective effects, EPO has direct positive inotropic and lusitropic effects in single murine cardiomyocytes. These are mediated by PI3K and novel PKC isoforms and are independent of changes in intracellular Ca 2+ handling, suggesting a PKC δor PKC ϵ mediated increase in myofilament contractile function.

Postnatal growth rate and gonadal development in circadian tau mutant hamsters reared in constant dim red light

Reproduction ◽  
2000 ◽  
pp. 327-330 ◽  
Author(s):  
RJ Lucas ◽  
JA Stirland ◽  
YN Mohammad ◽  
AS Loudon
Keyword(s):  
Time Course ◽  
Red Light ◽  
Somatic Growth ◽  
Postnatal Growth ◽  
Gonadal Development ◽  
Testicular Development ◽  
Wild Type ◽  
Similar Time ◽  
Syrian Hamsters ◽  
Body Weights

The role of the circadian clock in the reproductive development of Syrian hamsters (Mesocricetus auratus was examined in wild type and circadian tau mutant hamsters reared from birth to 26 weeks of age under constant dim red light. Testis diameter and body weights were determined at weekly intervals in male hamsters from 4 weeks of age. In both genotypes, testicular development, subsequent regression and recrudescence exhibited a similar time course. The age at which animals displayed reproductive photosensitivity, as exhibited by testicular regression, was unrelated to circadian genotype (mean +/- SEM: 54 +/- 3 days for wild type and 59 +/- 5 days for tau mutants). In contrast, our studies revealed a significant impact of the mutation on somatic growth, such that tau mutants weighed 18% less than wild types at the end of the experiment. Our study reveals that the juvenile onset of reproductive photoperiodism in Syrian hamsters is not timed by the circadian system.

scholarly journals Neurofilament Proteolysis after Focal Ischemia; When Do Cells Die after Experimental Stroke?

1999 ◽  
Vol 19 (6) ◽  
pp. 652-660 ◽  
Author(s):  
Jaroslaw Aronowski ◽  
Ki-Hyun Cho ◽  
Roger Strong ◽  
James C. Grotta
Keyword(s):  
Time Course ◽  
Infarct Volume ◽  
Focal Ischemia ◽  
Cellular Damage ◽  
Experimental Stroke ◽  
Infarct Core ◽  
Similar Time ◽  
The Core ◽  
Tetrazolium Staining ◽  
Previous Demonstration

To determine the occurrence and time-course of presumably irreversible subcellular damage after moderate focal ischemia, rats were subjected to 1, 3, 6, 9, or 24 hours of permanent unilateral middle cerebral and common carotid occlusion or 3 hours of reversible occlusion followed by 3, 6, or 21 hours of reperfusion. The topography and the extent of damage were analyzed with tetrazolium staining and immunoblot using an antibody capable of detecting breakdown of neurofilament. Neurofilament proteolysis began after 3 hours in the infarct core but was still incomplete in penumbral regions up to 9 hours. Similarly, tetrazolium-staining abnormalities were observed in the core of 50% of animals after 3 hours of ischemia. At 6 hours of permanent ischemia, infarct volume was maximal, and further prolongation of occlusion to 9 or 24 hours did not increase abnormal tetrazolium staining. In contrast to permanent ischemia and in agreement with the authors' previous demonstration of “reperfusion injury” in this model, prolongation of reperfusion from 3 hours to 6 and 21 hours after 3 hours of reversible occlusion gradually augmented infarct volume by 203% and 324%, respectively. Neurofilament proteolysis initiated approximately 3 hours after ischemia was quantitatively greatest in the core and extended during reperfusion to incorporate penumbra with a similar time course to that of tetrazolium abnormalities. These data demonstrate that, at least as measured by neurofilament breakdown and mitochondrial failure, extensive cellular damage is not present in penumbral regions for up to 9 hours, suggesting the potential for rescuing these regions by appropriate and timely neuroprotective strategies.

scholarly journals TGF-β1 increases permeability of ciliated airway epithelia via redistribution of claudin 3 from tight junction into cell nuclei

2021 ◽  
Author(s):  
Carolin Schilpp ◽  
Robin Lochbaum ◽  
Peter Braubach ◽  
Danny Jonigk ◽  
Manfred Frick ◽  
...  
Keyword(s):  
Time Course ◽  
Atopic Asthma ◽  
Ciliated Cells ◽  
Cell Nuclei ◽  
Tissue Remodelling ◽  
Similar Time ◽  
Airway Epithelia ◽  
Epithelial Integrity ◽  
Motile Cilia ◽  
Tgf Β1

AbstractTGF-β1 is a major mediator of airway tissue remodelling during atopic asthma and affects tight junctions (TJs) of airway epithelia. However, its impact on TJs of ciliated epithelia is sparsely investigated. Herein we elaborated effects of TGF-β1 on TJs of primary human bronchial epithelial cells. We demonstrate that TGF-β1 activates TGF-β1 receptors TGFBR1 and TGFBR2 resulting in ALK5-mediated phosphorylation of SMAD2. We observed that TGFBR1 and -R2 localize specifically on motile cilia. TGF-β1 activated accumulation of phosphorylated SMAD2 (pSMAD2-C) at centrioles of motile cilia and at cell nuclei. This triggered an increase in paracellular permeability via cellular redistribution of claudin 3 (CLDN3) from TJs into cell nuclei followed by disruption of epithelial integrity and formation of epithelial lesions. Only ciliated cells express TGF-β1 receptors; however, nuclear accumulations of pSMAD2-C and CLDN3 redistribution were observed with similar time course in ciliated and non-ciliated cells. In summary, we demonstrate a role of motile cilia in TGF-β1 sensing and showed that TGF-β1 disturbs TJ permeability of conductive airway epithelia by redistributing CLDN3 from TJs into cell nuclei. We conclude that the observed effects contribute to loss of epithelial integrity during atopic asthma.

scholarly journals Frequency of myelin oligodendrocyte glycoprotein antibodies in a large cohort of neurological patients

2021 ◽  
Vol 7 (2) ◽  
pp. 205521732110227
Author(s):  
Friederike Held ◽  
Sudhakar Reddy Kalluri ◽  
Achim Berthele ◽  
Ana-Katharina Klein ◽  
Markus Reindl ◽  
...  
Keyword(s):  
Time Course ◽  
Neurological Diseases ◽  
External Validation ◽  
Diagnostic Value ◽  
Myelin Oligodendrocyte Glycoprotein ◽  
Large Cohort ◽  
Wide Range ◽  
Neurological Patients ◽  
A Cell ◽  
Nosological Entity

Background Myelin oligodendrocyte glycoprotein (MOG) antibody disease (MOG-AD) is recognized as a distinct nosological entity. IgG antibodies against MOG (MOG-Ab) overlap with neuromyelitis optica spectrum disorders (NMOSD) phenotype in adults. However, an increasing number of clinical phenotypes have been reported to be associated with MOG-Ab. Objective To investigate the seroprevalence of MOG-Ab under consideration of demographics, disease entities and time course in a large cohort of unselected neurological patients. Methods Blood samples of 2.107 consecutive adult neurologic patients admitted to our department between 2016-2017 were tested for MOG-Ab using a cell-based assay. MOG-Ab persistence was analyzed in follow-up samples. External validation was performed in two independent laboratories. Results We found MOG-Ab in 25 of 2.107 (1.2%) patients. High antibody ratios were mostly associated with NMOSD and MOG-AD phenotype (5/25). Low ratios occurred in a wide range of neurological diseases, predominantly in other demyelinating CNS diseases (5/25) and stroke (6/25). MOG-Ab persistence over time was not confined to NMOSD and MOG-AD phenotype. Conclusion The present study demonstrates the occurrence of MOG-Ab in a wide range of neurological diseases. Only high MOG-Ab ratios were associated with a defined clinical phenotype, but low MOG-Ab ratios were not. The diagnostic value of low MOG-Ab is thus highly limited.

scholarly journals Intracellular Ionic Strength Sensing Using NanoLuc

2021 ◽  
Vol 22 (2) ◽  
pp. 677
Author(s):  
Tausif Altamash ◽  
Wesam Ahmed ◽  
Saad Rasool ◽  
Kabir H. Biswas
Keyword(s):  
Thermal Stability ◽  
Phase Separation ◽  
Drug Discovery ◽  
Ionic Strength ◽  
Cellular Signaling ◽  
Live Cells ◽  
Protein Activity ◽  
Cellular Survival ◽  
Cellular Processes ◽  
Wide Range

Intracellular ionic strength regulates myriad cellular processes that are fundamental to cellular survival and proliferation, including protein activity, aggregation, phase separation, and cell volume. It could be altered by changes in the activity of cellular signaling pathways, such as those that impact the activity of membrane-localized ion channels or by alterations in the microenvironmental osmolarity. Therefore, there is a demand for the development of sensitive tools for real-time monitoring of intracellular ionic strength. Here, we developed a bioluminescence-based intracellular ionic strength sensing strategy using the Nano Luciferase (NanoLuc) protein that has gained tremendous utility due to its high, long-lived bioluminescence output and thermal stability. Biochemical experiments using a recombinantly purified protein showed that NanoLuc bioluminescence is dependent on the ionic strength of the reaction buffer for a wide range of ionic strength conditions. Importantly, the decrease in the NanoLuc activity observed at higher ionic strengths could be reversed by decreasing the ionic strength of the reaction, thus making it suitable for sensing intracellular ionic strength alterations. Finally, we used an mNeonGreen–NanoLuc fusion protein to successfully monitor ionic strength alterations in a ratiometric manner through independent fluorescence and bioluminescence measurements in cell lysates and live cells. We envisage that the biosensing strategy developed here for detecting alterations in intracellular ionic strength will be applicable in a wide range of experiments, including high throughput cellular signaling, ion channel functional genomics, and drug discovery.

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