scholarly journals Role of store-operated Ca2+ entry in adenosine-induced vasodilatation of rat small mesenteric artery

2009 ◽  
Vol 297 (1) ◽  
pp. H347-H354 ◽  
Author(s):  
Shengpeng Wang ◽  
Yan Zhang ◽  
W. Gil Wier ◽  
Xiaojiang Yu ◽  
Ming Zhao ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Protective Role ◽  
Dependent Manner ◽  
Resistance Arteries ◽  
Concentration Dependent Manner ◽  
Scanning Confocal Microscopy ◽  
Store Depletion ◽  
Intracellular Ca ◽  
Isolated Artery

Store-operated Ca2+ entry (SOCE) has recently been proposed to contribute to Ca2+ influx in vascular smooth muscle cells (VSMCs). Adenosine is known for its protective role against hypoxia and ischemia by increasing nutrient and oxygen supply through vasodilation. This study was designed to examine the hypothesis that SOCE have a functional role in adenosine-induced vasodilation. Small mesenteric resistance arteries and mesenteric VSMCs were obtained from rats. Isometric tensions of isolated artery rings were measured by a sensitive myograph system. Laser-scanning confocal microscopy was used to determine the intracellular Ca2+ concentration of fluo 3-loaded VSMCs. Adenosine (0.1–100 μM) relaxed artery rings that were precontracted by phenylephrine in a concentration-dependent manner. In cultured mesenteric VSMCs, passive store depletion by thapsigargin and active store depletion by phenylephrine both induced Ca2+ influx due to SOCE. Adenosine inhibited SOCE-mediated increases in cytosolic Ca2+ levels evoked by the emptying of the stores. In isolated artery rings, adenosine inhibited SOCE-induced contractions due to store depletion. A2A receptor antagonism with SCH-58261 and adenylate cyclase inhibition with SQ-22536 largely attenuated adenosine responses. The cAMP analog 8-bromo-cAMP mimicked the effects of adenosine on SOCE. Our results indicate a novel mechanism of vasodilatation by adenosine that involves regulation of SOCE through the cAMP signaling pathway due to activation of adenosine A2A receptors.

Choline produces antiarrhythmic actions in animal models by cardiac M3 receptors: improvement of intracellular Ca2+ handling as a common mechanism

2008 ◽  
Vol 86 (12) ◽  
pp. 860-865 ◽  
Author(s):  
Yan Liu ◽  
Hong-li Sun ◽  
Dan-lu Li ◽  
Li-yan Wang ◽  
Yang Gao ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Common Mechanism ◽  
Protective Effects ◽  
Patch Clamp Technique ◽  
Beneficial Effects ◽  
Scanning Confocal Microscopy ◽  
M3 Receptors ◽  
Intracellular Ca ◽  
Antiarrhythmic Actions

It is well known that choline has protective effects on ischemic arrhythmias. We designed the present study to evaluate the antiarrhythmic effects of choline and to detect its related mechanisms in aconitine-induced rat and ouabain-induced guinea pig models of arrhythmia. Laser scanning confocal microscopy and patch-clamp technique were utilized to study the action of choline on intracellular calcium concentration and L-type calcium current (ICa-L) of cardiac myocytes. M3 receptor antagonist 4-DAMP (4-diphenylacetoxy-N-methylpiperidine-methiodide) was applied preliminarily to evaluate the role of the M3 receptor. Choline significantly increased the survival time of arrhythmic rats and guinea pigs, delayed the onset of arrhythmias and ventricular tachycardia, and decreased the arrhythmia score. The overload of intracellular Ca2+ induced by aconitine or ouabain was reduced in isolated myocytes pretreated with choline. Choline reduced the increased density of ICa-L induced by aconitine or ouabain. Moreover, the beneficial effects of choline were reversed by 4-DAMP. Choline produced antiarrhythmic actions on arrhythmia models by stimulating the cardiac M3 receptor. The mechanism may be related to the improvement of Ca2+ handling.

Acidosis and ischemia increase cellular Ca2+ transient alternans and repolarization alternans susceptibility in the intact rat heart

2009 ◽  
Vol 296 (5) ◽  
pp. H1491-H1512 ◽  
Author(s):  
Sunil Kapur ◽  
J. Andrew Wasserstrom ◽  
James E. Kelly ◽  
Alan H. Kadish ◽  
Gary L. Aistrup
Keyword(s):  
Myocardial Ischemia ◽  
Laser Scanning ◽  
Close Association ◽  
Late Phase ◽  
Laser Scanning Confocal Microscopy ◽  
Low Flow ◽  
Heart Rates ◽  
Scanning Confocal Microscopy ◽  
Intracellular Ca ◽  
Whole Heart

Cardiac cellular Ca2+ transient (CaT) alternans and electrocardiographic T-wave alternans (TWA) often develop in myocardial ischemia, but the mechanisms for this relationship have not been elucidated. Acidosis is a major component of ischemia, but there is no direct evidence linking acidosis-induced cellular CaT alternans to ischemia-induced CaT alternans and TWA in whole heart. We used laser-scanning confocal microscopy to measure intracellular Ca2+ (Cai2+) cycling in individual myocytes of fluo-4 AM-loaded rat hearts and simultaneously recorded pseudo-ECGs to investigate changes in CaTs and late-phase repolarization, respectively, during baseline and rapid pacing under control and either globally acidic or globally ischemic conditions. Acidosis (hypercapnia; pH 6.6) increased diastolic Cai2+ levels, prolonged CaT duration, and shifted to slower heart rates both the development of pacing-induced acidosis-induced CaT alternans (both concordant and discordant) and of repolarization alternans (RPA, a measure of TWA in rat ECGs). The magnitudes of these shifts were equivalent for both CaT alternans and RPA, suggesting a close association between them. Nearly identical results were found in low-flow global ischemia. Additionally, ischemic preconditioning reduced the increased propensity for CaT alternans and RPA development and was mimicked by preconditioning by acidosis alone. Our results demonstrate that global acidosis or ischemia modifies Cai2+ cycling in myocytes such that the diastolic Cai2+ rises and the cellular CaT duration is prolonged, causing spatially concordant as well as spatially discordant cellular CaT alternans to develop at slower heart rates than in controls. Since RPA also developed at slower heart rates, our results suggest that acidosis is a major contributor to CaT alternans, which underlies the proarrhythmic state induced by myocardial ischemia and therefore may play a role in its modulation and prevention.

scholarly journals The role of Ca2+-activated Cl− current in tone generation in the rabbit corpus cavernosum

2017 ◽  
Vol 313 (5) ◽  
pp. C475-C486 ◽  
Author(s):  
Karen I. Hannigan ◽  
Caoimhin S. Griffin ◽  
Roddy J. Large ◽  
Gerard P. Sergeant ◽  
Mark A. Hollywood ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Corpus Cavernosum ◽  
Laser Scanning Confocal Microscopy ◽  
Isometric Tension ◽  
Scanning Confocal Microscopy ◽  
Rabbit Corpus Cavernosum ◽  
Intracellular Ca ◽  
Low Sensitivity ◽  
Tone Generation

Rabbit corpus cavernosum smooth muscle (RCCSM) cells express ion channels that produce Ca2+-activated Cl− ( IClCa) current, but low sensitivity to conventional antagonists has made its role in tone generation difficult to evaluate. We have reexamined this question using two new generation IClCa blockers, T16Ainh-A01 and CaCCinh-A01. Isolated RCCSM cells were studied using the perforated patch method. Current-voltage protocols revealed that both L-type Ca2+ current and IClCa. T16Ainh-A01 and CaCCinh-A01 (10 μM) reduced IClCa by ~85%, while 30 μM abolished it. L-type Ca2+ current was unaffected by 10 μM CaCCinh-A01 but was reduced by 50% at 30 μM CaCCinh-A01, 46% at 10 μM T16Ainh-A01, and 78% at 30 μM T16Ainh-A01. Both drugs reduced spontaneous isometric tension in RCCSM strips, by 60–70% at 10 μM and >90% at 30 μM. Phenylephrine (PE)-enhanced tension was also reduced (ED50 = 3 μM, CaCCinh-A01; 14 μM, T16Ainh-A01). CaCCinh-A01 at 10 μM had little effect on 60 mM KCl contractures, though they were reduced by 30 μM CaCCinh-A01 and T16Ainh-A01 (10 μM and 30 μM) consistent with their effects on L-type Ca2+ current. Both drugs also reversed the stimulatory effect of PE on intracellular Ca2+ waves, studied with laser scanning confocal microscopy in isolated RCCSM cells. In conclusion, although both drugs were effective blockers of IClCa, the effect of T16Ainh-A01 on L-type Ca2+ current precludes its use for evaluating the role of IClCa in tone generation. However, 10 μM CaCCinh-A01 selectively blocked IClCa versus L-type Ca2+ current and reduced spontaneous and PE-induced tone, suggesting that IClCa is important in maintaining penile detumescence.

Laser Scanning Confocal Microscopy and Three-Dimesnsional Reconstruction of the Mitotic Spindles of Unequally Dividing Embryonic Cells

1990 ◽  
Vol 48 (3) ◽  
pp. 166-167
Author(s):  
J. Holy ◽  
G. Schatten
Keyword(s):  
Confocal Microscopy ◽  
Mitotic Spindle ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Confocal Microscopy ◽  
Two Dimensions ◽  
Embryonic Cells ◽  
Mitotic Spindles ◽  
Cleavage Division ◽  
Scanning Confocal Microscopy

One of the classic limitations of light microscopy has been the fact that three dimensional biological events could only be visualized in two dimensions. Recently, this shortcoming has been overcome by combining the technologies of laser scanning confocal microscopy (LSCM) and computer processing of microscopical data by volume rendering methods. We have employed these techniques to examine morphogenetic events characterizing early development of sea urchin embryos. Specifically, the fourth cleavage division was examined because it is at this point that the first morphological signs of cell differentiation appear, manifested in the production of macromeres and micromeres by unequally dividing vegetal blastomeres.The mitotic spindle within vegetal blastomeres undergoing unequal cleavage are highly polarized and develop specialized, flattened asters toward the micromere pole. In order to reconstruct the three-dimensional features of these spindles, both isolated spindles and intact, extracted embryos were fluorescently labeled with antibodies directed against either centrosomes or tubulin.

Automated 3-D cell population analysis in thick tissue sections using laser-scanning confocal-microscopy data

1993 ◽  
Vol 51 ◽  
pp. 562-563
Author(s):  
Hakan Ancin
Keyword(s):  
Laser Scanning ◽  
Population Analysis ◽  
Three Dimensional ◽  
Large Data ◽  
Laser Scanning Confocal Microscopy ◽  
Tissue Slices ◽  
Scanning Confocal Microscopy ◽  
Tissue Sections ◽  
Spatially Adaptive ◽  
Microscopy Data

This paper presents methods for performing detailed quantitative automated three dimensional (3-D) analysis of cell populations in thick tissue sections while preserving the relative 3-D locations of cells. Specifically, the method disambiguates overlapping clusters of cells, and accurately measures the volume, 3-D location, and shape parameters for each cell. Finally, the entire population of cells is analyzed to detect patterns and groupings with respect to various combinations of cell properties. All of the above is accomplished with zero subjective bias.In this method, a laser-scanning confocal light microscope (LSCM) is used to collect optical sections through the entire thickness (100 - 500μm) of fluorescently-labelled tissue slices. The acquired stack of optical slices is first subjected to axial deblurring using the expectation maximization (EM) algorithm. The resulting isotropic 3-D image is segmented using a spatially-adaptive Poisson based image segmentation algorithm with region-dependent smoothing parameters. Extracting the voxels that were labelled as "foreground" into an active voxel data structure results in a large data reduction.

scholarly journals Advanced Static and Dynamic Fluorescence Microscopy Techniques to Investigate Drug Delivery Systems

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 861
Author(s):  
Jacopo Cardellini ◽  
Arianna Balestri ◽  
Costanza Montis ◽  
Debora Berti
Keyword(s):  
Drug Delivery ◽  
Fluorescence Microscopy ◽  
Drug Delivery Systems ◽  
Laser Scanning ◽  
Super Resolution ◽  
Laser Scanning Confocal Microscopy ◽  
Delivery Systems ◽  
Scanning Confocal Microscopy ◽  
Biological Phenomena

In the past decade(s), fluorescence microscopy and laser scanning confocal microscopy (LSCM) have been widely employed to investigate biological and biomimetic systems for pharmaceutical applications, to determine the localization of drugs in tissues or entire organisms or the extent of their cellular uptake (in vitro). However, the diffraction limit of light, which limits the resolution to hundreds of nanometers, has for long time restricted the extent and quality of information and insight achievable through these techniques. The advent of super-resolution microscopic techniques, recognized with the 2014 Nobel prize in Chemistry, revolutionized the field thanks to the possibility to achieve nanometric resolution, i.e., the typical scale length of chemical and biological phenomena. Since then, fluorescence microscopy-related techniques have acquired renewed interest for the scientific community, both from the perspective of instrument/techniques development and from the perspective of the advanced scientific applications. In this contribution we will review the application of these techniques to the field of drug delivery, discussing how the latest advancements of static and dynamic methodologies have tremendously expanded the experimental opportunities for the characterization of drug delivery systems and for the understanding of their behaviour in biologically relevant environments.

Observation of Fine Structure in Bicontinuous Phase-Separated Domains of a Polymer Blend by Laser Scanning Confocal Microscopy

2001 ◽  
Vol 34 (15) ◽  
pp. 5186-5191 ◽  
Author(s):  
Hiroshi Jinnai ◽  
Hiroshi Yoshida ◽  
Kohtaro Kimishima ◽  
Yoshinori Funaki ◽  
Yoshitsugu Hirokawa ◽  
...  
Keyword(s):  
Fine Structure ◽  
Confocal Microscopy ◽  
Polymer Blend ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Scanning Confocal Microscopy

scholarly journals The DNA content of trophozoites and cysts of Giardia lamblia by microdensitometric quantitation of Feulgen staining and examination by laser scanning confocal microscopy.

1994 ◽  
Vol 42 (11) ◽  
pp. 1413-1416 ◽  
Author(s):  
S L Erlandsen ◽  
E M Rasch
Keyword(s):  
Confocal Microscopy ◽  
Genome Size ◽  
Dna Content ◽  
Giardia Lamblia ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Evolutionary Development ◽  
Scanning Confocal Microscopy ◽  
Dividing Cells ◽  
C Value

We investigated direct measurement of the DNA content of the parasitic intestinal flagellate Giardia lamblia through quantitation by Feulgen microspectrophotometry and also by visualization of Feulgen-stained DNA chromosomes within dividing cells by laser scanning confocal microscopy. Individual trophozoites of Giardia (binucleate) contained 0.144 +/- 0.018 pg of DNA/cell or 0.072 pg DNA/nucleus. Giardia lamblia cysts (quadranucleate) contained 0.313 +/- 0.003 pg DNA or 0.078 pg DNA/nucleus. The genome size (C) value per nucleus ranged between 6.5-7.1 x 10(7) BP for trophozoites and cysts, respectively. Confocal microscopic examination of Giardia trophozoites undergoing binary fission revealed five chromosome-like bodies within each nucleus. Further information about genome size and DNA content within different Giardia species may help to clarify the pivotal role of these primitive eukaryotic cells in evolutionary development.

The Effect of Chloroquine on the Apoptosis Induced by Cisplatin in Human Gastric Cancer BGC823 Cells

2014 ◽  
Vol 926-930 ◽  
pp. 1124-1127
Author(s):  
Zhen Xun Jin ◽  
Li Li Zhang ◽  
Yan Wang ◽  
Lin Chuan Zeng ◽  
Yang Yu ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Confocal Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Combination Group ◽  
Human Gastric Cancer ◽  
Apoptotic Cells ◽  
Toxic Dose ◽  
Scanning Confocal Microscopy ◽  
Mtt Tests

The aim of this study is to investigate the effects and mechanism of chloroquine (CQ) on the apoptosis induced by cisplatin in human gastric cancer BGC823 cells. MTT assay was used to detect the state of cell growth. The appearances of cellular apoptosis were detected by laser scanning confocal microscopy and light microscopy. The expressions of LC3 and p62 were detected by laser scanning confocal microscopy. MTT tests showed that the non-toxic dose of CQ could increase the inhibition rate of BGC823 cells induced by cisplatin. Under the light microscope, the ratio of apoptotic cells in the group treated with non-toxic dose of CQ combined with cisplatin was higher than that in the group treated with cisplatin alone. Hoechst33342 staining showed that the ratio of apoptotic cells in the combination group was higher than that in the cisplatin group. The expression and colocalization of LC3 and p62 proteins were significantly increased in the combination group. These results indicate that CQ can enhance the cell apoptosis induced by cisplatin in BGC823 cells, which is through the inhibition of autophagy.

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