scholarly journals Detection of differentially regulated subsarcolemmal calcium signals activated by vasoactive agonists in rat pulmonary artery smooth muscle cells

2014 ◽  
Vol 306 (7) ◽  
pp. C659-C669 ◽  
Author(s):  
Krishna P. Subedi ◽  
Omkar Paudel ◽  
James S. K. Sham
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Laser Scanning ◽  
Muscle Cells ◽  
Laser Scanning Confocal Microscopy ◽  
Cellular Functions ◽  
Permeabilized Cells ◽  
Scanning Confocal Microscopy ◽  
Subcellular Compartments ◽  
Transient Elevation

Intracellular calcium (Ca2+) plays pivotal roles in distinct cellular functions through global and local signaling in various subcellular compartments, and subcellular Ca2+ signal is the key factor for independent regulation of different cellular functions. In vascular smooth muscle cells, subsarcolemmal Ca2+ is an important regulator of excitation-contraction coupling, and nucleoplasmic Ca2+ is crucial for excitation-transcription coupling. However, information on Ca2+ signals in these subcellular compartments is limited. To study the regulation of the subcellular Ca2+ signals, genetically encoded Ca2+ indicators (cameleon), D3cpv, targeting the plasma membrane (PM), cytoplasm, and nucleoplasm were transfected into rat pulmonary arterial smooth muscle cells (PASMCs) and Ca2+ signals were monitored using laser scanning confocal microscopy. In situ calibration showed that the Kd for Ca2+ of D3cpv was comparable in the cytoplasm and nucleoplasm, but it was slightly higher in the PM. Stimulation of digitonin-permeabilized cells with 1,4,5-trisphosphate (IP3) elicited a transient elevation of Ca2+ concentration with similar amplitude and kinetics in the nucleoplasm and cytoplasm. Activation of G protein-coupled receptors by endothelin-1 and angiotensin II preferentially elevated the subsarcolemmal Ca2+ signal with higher amplitude in the PM region than the nucleoplasm and cytoplasm. In contrast, the receptor tyrosine kinase activator, platelet-derived growth factor, elicited Ca2+ signals with similar amplitudes in all three regions, except that the rise-time and decay-time were slightly slower in the PM region. These data clearly revealed compartmentalization of Ca2+ signals in the subsarcolemmal regions and provide the basis for further investigations of differential regulation of subcellular Ca2+ signals in PASMCs.

Preparation of recombinant human-like collagen/fibroin scaffold and its promoting effect on vascular cells biocompatibility

2018 ◽  
Vol 33 (4) ◽  
pp. 416-425 ◽  
Author(s):  
Jia Yan ◽  
Kun Hu ◽  
YongHao Xiao ◽  
Fan Zhang ◽  
Lu Han ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Laser Scanning ◽  
Muscle Cells ◽  
Laser Scanning Confocal Microscopy ◽  
Scanning Confocal Microscopy ◽  
Scanning Electron

A novel recombinant human-like collagen/fibroin scaffold has been prepared previously, which has high porosity, controllable pore size, and much better mechanical properties than the reported fibroin-based scaffold. In this research, the cell responses of vascular smooth muscle cells to this blend scaffold were examined in vitro. Cell morphology, adherence, and growth in scaffolds were observed by scanning electron microscopy, laser scanning confocal microscopy after staining of the cells with propidium iodide at 1, 3, 5, and 7 days, respectively. A wide range of measurements, including 3-[4,5–dimethylthiazol-2-yl]-2, 5-diphenyl tetrasodium bromide assay, and total intracellular protein content at the end of 7 days culture, were conducted. An increase of viability and protein content of vascular smooth muscle cells cultured in recombinant human-like collagen/fibroin scaffold was found. The laser scanning confocal microscopy and scanning electron microscopy results confirm that the cells readily adhered and proliferation in the blend than in fibroin scaffold, and indicate a better adhesion process. The positive effects were especially significant for vascular smooth muscle cells. The recombinant human-like collagen/fibroin scaffold could be a promising biomaterial for vascular tissue engineering.

Propagation of calcium waves along endothelium of hamster feed arteries

2007 ◽  
Vol 292 (3) ◽  
pp. H1634-H1640 ◽  
Author(s):  
Torben R. Uhrenholt ◽  
Timothy L. Domeier ◽  
Steven S. Segal
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Laser Scanning ◽  
Muscle Cells ◽  
Laser Scanning Confocal Microscopy ◽  
Retractor Muscle ◽  
Tissue Blood Flow ◽  
Vasomotor Tone ◽  
Feed Arteries

An increase in tissue blood flow requires relaxation of smooth muscle cells along entire branches of the resistance vasculature. Whereas the spread of hyperpolarization along the endothelium can coordinate smooth muscle cell relaxation, complementary signaling events have been implicated in the conduction of vasodilation. We tested the hypothesis that Ca2+ waves propagate from cell to cell along the endothelium of feed arteries exhibiting spontaneous vasomotor tone. Feed arteries of the hamster retractor muscle were isolated, pressurized to 75 mmHg at 37°C, and developed myogenic tone spontaneously. Smooth muscle cells and endothelial cells were loaded with the Ca2+ indicator Fluo-4. An acetylcholine stimulus was delivered locally using microiontophoresis (1-μm pipette tip, 1 μA, 1 s), and Ca2+ signaling within and along respective cell layers was determined using laser-scanning confocal microscopy. Acetylcholine triggered an increase in intracellular Ca2+ concentration ([Ca2+]i) of endothelial cells at the site of stimulation that preceded two distinct events: 1) a rapid synchronous decrease in smooth muscle [Ca2+]i along the entire vessel and 2) an ensuing Ca2+ wave that propagated bidirectionally along the endothelium at ∼111 μm/s for distances exceeding 1 mm. Maximal dilation of vessels with either nifedipine (1 μM) or sodium nitroprusside (SNP, 100 μM) reduced the distance that Ca2+ waves traveled to ∼300 μm ( P < 0.05). Thus Ca2+ waves propagate along the endothelium of resistance vessels with vasomotor tone, and this signaling pathway is compromised during maximal dilation with nifedipine or SNP.

scholarly journals The Contraction of Smooth Muscle Cells of Intrapulmonary Arterioles Is Determined by the Frequency of Ca2+ Oscillations Induced by 5-HT and KCl

2005 ◽  
Vol 125 (6) ◽  
pp. 555-567 ◽  
Author(s):  
Jose F. Perez ◽  
Michael J. Sanderson
Keyword(s):  
Pulmonary Hypertension ◽  
Smooth Muscle ◽  
Confocal Microscopy ◽  
Smooth Muscle Cells ◽  
Phase Contrast ◽  
Muscle Cells ◽  
Low Frequencies ◽  
Scanning Confocal Microscopy ◽  
Agonist Concentration ◽  
Mouse Lungs

Increased resistance of the small blood vessels within the lungs is associated with pulmonary hypertension and results from a decrease in size induced by the contraction of their smooth muscle cells (SMCs). To study the mechanisms that regulate the contraction of intrapulmonary arteriole SMCs, the contractile and Ca2+ responses of the arteriole SMCs to 5-hydroxytrypamine (5-HT) and KCl were observed with phase-contrast and scanning confocal microscopy in thin lung slices cut from mouse lungs stiffened with agarose and gelatin. 5-HT induced a concentration-dependent contraction of the arterioles. Increasing concentrations of extracellular KCl induced transient contractions in the SMCs and a reduction in the arteriole luminal size. 5-HT induced oscillations in [Ca2+]i within the SMCs, and the frequency of these Ca2+ oscillations was dependent on the agonist concentration and correlated with the extent of sustained arteriole contraction. By contrast, KCl induced Ca2+ oscillations that occurred with low frequencies and were preceded by small, localized transient Ca2+ events. The 5-HT–induced Ca2+ oscillations and contractions occurred in the absence of extracellular Ca2+ and were resistant to Ni2+ and nifedipine but were abolished by caffeine. KCl-induced Ca2+ oscillations and contractions were abolished by the absence of extracellular Ca2+ and the presence of Ni2+, nifedipine, and caffeine. Arteriole contraction was induced or abolished by a 5-HT2–specific agonist or antagonist, respectively. These results indicate that 5-HT, acting via 5-HT2 receptors, induces arteriole contraction by initiating Ca2+ oscillations and that KCl induces contraction via Ca2+ transients resulting from the overfilling of internal Ca2+ stores. We hypothesize that the magnitude of the sustained intrapulmonary SMC contraction is determined by the frequency of Ca2+ oscillations and also by the relaxation rate of the SMC.

Altered Cytoskeleton in Smooth Muscle of Aganglionic Bowel

2002 ◽  
Vol 126 (6) ◽  
pp. 692-696
Author(s):  
Laszlo Nemeth ◽  
Udo Rolle ◽  
Prem Puri
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Laser Scanning ◽  
Hirschsprung Disease ◽  
Muscle Cells ◽  
Cytoskeletal Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Normal Bowel ◽  
Pull Through

Abstract Context.—Intestinal motility is under the control of smooth muscle cells, enteric plexus, and hormonal factors. In Hirschsprung disease (HD), the aganglionic colon remains spastic or tonically enhanced and unable to relax. The smooth muscle cell's cytoskeleton consists of proteins or structures whose primary function is to link or connect protein filaments to each other or to the anchoring sites. Dystrophin is a subsarcolemmal protein with a double adhesion property, one between the membrane elements and the contractile filaments of the cytoskeleton and the other between the cytoskeletal proteins and the extracellular matrix. Desmin and vinculin are functionally related proteins that are present in the membrane-associated dense bodies in the sarcolemma of the smooth muscle cells. Objective.—To examine the distribution of the cytoskeletal proteins in the smooth muscle of the aganglionic bowel. Design.—Bowel specimens from ganglionic and aganglionic sections of the colon were collected at the time of pull-through surgery from 8 patients with HD. Colon specimens collected from 4 patients at the time of bladder augmentation acted as controls. Anti-dystrophin, anti-desmin, and anti-vinculin antibodies were used for fluorescein immunostaining using confocal laser scanning microscopy. Results.—Moderate to strong dystrophin immunoreactivity was observed at the periphery of smooth muscle fibers in normal bowel and ganglionic bowel from patients with HD, whereas dystrophin immunoreactivity was either absent or weak in the smooth muscle of aganglionic colon. Moderate to strong cytoplasmic immunostaining for vinculin and desmin was seen in the smooth muscle of normal bowel and ganglionic bowel from patients with HD, whereas vinculin and desmin staining in the aganglionic colon was absent or weak. Conclusion.—This study demonstrates that the cytoskeletal proteins are abundant in the smooth muscle of normal bowel, but are absent or markedly reduced in the aganglionic bowel of HD. As cytoskeletal proteins are required for the coordinated contraction of muscle cells, their absence may be responsible for the motility dysfunction in the aganglionic segment.

Intravascular pressure regulates local and global Ca2+ signaling in cerebral artery smooth muscle cells

2001 ◽  
Vol 281 (2) ◽  
pp. C439-C448 ◽  
Author(s):  
Jonathan H. Jaggar
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Laser Scanning ◽  
Muscle Cells ◽  
Membrane Depolarization ◽  
Wave Frequency ◽  
Current Frequency ◽  
Frequency Wave ◽  
Intracellular Ca ◽  
Intravascular Pressure

The regulation of intracellular Ca2+ signals in smooth muscle cells and arterial diameter by intravascular pressure was investigated in rat cerebral arteries (∼150 μm) using a laser scanning confocal microscope and the fluorescent Ca2+ indicator fluo 3. Elevation of pressure from 10 to 60 mmHg increased Ca2+spark frequency 2.6-fold, Ca2+ wave frequency 1.9-fold, and global intracellular Ca2+ concentration ([Ca2+]i) 1.4-fold in smooth muscle cells, and constricted arteries. Ryanodine (10 μM), an inhibitor of ryanodine-sensitive Ca2+ release channels, or thapsigargin (100 nM), an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase, abolished sparks and waves, elevated global [Ca2+]i, and constricted pressurized (60 mmHg) arteries. Diltiazem (25 μM), a voltage-dependent Ca2+ channel (VDCC) blocker, significantly reduced sparks, waves, and global [Ca2+]i, and dilated pressurized (60 mmHg) arteries. Steady membrane depolarization elevated Ca2+ signaling similar to pressure and increased transient Ca2+-sensitive K+ channel current frequency e-fold for ∼7 mV, and these effects were prevented by VDCC blockers. Data are consistent with the hypothesis that pressure induces a steady membrane depolarization that activates VDCCs, leading to an elevation of spark frequency, wave frequency, and global [Ca2+]i. In addition, pressure induces contraction via an elevation of global [Ca2+]i, whereas the net effect of sparks and waves, which do not significantly contribute to global [Ca2+]i in arteries pressurized to between 10 and 60 mmHg, is to oppose contraction.

Effect of halothane on intracellular calcium oscillations in porcine tracheal smooth muscle cells

1999 ◽  
Vol 276 (1) ◽  
pp. L81-L89 ◽  
Author(s):  
Christina M. Pabelick ◽  
Y. S. Prakash ◽  
Mathur S. Kannan ◽  
Keith A. Jones ◽  
David O. Warner ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Ryanodine Receptor ◽  
Muscle Cells ◽  
Calcium Oscillations ◽  
Tracheal Smooth Muscle ◽  
Permeabilized Cells ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca ◽  
Trisphosphate Receptor

The effect of halothane on intracellular Ca2+ concentration ([Ca2+]i) regulation in porcine tracheal smooth muscle cells was examined with real-time confocal microscopy. Both 1 and 2 minimum alveolar concentration (MAC) halothane increased basal [Ca2+]iwhen Ca2+ influx and efflux were blocked, suggesting increased sarcoplasmic reticulum (SR) Ca2+ leak and/or decreased reuptake. In β-escin-permeabilized cells, heparin inhibition of inositol 1,4,5-trisphosphate-receptor channels blunted the halothane-induced increase in [Ca2+]i. Both 1 and 2 MAC halothane decreased the frequency and amplitude of ACh-induced [Ca2+]ioscillations (which represent SR Ca2+ release through ryanodine-receptor channels), abolishing oscillations in ∼20% of tracheal smooth muscle cells at 2 MAC. When Ca2+ influx and efflux were blocked, halothane increased the baseline and decreased the frequency and amplitude of [Ca2+]ioscillations, inhibiting oscillations in ∼70% of cells at 2 MAC. The fall time of [Ca2+]ioscillations and the rate of fall of the [Ca2+]iresponse to caffeine were both increased by halothane. These results suggest that halothane abolishes agonist-induced [Ca2+]ioscillations by 1) depleting SR Ca2+ via increased Ca2+ leak through inositol 1,4,5-trisphosphate-receptor channels, 2) decreasing Ca2+ release through ryanodine-receptor channels, and 3) inhibiting reuptake.

Thapsigargin stimulates Ca2+ entry in vascular smooth muscle cells: nicardipine-sensitive and -insensitive pathways

1992 ◽  
Vol 262 (5) ◽  
pp. C1258-C1265 ◽  
Author(s):  
Y. T. Xuan ◽  
O. L. Wang ◽  
A. R. Whorton
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Inositol Polyphosphate ◽  
Inositol Polyphosphates ◽  
Permeabilized Cells ◽  
Inositol 1,4,5 Trisphosphate

We have investigated the role of the sarcoplasmic reticulum Ca2+ pool in regulating Ca2+ entry in vascular smooth muscle cells using a receptor-independent means of mobilizing the intracellular Ca2+ pool. Thapsigargin (TG) has been shown to inhibit the endoplasmic reticulum Ca(2+)-ATPase, mobilize intracellular Ca2+, and activate Ca2+ entry in nonmuscle tissues. When smooth muscle cells were treated with 0.2 microM TG, cytosolic Ca2+ concentrations rose gradually over 8 min to a peak value of 365 +/- 18 nM. Cytosolic Ca2+ remained elevated for at least 20 min and was supported by continued entry of extracellular Ca2+. TG also stimulated entry of Mn2+ and 45Ca2+ from outside the cell. Importantly, TG-induced Ca2+ entry and Mn2+ entry were found to occur through mechanisms that were independent of L-type Ca2+ channel activation because influx was not inhibited by concentrations of nicardipine that were found to block either endothelin- or 100 mM extracellular K(+)-induced cation influx. The mechanism through which TG activates cation entry appears to involve mobilization of the inositol 1,4,5-trisphosphate-responsive intracellular Ca2+ pool. In permeabilized cells, TG prevented ATP-stimulated Ca2+ uptake into the sarcoplasmic reticulum and slowly released sequestered Ca2+. The Ca2+ pool involved was responsive to inositol 1,4,5-trisphosphate. However, TG did not initiate the formation of inositol polyphosphates. Thus TG mobilizes the sarcoplasmic reticulum Ca2+ pool and activates Ca2+ entry through a nicardipine-insensitive Ca2+ channel in vascular smooth muscle. The mechanism is independent of inositol polyphosphate formation.

scholarly journals Quantitative comparison of intracellular concentration and volume of Clara cell 10 KD protein in rat bronchi and bronchioles based on laser scanning confocal microscopy.

1993 ◽  
Vol 41 (8) ◽  
pp. 1171-1183 ◽  
Author(s):  
D E Dodge ◽  
R B Rucker ◽  
G Singh ◽  
C G Plopper
Keyword(s):  
Endoplasmic Reticulum ◽  
Confocal Microscopy ◽  
Cell Volume ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Clara Cell ◽  
Unit Surface Area ◽  
Ciliated Epithelium ◽  
Scanning Confocal Microscopy ◽  
Subcellular Compartments

We used an antibody to rat Clara cell 10 KD secretory protein (CC10) to compare the abundance of CC10 in non-ciliated epithelium of bronchioles and bronchi by immunohistochemistry and laser scanning confocal microscopy (LSCM) in the reflectance mode. Three zones of reflectance intensity (high, medium, low), directly related to CC10 content, were used to distinguish subcellular compartments of differing densities. Two major compartments contained CC10: granules and endoplasmic reticulum. Bronchiolar cell granules had relatively even reflectance, a high CC10 concentration (0.92 mg/ml), and occupied 7.5% of the cell volume. Bronchial cell granules were bi-zonal, lower in CC10 concentration (0.83 mg/ml), and occupied less cell volume (2.3%). Low-reflecting endoplasmic reticulum occupied a greater cell volume in bronchioles than in bronchi (32.8% and 22.1%, respectively). An intermediate-density zone consisted of endoplasmic reticulum close to granules. CC10 abundance, expressed as ng stored per unit surface area of epithelial basal lamina, was more than three times greater in bronchioles than in proximal bronchi (1.50 ng/mm2 vs 0.42 ng/mm2). These data demonstrate that the process of CC10 secretion differs markedly in non-ciliated epithelium of bronchi and bronchioles. Identifiable mucous-goblet cells did not contain CC10. The LSCM provides a method for quantifying intracellular CC10 pools in intact lung cells and has the potential for quantifying other proteins in subcellular compartments.

scholarly journals Recombinant prolylcarboxypeptidase activates plasma prekallikrein

Blood ◽  
2004 ◽  
Vol 103 (12) ◽  
pp. 4554-4561 ◽  
Author(s):  
Zia Shariat-Madar ◽  
Fakhri Mahdi ◽  
Alvin H. Schmaier
Keyword(s):  
Angiotensin Ii ◽  
Laser Scanning ◽  
Ethylenediaminetetraacetic Acid ◽  
Umbilical Vein ◽  
Neutralizing Antibody ◽  
Antigen Expression ◽  
Laser Scanning Confocal Microscopy ◽  
Permeabilized Cells ◽  
Scanning Confocal Microscopy ◽  
Factor Xiia

Abstract The serine protease prolylcarboxypeptidase (PRCP), isolated from human umbilical vein endothelial cells (HUVECs), is a plasma prekallikrein (PK) activator. PRCP cDNA was cloned in pMT/BIP/V5-HIS-C, transfected into Schneider insect (S2) cells, and purified from serum-free media. Full-length recombinant PRCP (rPRCP) activates PK when bound to high-molecular-weight kininogen (HK). Recombinant PRCP is inhibited by leupeptin, angiotensin II, bradykinin, anti-PRCP, diisopropyl-fluorophosphonate (DFP), phenylmethylsulfonyl fluoride (PMSF), and Z-Pro-Proaldehyde-dimethyl acetate, but not by 1 mM EDTA (ethylenediaminetetraacetic acid), bradykinin 1-5, or angiotensin 1-7. Corn trypsin inhibitor binds to prekallikrein to prevent rPRCP activation, but it does not directly inhibit the active site of either enzyme. Unlike factor XIIa, the ability of rPRCP to activate PK is blocked by angiotensin II, not by neutralizing antibody to factor XIIa. PRCP antigen is detected on HUVEC membranes using flow cytometry and laser scanning confocal microscopy. PRCP antigen does not colocalize with LAMP1 on nonpermeabilized HUVECs, but it partially colocalizes in permeabilized cells. PRCP colocalizes with all the HK receptors, gC1qR, uPAR, and cytokeratin 1 antigen, on nonpermeabilized HUVECs. PRCP activity and antigen expression on cultured HUVECs are blocked by a morpholino antisense oligonucleotide. These investigations indicate that rPRCP is functionally identical to isolated HUVEC PRCP and is a major HUVEC membrane-expressed, PK-activating enzyme detected in the intravascular compartment. (Blood. 2004;103:4554-4561)

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