scholarly journals Agonist-induced calcium regulation in freshly isolated renal microvascular smooth muscle cells.

1997 ◽  
Vol 8 (4) ◽  
pp. 569-579
Author(s):  
E W Inscho ◽  
M J Mason ◽  
A C Schroeder ◽  
P C Deichmann ◽  
K D Stiegler ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Single Cells ◽  
Control Level ◽  
Muscle Cells ◽  
Detectable Effect ◽  
Sprague Dawley ◽  
Sprague Dawley Rats ◽  
Transduction Mechanisms ◽  
K Concentration

The studies presented here were performed to determine the effect of agonist stimulation on the cytosolic free Ca2+ concentration ([Ca2+]i) in single smooth muscle cells, freshly isolated from afferent arterioles and interlobular arteries averaging between 10 to 40 microns in diameter. Microvessels were obtained from male Sprague-Dawley rats using an iron oxide collection technique followed by collagenase digestion. Freshly isolated microvascular smooth muscle cells (MVSMC) were loaded with fura 2 and studied using fluorescence photometry techniques. The resting [Ca2+]i averaged 67 +/- 3 nM (N = 82 cells). Increasing the extracellular K+ concentration significantly increased [Ca2+]i dose-dependently (P < 0.05). Involvement of extracellular Ca2+ in the response to KCl-induced depolarization was also evaluated. Resting [Ca2+]i increased approximately 132% from 40 +/- 5 nM to 93 +/- 26 nM in response to 90 mM extracellular KCl. This change was abolished in nominally Ca(2+)-free conditions and markedly attenuated by diltiazem. Inhibition of K+ channels with charybdotoxin or tetraethylammonium chloride produced a modest transient increase in [Ca2+]i during the response to 30 mM K+ and had no detectable effect on responses to 90 mM K+. Studies were also performed to establish whether freshly isolated renal MVSMC exhibit appropriate responses to receptor-dependent physiological agonists. Angiotensin II (100 nM) increased cell Ca2+ from 97 +/- 10 nM to 265 +/- 47 nM (N = 12 cells). Similarly, 100 microM ATP increased MVSMC [Ca2+]i from a control level of 71 +/- 14 nM to 251 +/- 47 nM (N = 11 cells). Norepinephrine administration caused [Ca2+]i to increase from 63 +/- 4 nM to 212 +/- 47 nM (N = six cells), and vasopressin increased [Ca2+]i from 86 +/- 10 nM to 352 +/- 79 nM (N = five cells). These data demonstrate that receptor-dependent and -independent vasoconstrictor agonists increase [Ca2+]i in MVSMC, freshly isolated from rat preglomerular vessels. Furthermore, the ability to measure [Ca2+]i in responses to physiological stimuli in these single cells permits investigation of signal transduction mechanisms involved in regulating renal microvascular resistance.

scholarly journals Yixintongmai Inhibits Proliferation, Migration and Promotes Apoptosis of Vascular Smooth Muscle Cells Cultured with High Glucose

2020 ◽  
Author(s):  
JingJing Guo ◽  
Di Zhao ◽  
Pingshuan Dong
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
High Glucose ◽  
Muscle Cells ◽  
Cell Counting ◽  
Ros Generation ◽  
Sprague Dawley ◽  
Sprague Dawley Rats

Abstract Background: This study was designed to evaluate the effects of yixintongmai on proliferation, migration, and apoptosis of vascular smooth muscle cells (VSMCs) cultured with high glucose. Methods: VSMCs of the thoracic aorta from 5–8 weeks male Sprague-Dawley rats were cultured with normal (4.5 mM) or high (25 mM) glucose, respectively. The effects of yixintongmai on proliferation, migration, and apoptosis of VSMCs cultured with high glucose were evaluated. The concentration of yixintongmai powder at 360 μg/ml was chosen for this study according to pre-experimental results. Results: Yixintongmai inhibited the proliferation of VSMCs (CCK-8 assay: 0.75 ± 0.04 versus 0.98 ± 0.09 OD, P<0.001; cell counting: 37533 ± 1861 versus 56009 ± 3779 cells/well, P<0.001) and the expression of PCNA (0.74 ± 0.08 folds, P<0.001) as compared with high glucose. Yixintongmai inhibited the migration of VSMCs (transwell assay: 146 ± 16 versus 265 ± 62 cells; P<0.001; scratch wound assay: 2.69 ± 0.22 folds, P<0.001) and the expression of MMP-9 (0.87 ± 0.03 folds, P<0.001) as compared with high glucose. Yixintongmai promoted the apoptosis of VSMCs (0.36 ± 0.12 folds, P<0.001) and inhibited the expression of bcl-2 (0.83 ± 0.07 folds, P<0.01) as compared with high glucose. Yixintongmai inhibited ROS generation (0.58 ± 0.01 folds, P<0.001) and the expression of NF-κB (0.71 ± 0.07 folds, P<0.001) of VSMCs as compared with high glucose. Conclusions: Yixintongmai inhibits the proliferation, migration and promotes the apoptosis of VSMCs cultured with high glucose, which suggests the potential anti-atherosclerotic effects of this traditional Chinese medicine.

Growth characteristics of pulmonary artery smooth muscle cells from fawn-hooded rats

1995 ◽  
Vol 268 (3) ◽  
pp. L465-L470 ◽  
Author(s):  
K. Janakidevi ◽  
C. Tiruppathi ◽  
P. J. Del Vecchio ◽  
J. M. Pinheiro ◽  
A. B. Malik
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Specific Binding ◽  
Pulmonary Arteries ◽  
Muscle Cells ◽  
Serum Concentrations ◽  
Egf Receptors ◽  
Sprague Dawley ◽  
Sprague Dawley Rats ◽  
Epidermal Growth

We compared the proliferative rates of vascular smooth muscle cells (VSMC) from pulmonary arteries of pulmonary hypertensive fawn-hooded rats (FHR) with VSMC from normotensive Sprague-Dawley rats (SDR). VSMC from FHR grew at increased rates and reached higher densities at all serum concentrations studied (5-20%) than the VSMC from SDR. The VSMC from FHR also responded to epidermal growth factor (EGF) at low serum concentrations, as evidenced by significantly greater DNA synthetic rates, than the control VSMC. The increased growth in these cells could be due to increased number and/or affinity of EGF receptors because of the higher specific binding of 125I-EGF to the VSMC from FHR. The VSMC from FHR and SDR were equally sensitive to the antiproliferative effects of heparin, suggesting that the heparin-sensitive pathways are not altered in the VSMC from FHR. These results suggest that the development of pulmonary hypertension in FHR may be related to the higher proliferative capacity of the pulmonary VSMC, which may be coupled to increased activity of the EGF receptors on these cells.

Metabolic modulation of hexokinase association with mitochondria in living smooth muscle cells

1996 ◽  
Vol 270 (2) ◽  
pp. C488-C499 ◽  
Author(s):  
R. M. Lynch ◽  
W. Carrington ◽  
K. E. Fogarty ◽  
F. S. Fay
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Energy State ◽  
Single Cells ◽  
Inverse Correlation ◽  
Muscle Cells ◽  
Cell Types ◽  
Differential Sensitivity ◽  
Similar Distribution ◽  
Apparent Difference

Hexokinase isoform I binds to mitochondria of many cell types. It has been hypothesized that this association is regulated by changes in the concentrations of specific cellular metabolites. To study the distribution of hexokinase in living cells, fluorophore-labeled functional hexokinase I was prepared. After microinjection into A7r5 smooth muscle cells, hexokinase localized to distinct structures identified as mitochondria. The endogenous hexokinase demonstrated a similar distribution with the use of immunocytochemistry. 2-Deoxyglucose elicited an increase in glucose 6-phosphate (G-6-P) and a decrease in ATP levels and diminished hexokinase binding to mitochondria in single cells. 3-O-methylglucose elicited slowly developing decreases in all three parameters. In contrast, cyanide elicited a rapid decrease in both ATP and hexokinase binding. Analyses of changes in metabolite levels and hexokinase binding indicate a positive correlation between binding and cell energy state as monitored by ATP. On the other hand, only in the presence of 2-deoxyglucose was the predicted inverse correlation between binding and G-6-P observed. Unlike the relatively large changes in distribution observed with the fluorescent-injected hexokinase, cyanide caused only a small decrease in the localization of endogenous hexokinase with mitochondria. These findings suggest that changes in the concentrations of specific metabolites can alter the binding of hexokinase I to specific sites on mitochondria. Moreover, the apparent difference in sensitivity of injected and endogenous hexokinase to changes in metabolites may reflect the presence of at least two classes of binding mechanisms for hexokinase, with differential sensitivity to metabolites.

Role of HERG-like K+ currents in opossum esophageal circular smooth muscle

1999 ◽  
Vol 277 (6) ◽  
pp. C1284-C1290 ◽  
Author(s):  
Hamid I. Akbarali ◽  
Hemant Thatte ◽  
Xue Dao He ◽  
Wayne R. Giles ◽  
Raj K. Goyal
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Smooth Muscle Cells ◽  
Single Cells ◽  
Circular Muscle ◽  
Muscle Cells ◽  
Resting Membrane Potential ◽  
Channel Blockers ◽  
Circular Smooth Muscle ◽  
Inward Currents

An inwardly rectifying K+ conductance closely resembling the human ether-a-go-go-related gene (HERG) current was identified in single smooth muscle cells of opossum esophageal circular muscle. When cells were voltage clamped at 0 mV, in isotonic K+ solution (140 mM), step hyperpolarizations to −120 mV in 10-mV increments resulted in large inward currents that activated rapidly and then declined slowly (inactivated) during the test pulse in a time- and voltage- dependent fashion. The HERG K+ channel blockers E-4031 (1 μM), cisapride (1 μM), and La3+ (100 μM) strongly inhibited these currents as did millimolar concentrations of Ba2+. Immunoflourescence staining with anti-HERG antibody in single cells resulted in punctate staining at the sarcolemma. At membrane potentials near the resting membrane potential (−50 to −70 mV), this K+ conductance did not inactivate completely. In conventional microelectrode recordings, both E-4031 and cisapride depolarized tissue strips by 10 mV and also induced phasic contractions. In combination, these results provide direct experimental evidence for expression of HERG-like K+ currents in gastrointestinal smooth muscle cells and suggest that HERG plays an important role in modulating the resting membrane potential.

Isolation of smooth muscle cells from swine carotid artery by digestion with papain

1986 ◽  
Vol 251 (3) ◽  
pp. C474-C481 ◽  
Author(s):  
S. P. Driska ◽  
R. Porter
Keyword(s):  
Smooth Muscle ◽  
Carotid Artery ◽  
Smooth Muscle Cells ◽  
Single Cells ◽  
Calcium Ionophore ◽  
Muscle Cells ◽  
Ionophore A23187 ◽  
Free Solution ◽  
Isolated Cells ◽  
Viable Cells

A new method is described for the preparation of viable, elongated smooth muscle cells from the swine carotid artery. Cells were prepared by papain digestion of pressurized arteries in calcium-free solution. After digestion, the arteries were everted, and fine strips were teased from the intimal surface of the media in calcium-free solution, releasing single cells. Viability was assessed by exclusion of trypan blue and by appearance under phase-contrast microscopy. By these criteria, approximately 20% of the isolated cells were viable. The most distinguishing and unexpected characteristic of these cells was their length. Mean length of the relaxed viable cells was 240.4 +/- 47.4 microns (SD, n = 76), which is much longer than previously reported for arterial smooth muscle cells. Calcium (1.6 mM) caused most of the viable cells to contract slightly, and the mean cell length in calcium was 194.4 +/- 57.7 microns. Cells in 1.6 mM calcium contracted substantially in response to 10 microM histamine or the calcium ionophore A23187 (10 microM), demonstrating that histamine receptors and the contractile apparatus were still functional.

Trimethylamine But Not Trimethylamine Oxide Increases With Age in Rat Plasma and Affects Smooth Muscle Cells Viability

2019 ◽  
Vol 75 (7) ◽  
pp. 1276-1283 ◽  
Author(s):  
Kinga Jaworska ◽  
Marek Konop ◽  
Tomasz Hutsch ◽  
Karol Perlejewski ◽  
Marek Radkowski ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Cardiovascular Risk ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Biological Effects ◽  
Muscle Cells ◽  
Gut Bacteria ◽  
Sprague Dawley ◽  
Trimethylamine Oxide

Abstract It has been suggested that trimethylamine oxide (TMAO), a liver oxygenation product of gut bacteria-produced trimethylamine (TMA), is a marker of cardiovascular risk. However, mechanisms of the increase and biological effects of TMAO are obscure. Furthermore, the potential role of TMAO precursor, that is TMA, has not been investigated. We evaluated the effect of age, a cardiovascular risk factor, on plasma levels of TMA and TMAO, gut bacteria composition, gut-to-blood penetration of TMA, histological and hemodynamic parameters in 3-month-old and 18-month-old, male, Sprague–Dawley and Wistar–Kyoto rats. Cytotoxicity of TMA and TMAO was studied in human vascular smooth muscle cells. Older rats showed significantly different gut bacteria composition, a significantly higher gut-to-blood TMA penetration, and morphological and hemodynamic alterations in intestines. In vitro, TMA at concentration of 500 µmol/L (2-fold higher than in portal blood) decreased human vascular smooth muscle cells viability. In contrast, TMAO at 1,000-fold higher concentration than physiological one had no effect on human vascular smooth muscle cells viability. In conclusion, older rats show higher plasma level of TMA due to a “leaky gut”. TMA but not TMAO affects human vascular smooth muscle cells viability. We propose that TMA but not TMAO may be a marker and mediator of cardiovascular risk.

Density-dependent hyperpolarization in cultured aortic smooth muscle cells

1989 ◽  
Vol 256 (3) ◽  
pp. C644-C651 ◽  
Author(s):  
M. G. Blennerhassett ◽  
M. S. Kannan ◽  
R. E. Garfield
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Continuous Process ◽  
Primary Cultures ◽  
Muscle Cells ◽  
Sprague Dawley ◽  
Aortic Smooth Muscle Cells ◽  
Aortic Smooth Muscle ◽  
Wistar Kyoto

The membrane potential (Em) of cultured aortic smooth muscle cells from Sprague-Dawley (SD), Wistar-Kyoto (WKY), and spontaneously hypertensive (SHR) rats was measured in proliferating primary cultures. Em of SD cells in high-density cultures was -51 to -58 mV, whereas that of low-density cultures (1-2 days) was -30 mV. This difference was due to a continuous process of hyperpolarization during proliferation in culture. Em of WKY and SHR hyperpolarized similarly, from -12 to -42 and -38 mV, respectively. Hyperpolarization of Em of SD, WKY, and SHR cells was related to cell density rather than time in culture. Em may be a sensitive and significant indicator of the changes in the differentiated state expressed by proliferating smooth muscle in vitro.

scholarly journals Dedifferentiation, redifferentiation and bundle formation of smooth muscle cells in tissue culture: the influence of cell number and nerve fibres

Development ◽  
1974 ◽  
Vol 32 (2) ◽  
pp. 297-323
Author(s):  
Julie H. Chamley ◽  
Gordon R. Campbell ◽  
Geoffrey Burnstock
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vas Deferens ◽  
Close Association ◽  
Single Cells ◽  
Muscle Cells ◽  
Enzyme Treatment ◽  
Nerve Fibres ◽  
Isolated Cells

Smooth muscle from newborn guinea-pig vas deferens was enzymically dispersed into single cells or small clumps and grown in culture in the presence or absence of sympathetic ganglion explants. Most single smooth muscle cells gradually lost their typical ultrastructural features and contractile properties during the first few days in culture. At 7 days of culture these dedifferentiated smooth muscle cells underwent extensive proliferation. If sufficient cells were present in the culture inoculate, a continuous monolayer formed at about 9 days of culture and redifferentiation of smooth muscle began. At 11–12 days of culture the cells reaggregated into clumps, began to contract spontaneously, and formed into well-organized muscle bundles in two layers at right angles, resembling the muscle layer organization of the in vivo vas deferens. In cultures where a continuous monolayer was not formed at 9 days, isolated cells did not redifferentiate. The process of dedifferentiation and proliferation was delayed in those smooth muscle cells which had sympathetic nerve fibres in close association. Clumps of vas deferens tissue which were not fully dispersed by the enzyme treatment did not dedifferentiate with time in culture but muscle bundles were disrupted and asynchronous contractions resulted. After 8–12 days of culture the muscle bundles reformed and foci of synchronous contractions developed. Nerve fibres appeared to accelerate bundle and nexus formation in this situation, with synchronous contractions resuming at 3–5 days. The relation of these findings to the process of wound healing in smooth muscle tissues in vivo is discussed.

scholarly journals SIRT1 Inhibition Impairs Ca2+ Buffering in Coronary Smooth Muscle Cells of Ossabaw Miniature Swine

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
John Reed ◽  
Aish Thamba ◽  
John Strobel ◽  
James Byrd ◽  
Mouhamad Alloosh ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Coronary Artery ◽  
Smooth Muscle Cells ◽  
Single Cells ◽  
Muscle Cells ◽  
Atherogenic Diet ◽  
Miniature Swine ◽  
Rapid Phase ◽  
Coronary Smooth Muscle ◽  
Normal Chow

Background: SIRT1 is a deacetylase that has diverse roles in intracellular Ca2+ signaling, metabolism, and cardiovascular disease. SIRT1 increases sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) activity that is essential to buffer the increase in Ca2+ induced by release from the sarcoplasmic reticulum (SR). Our lab has shown that metabolic syndrome (MetS) impairs SERCA activity in coronary smooth muscle cells and causes coronary artery disease in Ossabaw miniature swine. We hypothesized that  SIRT1 inhibition and MetS would impair Ca2+ buffering.   Methods: CRISPR/Cas9 methods delivered a leucine to proline point mutation in SIRT1 (SIRT1L100P) into the Ossabaw swine genome to compare to wild type (WT) and mimic the naturally occurring mutation in humans and decrease SIRT1 activity. Four treatment groups of juvenile swine were based on genotype and diet: WT Lean, SIRT1 Lean, WT MetS, and SIRT1 MetS. Lean swine were fed normal chow and MetS were fed a hypercaloric, atherogenic diet for 7 months. The left anterior descending coronary artery was harvested and enzymatically digested to obtain cells. Fluorescence microscopy measured the Ca2+ indicator fura-2 in single cells. The cells were exposed to 5 mM caffeine to maximally release stores of Ca2+ from the SR. Ca2+ buffering capacity of each cell was analyzed after the caffeine-induced peak increase to assess Ca2+ efflux and SERCA activity.   Results: MetS was confirmed by increased body weight, impaired glucose tolerance, hyperinsulinemia, and hypercholesterolemia. Coronary atherosclerosis was shown by angiography, intravascular ultrasound, and gross imaging. The rapid phase of Ca2+ buffering due to Ca2+ efflux was not affected by SIRT1 mutation or MetS. The slower phase of Ca2+ buffering due to SERCA activity was impaired only by SIRT1 mutation (p<0.0005), not by MetS.   Conclusion:  SIRT1 mutation alone inhibited SERCA buffering of Ca2+ in coronary smooth muscle. (Support: NIH T35HL110854, DK120240, DK09751.) 

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