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.

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 Leukotrienes stimulate initiation of DNA synthesis in cultured arterial smooth muscle cells

1987 ◽  
Vol 88 (2) ◽  
pp. 151-159
Author(s):  
L. Palmberg ◽  
H.E. Claesson ◽  
J. Thyberg
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Dna Synthesis ◽  
Calcium Ionophore ◽  
Muscle Cells ◽  
Cyclooxygenase Inhibitors ◽  
Ionophore A23187 ◽  
Maximal Effect ◽  
Arterial Smooth Muscle ◽  
Arterial Smooth Muscle Cells

The effects of leukotrienes on initiation of DNA synthesis in growth-arrested arterial smooth muscle cells cultivated in a defined, serum-free medium were studied. The results showed that LTB4, LTC4 and LTD4 were all stimulatory with a distinct effect already at 0.01 pM and a maximal effect at 10 pM; in contrast, LTE4 lacked effect. 5S,12S-DHETE, an isomer of LTB4, was inactive in itself but blocked the effect of LTB4. Treatment of the cells with indomethacin or acetylsalicylic acid, two cyclooxygenase inhibitors, blocked induction of DNA synthesis by LTB4, indicating that the effect of this compound was mediated by a cyclooxygenase product. Up to 10 pM, the leukotrienes stimulated initiation of DNA synthesis with similar potency to platelet-derived growth factor (PDGF). The maximum labelling index obtained with PDGF was, however, about twice that obtained with the leukotrienes. At suboptimal concentrations of PDGF, the leukotrienes had an additive effect. The prereplicative lag phase was 16–20 h with LTB4, 12–16 h with LTC4, and 8–12 h with PDGF. In metabolic experiments no signs of synthesis of leukotrienes were detected by cells stimulated with the calcium ionophore A23187, arachidonic acid, or LTA4 for 10–30 min, neither was any apparent degradation of LTB4 observed. On the other hand, LTC4 was transformed into LTD4 and LTE4. Taken together, the results indicate that leukotrienes are able to stimulate quiescent arterial smooth muscle cells to enter the cell cycle and to replicate their DNA. In vivo, stimulation of cell growth by leukotrienes could add to other established functions of these substances in tissue repair, inflammation, and atherogenesis.

Production Of PGI2 By Cultured Endothelial Cells And Smooth Muscle Cells

1981 ◽  
Author(s):  
E A Jaffe
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Culture Media ◽  
Calcium Ionophore ◽  
Muscle Cells ◽  
Ionophore A23187 ◽  
Cultured Endothelial Cells ◽  
Camp Levels

Prostacyclin (PGI2) is the main product of the cyclooxygenase pathway in endothelial cells and is a short-lived vasodilator and inhibitor of platelet aggregation. Cultured endothelial cells (EC) make PGI2 from exogenous arachidonic acid (AA) and the unstable endoperoxide PGH2. EC have also been shown to make PGI2 from endogenous AA when stimulated by the calcium ionophore A23187, thrombin, histamine, bradykinin, trypsin, platelet derived growth factor, and uremic plasma. PGI2 synthesis in EC is blocked by aspirin, indomethacin, or 15-HPAA. PGI2 synthesis is partially self-controlled and self-limiting at least in vitro since PGI2 elevates EC cAMP levels and elevated cAMP levels decrease EC PGI2 production. While the proteolytic site of thrombin (and trypsin) is involved in the stimulation of PGI2 production, high affinity binding of thrombin to endothelial cells appears not to be necessary. Alteration of the fatty acid composition of EC by manipulation of the culture media can decrease PGI2 production in response to thrombin or A23187 by 60-751. Cultured aortic smooth muscle cells (SMC) also synthesize PGI2 but at significantly lower levels than EC. SMC PGI2 production is stimulated by PDGF and in some SMC lines by thrombin. SMC grown from atherosclerotic rabbit aortas produce considerably less PGI2 from exogenous AA than do SMC from noimal aortas. Future investigations into this area will further clarify normal biochemical and physiologic pathways and perhaps suggest means by which PGI2 production in EC and SMC may be modulated in order to treat human disease.

Cholecystokinin-induced contraction of dispersed smooth muscle cells

1982 ◽  
Vol 243 (6) ◽  
pp. G497-G504
Author(s):  
S. M. Collins ◽  
J. D. Gardner
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Calcium Ionophore ◽  
Muscle Cells ◽  
Threshold Concentration ◽  
Calcium Ionophore A23187 ◽  
Cell Length ◽  
Ionophore A23187 ◽  
Guinea Pig Stomach ◽  
Percent Decrease

Isolated smooth muscle cells were prepared from the fundus of guinea pig stomach by incubation with collagenase. Incubating the cells with the C-terminal octapeptide of cholecystokinin induced contraction, which was measured by micrometry and expressed as percent decrease in mean cell length. Cholecystokinin-induced contraction was maximal within 30 s and reduced cell length by approximately 37%. The threshold concentration of cholecystokinin was 0.1 pM, and the maximally effective concentration was 0.3 nM. Contraction caused by cholecystokinin could be inhibited by proglumide and by glucagon. Inhibition by proglumide was competitive and resulted in a parallel rightward shift of the cholecystokinin dose-response curve. In contrast, inhibition by glucagon was noncompetitive and resulted in a reduction in the efficacy of cholecystokinin without a change in its potency. Furthermore, proglumide-induced inhibition was specific for cholecystokinin, whereas glucagon-induced inhibition of contraction was nonspecific and reduced the contraction caused by carbamylcholine and the calcium ionophore A23187.

Gap junction-mediated intercellular diffusion of Ca2+ in cultured human corporal smooth muscle cells

1992 ◽  
Vol 263 (2) ◽  
pp. C373-C383 ◽  
Author(s):  
G. J. Christ ◽  
A. P. Moreno ◽  
A. Melman ◽  
D. C. Spray
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Gap Junctions ◽  
Intracellular Calcium ◽  
Gap Junction ◽  
Calcium Ionophore ◽  
Muscle Cells ◽  
Calcium Flux ◽  
Ionophore A23187 ◽  
Fura 2

Ratio imaging using the calcium-sensitive probe fura-2 was employed to study intracellular calcium concentrations and intercellular calcium flux through gap junctions in homogeneous vascular smooth muscle cell cultures derived from the human corpora cavernosa. Microinjection techniques demonstrated that fura-2 free acid was freely diffusible through gap junctions between cultured cells. The resting intracellular calcium level in fura-2-loaded cells was 176.9 +/- 10.5. A robust increase in intracellular calcium was seen in response to both phenylephrine and the calcium ionophore A23187. Microinjection of Ca2+ into individual smooth muscle cells always resulted in significant, although temporally delayed, increases in intracellular calcium levels in adjacent cells; this intercellular calcium flux was reversibly blocked by inhibition of gap junctional communication with 2 mM heptanol. However, although microinjection of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] into individual smooth muscle cells always produced significant increases in intracellular calcium levels in the injected cell, the intercellular spread of Ca2+ in response to Ins(1,4,5)P3 was more variable than for Ca2+ injections. These studies demonstrate that Ca2+, and perhaps Ins(1,4,5)P3 as well, can diffuse between smooth muscle cells through gap junction channels.

Characterization and confocal imaging of calponin in gastrointestinal smooth muscle

1993 ◽  
Vol 265 (5) ◽  
pp. C1371-C1378 ◽  
Author(s):  
M. P. Walsh ◽  
J. D. Carmichael ◽  
G. J. Kargacin
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Smooth Muscle Cells ◽  
Thin Filament ◽  
Muscle Cells ◽  
Biochemical Properties ◽  
Confocal Imaging ◽  
Isolated Cells ◽  
Independent Manner

Calponin isolated from chicken gizzard smooth muscle binds in vitro to actin in a Ca(2+)-independent manner and thereby inhibits the actin-activated Mg(2+)-adenosinetriphosphatase of smooth muscle myosin. This inhibition is relieved when calponin is phosphorylated by protein kinase C or Ca2+/calmodulin-dependent protein kinase II, suggesting that calponin is involved in thin filament-associated regulation of smooth muscle contraction. To further examine this possibility, calponin was isolated from toad stomach smooth muscle, characterized biochemically, and localized in intact isolated cells. Toad stomach calponin had the same basic biochemical properties as calponin from other sources. Confocal immunofluorescence microscopy revealed that calponin in intact smooth muscle cells was localized to long filamentous structures that were colabeled by antibodies to actin or tropomyosin. Preservation of the basic biochemical properties of calponin from species to species suggests that these properties are relevant for its in vivo function. Its colocalization with actin and tropomyosin indicates that calponin is associated with the thin filament in intact smooth muscle 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.

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