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  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    ISSN: 1749-6632
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Natural Sciences in General
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    [s.l.] : Nature Publishing Group
    Nature 288 (1980), S. 585-587 
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] Strips of dog antral circular smooth muscle were prepared as described previously8. In each preparation aequorin9'10, dissolved in 150 mM KC1, 5 mM HEPES, pH 8.0, was pressure-injected through glass micropipettes into 20-60 cells over an approximately 1-mm2 area. Light from the aequorin-injected ...
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  • 3
    ISSN: 1573-7241
    Keywords: calcium ; vascular smooth muscle ; protein kinase C ; vasoconstriction ; calcium indicators
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary An overview is given of the current understanding of the mechanism of contraction of vascular smooth muscle. The regulation of vascular tone by intracellular ionized calcium levels appears to occur primarily through calciumdependent phosphorylation of the myosin light chains by the enzyme myosin light chain kinase. Evidence is presented that additional relatively calcium-independent processes also exist and contribute to the regulation of vascular tone. A scheme is presented whereby vasoconstriction may occur in the absence of any change in cytoplasmic ionized calcium levels. The multiplicity of excitation-contraction coupling pathways in vascular smooth muscle predicts a multitude of rational therapeutic approaches to vascular pathologies.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Pflügers Archiv 395 (1982), S. 75-77 
    ISSN: 1432-2013
    Keywords: Excitation-contraction coupling ; Cell calcium ; Vascular smooth muscle ; Reversibly hyperpermeable cells ; Calcium indicators ; Aequorin ; Phenylephrine ; Angiotensin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The bioluminescent calcium indicator aequor in was successfully loaded into vascular smooth muscle cells ofAmphiuma tridactylum by either microinjection or a new method which makes the cells reversibly hyperpermeable. Both gave similar results; however, the latter method produced larger signals. Vasoconstrictors produced a sustained contraction and a light (calcium) response consisting of two component: a large transient followed by a smaller, sustained response. Electrical stimulation produced a light transient that was much briefer than the contraction. These results suggest that tension can be maintained in smooth muscle in the presence of lower calcium levels than those present during force development.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1432-2013
    Keywords: Excitation-contraction coupling ; Cell calcium ; Mammalian cardiac muscles ; Reversibly hyperpermeable cells ; Calcium indicators ; Aequorin ; Isoproterenol ; Caffeine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The bioluminescent calcium indicator aequorin was successfully loaded into mammalian working myocardium of ferrets by a chemical procedure which makes the cells reversibly hyperpermeable through exposure to Ethylenebis-(oxyethylenenitrilo) tetraacetic acid (EGTA). After undergoing the loading procedure, developed tension at Lmax was 103±26% of the control, which indicates that the muscles regained normal function. The configurations of the aequorin signals (i.e., calcium transients) and their responses to drugs were the same as reported after microinjection of aequorin. The peak of the Ca++ transient determined by the method of fractional luminescence at 3s intervals of stimulation, 2.5mM [Ca++]o, 22.5°C was 1.1μM; this value is similar to that reported for microinjection. These results indicate that the chemical loading procedure is a useful alternative to microinjection for loading aequorin into mammalian working myocardium.
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  • 6
    ISSN: 1432-2013
    Keywords: Quin 2 ; Aequorin ; Vascular Smooth Muscle ; Mammalian Isolated Cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract A comparison between the fluorescent indicator quin 2 and the bioluminescent indicator aequorin was performed in the same smooth muscle cell type. Aequorin was loaded into intact strips and quin was loaded into enzymatically isolated single cells from ferret portal vein. Both indicators gave qualitatively the same calcium profiles when the tissue was challenged with agonists. Quin loading caused a dramatic shift to the right in dose response curves to potassium and phenylephrine. The ED50 values for quin loaded cells were significantly different from those for control cells for both agonists. Intracellular calcium levels at rest were not significantly different with quin and aequorin. Cells stimulated with potassium gave significantly different intracellular calcium values with the two indicators suggesting a change in the stimulated steady state level due to the introduction of an additional calcium buffer (quin2) into the cell.
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  • 7
    ISSN: 1432-2013
    Keywords: Vascular smooth muscle ; Phosphorylation ; Aequorin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The relationship between phosphorylation of the 20-kDa myosin light chain, intracellular calcium levels ([Ca2+]i), and isometric force was studied during prolonged activation of arterial smooth muscle. Aequorin, preloaded into ferret aortic strips, was used as a [Ca2+]i indicator. Two dimensional polyacrylamide gel electrophoresis was used to determine the phosphorylation levels of the 20-kDa myosin light chain (LC20). During the 30-min depolarization of arterial smooth muscle by K+ (21 mM), both LC20 phosphorylation and [Ca2+]i increased significantly at all time points examined as did the steady state stress. A transient rise in LC20 phosphorylation and [Ca2+]i occurred within 30 s, followed by suprabasal levels through the 10-min period during a sustained alpha1-mediated activation by 10−5 M phenylephrine whereas a higher force was developed at a shorter time compared to K+. An active phorbol ester 12-deoxyphorbol 13-isobutyrate 20-acetate (DPBA, 10−6 M) induced a slow contraction of similar magnitude to that induced by K+ without significantly changing either [Ca2+]i or LC20 phosphorylation over a 90-min period. These results demonstrate that the amount of LC20 phosphorylation correlates with the [Ca2+]i in all three types of activation. The initial levels of [Ca2+]i and LC20 phosphorylation correlate with the onset of force development but not the magnitude of steady state stress, suggesting a role for [Ca2+]i and LC20 phosphorylation in regulating the cross bridge cycling rate during tension development. The lack of a detectable increase in [Ca2+]i and LC20 phosphorylation during DPBA activation suggests that sites other than LC20, phosphorylated by protein kinase C, may be involved in regulating smooth muscle contraction.
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  • 8
    ISSN: 1432-2013
    Keywords: Mammalian vascular smooth muscle ; Enzymatically isolated cells ; Vasodilators
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract A modified method for enzymatically isolating mammalian vascular smooth muscle cells has been developed and tested for ferret portal vein smooth muscle. This method produces a high proportion of fully relaxed cells and these cells appear to have normal pharmacological responsiveness. The ED50 values for both alpha stimulation and potassium depolarization are not significantly different in the isolated cells from those obtained from intact strips of ferret portal vein, suggesting that the enzymatic treatment does not destroy receptors or alter the electrical responsiveness of the cells. It was also possible to demonstrate a vasodilatory action of papaverine, nitroprusside and adenosine directly on the isolated cells indicating that the pathways involved are intact in the isolated cells. This method should be of considerable usefulness, particularly in combination with the new fluorescent indicators and cell sorter techniques which require isolated cells.
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  • 9
    ISSN: 1432-2013
    Keywords: Smooth Muscle ; Calcium ; PDGF ; Aequorin ; Angiotensin ; Atherosclerosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We studied stimulus-specific alterations of the excitation-contraction coupling pathway in freshly isolated contractile and subcultured non-contractile vascular smooth muscle cells. Using the calcium indicator aequorin, we detected physiological increases in cytoplasmic free calcium ([Ca2+]i) in subcultured smooth muscle cells subjected to angiotensin or 33 mM potassium depolarization. These increases were qualitatively identical to those previously measured in intact vascular strips. Platelet-derived growth factor (PDGF) induced a slow, sustained [Ca2+]i increase when applied to the subcultured smooth muscle cells at low picomolar concentrations. Freshly isolated, contractile vascular smooth muscle cells, prepared by a novel technique, exhibited a slow shortening of 20% of resting length in response to PDGF. PDGF also markedly potentiated smooth muscle cell shortening in response to an ED50 dose of phenylephrine. This effect was PDGF concentration dependent. The time course of shortening induced by PDGF alone was consistent with the time course of the PDGF-induced [Ca2+]i increase in the cultured smooth muscle cells. These data suggest that agonists which induce [Ca2+]i changes in contractile smooth muscle cells may retain this ability with respect to cultured smooth muscle cells. PDGF, a peptide mitogen for proliferative smooth muscle cells, may also serve to modulate vascular tone by modestly raising [Ca2+]i in contractile smooth muscle cell and, therefore, sensitizing the cells to alpha adrenergic agonists.
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  • 10
    ISSN: 1432-2013
    Keywords: Vascular smooth muscle ; Protein kinase C ; Excitation/contraction coupling
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract An isolation technique was developed for single cells from the ferret aorta, which resulted in the isolation of long (87±27 μm; x±SD, n=62), relaxed, pharmacologically active smooth muscle cells. These cells were attached to microtools, one of which was connected to a force transducer. Force in maximally phenylephrine-stimulated contractions of the intact cells averaged 2.3 ±1.4 μN (n=17). After cell skinning with saponin, the threshold for force development was 0.05 μM [Ca2+], and force reached a maximum of 4.4±1.6 μN (n = 36) at 0.5 μM [Ca2+]. Plots of relative steady-state force vs pCa (−log10[Ca2+]) were fit to the Hill equation, which yielded a pCa at half-maximal force of 6.87 ± 0.30 and a Hill coefficient of 2.3±1.4 (n = 29). When 2.5 μM calmodulin was added to the solutions, the calcium sensitivity of force was significantly increased (P〈0.05) without changing the maximal force (P〉0.05). In a solution of pCa 7, the skinned cells developed 2.5±0.5 μN (n = 5) of force when stimulated with a phorbol ester. The addition of a specific inhibitor (17 kDa) of protein kinase C to the calcium buffers depressed (P〈0.05) the maximally Ca2+ -activated force without a change in the calcium sensitivity of force (P〉0.05). These data strongly suggest that in vascular smooth muscle, protein kinase C may be involved in a physiological, regulatory system for force.
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