Actin filament disruption inhibits L-type Ca2+ channel current in cultured vascular smooth muscle cells

To clarify interactions between the cytoskeleton and activity of L-type Ca2+ (CaL) channels in vascular smooth muscle (VSM) cells, we investigated the effect of disruption of actin filaments and microtubules on the L-type Ca2+ current [ I Ba(L)] of cultured VSM cells (A7r5 cell line) using whole cell voltage clamp. The cells were exposed to each disrupter for 1 h and then examined electrophysiologically and morphologically. Results of immunostaining using anti-α-actin and anti-α-tubulin antibodies showed that colchicine disrupted both actin filaments and microtubules, cytochalasin D disrupted only actin filaments, and nocodazole disrupted only microtubules. I Ba(L) was greatly reduced in cells that were exposed to colchicine or cytochalasin D but not to nocodazole. Colchicine even inhibited I Ba(L) by about 40% when the actin filaments were stabilized by phalloidin or when the cells were treated with phalloidin plus taxol to stabilize both cytoskeletal components. These results suggest that colchicine must also cause some inhibition of I Ba(L) due to another unknown mechanism, e.g., a direct block of CaLchannels. In summary, actin filament disruption of VSM cells inhibits CaL channel activity, whereas disrupting the microtubules does not.

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Actin filaments regulate epithelial Na+ channel activity

AJP Cell Physiology ◽

10.1152/ajpcell.1991.261.5.c882 ◽

1991 ◽

Vol 261 (5) ◽

pp. C882-C888 ◽

Cited By ~ 186

Author(s):

H. F. Cantiello ◽

J. L. Stow ◽

A. G. Prat ◽

D. A. Ausiello

Keyword(s):

Actin Filament ◽

Actin Filaments ◽

Functional Role ◽

Cytochalasin D ◽

Actin Binding ◽

Na Channel ◽

Na Channels ◽

Channel Activity ◽

Cortical Actin ◽

Excised Patches

The functional role of the cytoskeleton in the control of ion channel activity is unknown. In the present study, immunocolocalization of Na+ channels with specific antibodies and fluorescein isothiocyanate-phalloidin to stain the cortical cytoskeleton indicates that actin is always present in close proximity to apical Na+ channels in A6 cells. The patch-clamp technique was used to assess the effect of cortical actin networks on apical Na+ channels in these A6 epithelial cells. The actin filament disrupter, cytochalasin D (5 micrograms/ml), induced Na+ channel activity in cell-attached patches within 5 min of addition. Cytochalasin D also induced and/or increased Na+ channel activity in 90% of excised patches tested within 2 min. Addition of short actin filaments (greater than 5 microM) to excised patches also induced channel activity. This effect was enhanced by addition of ATP and/or cytochalasin D. The effect of actin on Na+ channel activity was reversed by addition of the G actin-binding protein DNase I or completely prevented by treatment of the excised patches with this enzyme. Addition of the actin-binding protein, filamin, reversibly inhibited both spontaneous and actin-induced Na+ channels. Thus actin filament networks, achieved by either depolymerizing endogenous actin filaments by treatment with cytochalasin D, the addition of exogenous short actin filaments plus ATP, or actin plus cytochalasin D, regulate apical Na+ channel activity. This conclusion was supported by the observation that the addition of short actin filaments in the form of actin-gelsolin complexes in molar ratios less than 8:1 was also effective in activating Na+ channels. We have thus demonstrated a functional role for the cortical actin network in the regulation of epithelial Na+ channels that may complement a structural role for membrane protein targetting and assembly.

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Involvement of actin filaments and integrins in the binding step in collagen phagocytosis by human fibroblasts

Journal of Cell Science ◽

10.1242/jcs.114.1.119 ◽

2001 ◽

Vol 114 (1) ◽

pp. 119-129 ◽

Cited By ~ 2

Author(s):

G. Segal ◽

W. Lee ◽

P.D. Arora ◽

M. McKee ◽

G. Downey ◽

...

Keyword(s):

Actin Filament ◽

Actin Filaments ◽

Human Fibroblasts ◽

Cytochalasin D ◽

Rhodamine Phalloidin ◽

Collagen Receptors ◽

Swinholide A ◽

Beta 1 Integrin ◽

Collagen Phagocytosis ◽

Collagen Receptor

In physiological conditions, collagen degradation by fibroblasts occurs primarily via phagocytosis, an intracellular pathway that is thought to require collagen receptors and actin assembly for fibril internalization and degradation. Currently it is unclear which specific steps of collagen phagocytosis in fibroblasts involve actin filament assembly. As studies of phagocytosis in fibroblasts are complicated by the relatively slow rate of particle internalization compared to professional phagocytes, we have examined the role of collagen receptors and actin only in the initial collagen binding step. Prior to the binding of collagen-coated fluorescent beads by human gingival fibroblasts, a cell type that is avidly phagocytic in vitro, cells were treated with cytochalasin D (actin filament barbed-end capping) or swinholide A (actin dimer sequestering and severing) or latrunculin B (actin monomer sequestering). Bead binding and immunostaining of (alpha)(2)(beta)(1) and (alpha)(3)(beta)(1) integrin collagen receptors were measured by flow cytometry. After 1–3 hours of coincubation with beads, cytochalasin D or swinholide A eliminated actin filaments stained by rhodamine-phalloidin and inhibited collagen bead binding (reductions of 25% and 50%, respectively), possibly because of cell rounding and restricted interactions with beads. In contrast, latrunculin enhanced binding dose-dependently over controls (twofold at 1 microM) and induced the formation of brightly staining aggregates of actin and the retention of long cytoplasmic extensions. Latrunculin also reduced surface (beta)(1), (alpha)(2) and (alpha)(3) integrin staining up to 40% in bead-free and bead-loaded cells, indicating that latrunculin enhanced collagen receptor internalization. As determined by fluorescence recovery after photobleaching, latrunculin increased the mobility of surface-bound (beta)(1) integrin. The stimulatory effect of latrunculin on collagen bead binding was reduced to control levels by treatment with a (beta)(1) integrin inactivating antibody while a (beta)(1) integrin blocking antibody abrogated both bead binding and the latrunculin-induced stimulation. Immunoblotting of bead-associated proteins showed that latrunculin completely eliminated binding of (beta)-actin to collagen beads but did not affect (beta)(1) integrin binding. These data indicate that latrunculin-induced sequestration of actin monomers facilitates the disengagement of actin from (beta)(1) integrin receptors, increases collagen bead binding and enhances collagen receptor mobility. We suggest that these alterations increase the probability of adhesive bead-to-cell interactions.

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Bundling of actin filaments by alpha-actinin depends on its molecular length.

The Journal of Cell Biology ◽

10.1083/jcb.110.6.2013 ◽

1990 ◽

Vol 110 (6) ◽

pp. 2013-2024 ◽

Cited By ~ 169

Author(s):

R K Meyer ◽

U Aebi

Keyword(s):

Smooth Muscle ◽

Actin Filament ◽

Actin Filaments ◽

Actin Binding ◽

Molar Ratio ◽

Cross Linking ◽

Actin Bundle ◽

Actin Bundles ◽

Chicken Gizzard ◽

Molecular Length

Cross-linking of actin filaments (F-actin) into bundles and networks was investigated with three different isoforms of the dumbbell-shaped alpha-actinin homodimer under identical reaction conditions. These were isolated from chicken gizzard smooth muscle, Acanthamoeba, and Dictyostelium, respectively. Examination in the electron microscope revealed that each isoform was able to cross-link F-actin into networks. In addition, F-actin bundles were obtained with chicken gizzard and Acanthamoeba alpha-actinin, but not Dictyostelium alpha-actinin under conditions where actin by itself polymerized into disperse filaments. This F-actin bundle formation critically depended on the proper molar ratio of alpha-actinin to actin, and hence F-actin bundles immediately disappeared when free alpha-actinin was withdrawn from the surrounding medium. The apparent dissociation constants (Kds) at half-saturation of the actin binding sites were 0.4 microM at 22 degrees C and 1.2 microM at 37 degrees C for chicken gizzard, and 2.7 microM at 22 degrees C for both Acanthamoeba and Dictyostelium alpha-actinin. Chicken gizzard and Dictyostelium alpha-actinin predominantly cross-linked actin filaments in an antiparallel fashion, whereas Acanthamoeba alpha-actinin cross-linked actin filaments preferentially in a parallel fashion. The average molecular length of free alpha-actinin was 37 nm for glycerol-sprayed/rotary metal-shadowed and 35 nm for negatively stained chicken gizzard; 46 and 44 nm, respectively, for Acanthamoeba; and 34 and 31 nm, respectively, for Dictyostelium alpha-actinin. In negatively stained preparations we also evaluated the average molecular length of alpha-actinin when bound to actin filaments: 36 nm for chicken gizzard and 35 nm for Acanthamoeba alpha-actinin, a molecular length roughly coinciding with the crossover repeat of the two-stranded F-actin helix (i.e., 36 nm), but only 28 nm for Dictyostelium alpha-actinin. Furthermore, the minimal spacing between cross-linking alpha-actinin molecules along actin filaments was close to 36 nm for both smooth muscle and Acanthamoeba alpha-actinin, but only 31 nm for Dictyostelium alpha-actinin. This observation suggests that the molecular length of the alpha-actinin homodimer may determine its spacing along the actin filament, and hence F-actin bundle formation may require "tight" (i.e., one molecule after the other) and "untwisted" (i.e., the long axis of the molecule being parallel to the actin filament axis) packing of alpha-actinin molecules along the actin filaments.

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Sphingolipids as mediators of effects of platelet-derived growth factor in vascular smooth muscle cells

AJP Cell Physiology ◽

10.1152/ajpcell.1993.265.3.c740 ◽

1993 ◽

Vol 265 (3) ◽

pp. C740-C747 ◽

Cited By ~ 45

Author(s):

L. S. Jacobs ◽

M. Kester

Keyword(s):

Smooth Muscle ◽

Growth Factor ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Dna Synthesis ◽

Vascular Smooth Muscle Cells ◽

Platelet Derived Growth Factor ◽

Sphingolipid Metabolism ◽

A7r5 Cell ◽

A7r5 Cells

The role of sphingolipids in mediating the action of platelet-derived growth factor (PDGF) has been investigated in the vascular smooth muscle-derived A7r5 cell line. L-Cycloserine (2 mM), an inhibitor of sphingolipid synthesis, caused time-dependent inhibition of [3H]serine incorporation into [3H]sphingomyelin in A7r5 cells. PDGF-AB (10 ng/ml), PDGF-BB (10 ng/ml), or sphingosine (10 microM) independently stimulated [3H]thymidine incorporation into DNA in A7r5 cells. L-Cycloserine (2 mM) inhibited stimulation of DNA synthesis by both PDGF-AB and PDGF-BB. L-Cycloserine (2 mM, 16 h) did not affect the ability of PDGF or sphingosine to increase intracellular free calcium ([Ca2+]i) in A7r5 cells loaded with the fluorescent indicator fura 2. Measurement of adenine nucleotide levels in A7r5 cell extracts by reverse-phase high-performance liquid chromatography indicated that treatment with L-cycloserine did not adversely affect cellular metabolism. To determine directly whether PDGF activates sphingolipid metabolism, A7r5 cells were labeled with [3H]serine for 48 h and then treated with PDGF-AB (10 ng/ml) for 1 h. Sphingolipids were separated by thin-layer chromatography and quantified by liquid scintillation counting. PDGF-AB stimulated an increase in [3H]sphingosine from 25.5 +/- 3.0 to 37.5 +/- 4.1 counts.min-1 (cpm).micrograms protein-1 and a concomitant decrease in [3H]ceramide from 24.3 +/- 3.2 to 18.5 +/- 2.9 cpm/micrograms protein. These data suggest that the PDGF-stimulated increase in [Ca2+]i is not sufficient for induction of DNA synthesis and that mitogenic effects of PDGF in vascular smooth muscle cells are mediated by sphingolipid metabolism.

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Depletion and filling of intracellular calcium stores in vascular smooth muscle

AJP Cell Physiology ◽

10.1152/ajpcell.1995.268.2.c503 ◽

1995 ◽

Vol 268 (2) ◽

pp. C503-C512 ◽

Cited By ~ 20

Author(s):

L. A. Blatter

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Surface Membrane ◽

Membrane Receptors ◽

Calcium Stores ◽

Fura 2 ◽

Vasoactive Substances ◽

Cytoplasmic Compartment ◽

A7r5 Cell Line ◽

Cellular Compartments

In vascular smooth muscle, binding of vasoactive substances to surface membrane receptors leads to a rise of intracellular cytoplasmic Ca2+ and to contraction. Cytoplasmic free Ca2+ concentration ([Ca2+]i) increases through release of Ca2+ from intracellular stores and Ca2+ entry through surface membrane ion channels. Membrane-permeant and membrane-impermeant forms of fura 2 were used to distinguish changes in intracellularly stored Ca2+ ([Ca2+]s) from changes in [Ca2+]i. The spatiotemporal patterns of the movement of Ca2+ between these two cellular compartments in cultured vascular smooth muscle cells (A7r5 cell line) were visualized with digital imaging fluorescence microscopy. The Ca2+ stores were localized by double staining with a fluorescent organelle-specific dye and the Ca2+ indicator. [Ca2+]s was measured after accumulation of the membrane-permeant form of fura 2 inside the stores and quenching of the fura 2 fluorescence in the cytoplasmic compartment with manganese. Stimulation with vasopressin led to a transient increase of [Ca2+]i and a concomitant decrease of [Ca2+]s. After stimulation with vasopressin, [Ca2+]i returned rapidly to normal resting levels, whereas the recovery of [Ca2+]s occurred on a much slower time scale. The refilling pathway of depleted stores involved Ca2+ entry into the bulk cytoplasmic compartment before uptake into the stores.

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Measurement of Contractile Properties and Intracellular Distribution of Actin Filaments in Freshly-isolated Vascular Smooth Muscle Cells

The proceedings of the JSME annual meeting ◽

10.1299/jsmemecjo.2004.5.0_113 ◽

2004 ◽

Vol 2004.5 (0) ◽

pp. 113-114

Author(s):

Kazuaki NAGAYAMA ◽

Shinichiro YANAGIHARA ◽

Takahiro SHIRONO ◽

Takeo MATSUMOTO

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Actin Filaments ◽

Muscle Cells ◽

Intracellular Distribution ◽

Contractile Properties

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ACTIN FILAMENTS REGULATE ANGIOTENSIN II-INDUCED Ca2+ INFLUX IN VASCULAR SMOOTH MUSCLE CELLS FROM SPONTANEOUS HYPERTENSIVE RATS

Anesthesiology ◽

10.1097/00000542-199809120-00100 ◽

1998 ◽

Vol 89 (Supplement) ◽

pp. 688A

Author(s):

H. Bouillier ◽

E. Samain ◽

J. Marty ◽

M. Safar ◽

G. Dagher

Keyword(s):

Smooth Muscle ◽

Angiotensin Ii ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Actin Filaments ◽

Muscle Cells ◽

Hypertensive Rats

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Calcium channel current of vascular smooth muscle cells: extracellular protons modulate gating and single channel conductance.

The Journal of General Physiology ◽

10.1085/jgp.103.4.665 ◽

1994 ◽

Vol 103 (4) ◽

pp. 665-678 ◽

Cited By ~ 75

Author(s):

U Klöckner ◽

G Isenberg

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Single Channel ◽

Muscle Cells ◽

Surface Charges ◽

Channel Conductance ◽

Channel Current ◽

Voltage Dependent

Modulation of L-type Ca2+ channel current by extracellular pH (pHo) was studied in vascular smooth muscle cells from bovine pial and porcine coronary arteries. Relative to pH 7.4, alkaline pH reversibly increased and acidic pH reduced ICa. The efficacy of pHo in modulating ICa was reduced when the concentration of the charge carrier was elevated ([Ca2+]o or [Ba2+]o varied between 2 and 110 mM). Analysis of whole cell and single Ca2+ channel currents suggested that more acidic pHo values shift the voltage-dependent gating (approximately 15 mV per pH-unit) and reduce the single Ca2+ channel conductance gCa due to screening of negative surface charges. pHo effects on gCa depended on the pipette [Ba2+] ([Ba2+]p), pK*, the pH providing 50% of saturating conductance, increased with [Ba2+]p according to pK* = 2.7-2.log ([Ba2+]p) suggesting that protons and Ba2+ ions complete for a binding site that modulates gCa. The above mechanisms are discussed in respect to their importance for Ca2+ influx and vasotonus.

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