Change of Ca2+ requirement for myosin phosphorylation by prostaglandin F2 alpha

1991 ◽  
Vol 261 (2) ◽  
pp. C253-C258 ◽  
Author(s):  
E. Suematsu ◽  
M. Resnick ◽  
K. G. Morgan
Keyword(s):  
Steady State ◽  
Myosin Light Chain ◽  
Light Signal ◽  
Tonic Contraction ◽  
Prostaglandin F2 Alpha ◽  
Force Curves ◽  
State Force ◽  
Mlc Phosphorylation ◽  
Small Spike ◽  
Sustained Increase

The mechanism of contraction of vascular smooth muscle by prostaglandin F2 alpha (PGF2 alpha) was examined by simultaneous measurement of the intracellular Ca2+ concentration [( Ca2+]i), force, and myosin light-chain (MLC) phosphorylation in ferret aorta. In the presence of 2.5 mM extracellular Ca2+, PGF2 alpha (10(-5)M) produced a tonic contraction with a transient spike in [Ca2+]i, followed by a relatively small sustained increase in [Ca2+]i (from a basal level of 2.32 +/- 0.07 x 10(-7) to 2.72 +/- 0.05 x 10(-7) M). In Ca(2+)-free bathing media, PGF2 alpha also produced a tonic contraction with a small spike in [Ca2+]i, indicating a release of Ca2+ from intracellular store sites, followed by no significant increase in [Ca2+]i. Ca(2+)-force curves were constructed by plotting the calibrated steady-state aequorin light signal against the resulting steady-state force. The curve was significantly shifted to the left by PGF2 alpha. PGF2 alpha also shifted the Ca(2+)-phosphorylation curve to the left. These results suggest that PGF2 alpha causes contraction by both elevating [Ca2+]i and decreasing the Ca2+ requirement for MLC phosphorylation. The data are consistent with a mechanism where there is either an increase in activity of MLC kinase or a decrease in phosphatase activity. Additionally, there was a smaller, but statistically significant, effect to increase force at any one phosphorylation level, pointing to the possibility of regulation of contractile force separate from MLC phosphorylation.

Myosin light chain phosphorylation associated with contraction in arterial smooth muscle

1981 ◽  
Vol 240 (5) ◽  
pp. C222-C233 ◽  
Author(s):  
S. P. Driska ◽  
M. O. Aksoy ◽  
R. A. Murphy
Keyword(s):  
Smooth Muscle ◽  
Steady State ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Isometric Force ◽  
Physiological Salt Solution ◽  
Physiological Control ◽  
The Media ◽  
Low Levels ◽  
State Force

The hypothesis that Ca2+ initiates contraction in smooth muscle by activating an endogenous myosin light chain kinase (MLCK) that phosphorylates the 20,000 dalton light chain (LC 20) of myosin was tested in tissues prepared from the media of swine carotid arteries. Unstimulated tissues with low levels of tone exhibited low levels of phosphorylated LC 20. On stimulation with a high-K+ physiological salt solution containing 1.6 mM CaCl2, LC 20 phosphorylation increased to 0.6 mol P/mol LC 20 within 30 s. This increase preceded force development, which required 2-4 min to attain a maximum steady-state value of 3.34 +/- 0.15 (SE) X 10(5) N/m2. These results support the hypothesis, as the stimulus was submaximal for the preparation. However, LC 20 phosphorylation declined significantly from its peak value before steady-state force was attained, reaching near control levels after 10 min of stimulation. The results suggest that Ca2+-stimulated LC 20 phosphorylation is an important physiological control mechanism but that additional factors are involved in the maintenance of tonic isometric force.

Transient myosin phosphorylation at constant Ca2+ during agonist activation of permeabilized arteries

1992 ◽  
Vol 263 (2) ◽  
pp. C540-C544 ◽  
Author(s):  
S. Moreland ◽  
J. Nishimura ◽  
C. van Breemen ◽  
H. Y. Ahn ◽  
R. S. Moreland
Keyword(s):  
Myosin Light Chain ◽  
Tonic Contraction ◽  
Mesenteric Arteries ◽  
Initial Increase ◽  
Alpha Toxin ◽  
Shortening Velocity ◽  
Myosin Phosphorylation ◽  
Temporal Relationships ◽  
Mlc Phosphorylation ◽  
Rabbit Mesenteric Arteries

Norepinephrine (NE) plus guanosine triphosphate (GTP) increases myofilament Ca2+ sensitivity in alpha-toxin-permeabilized smooth muscle. We used alpha-toxin-permeabilized rabbit mesenteric arteries to determine the temporal relationships among force, myosin light chain (MLC) phosphorylation, stiffness, and shortening velocity during contractions in response to Ca2+ alone and to the same [Ca2+] in the presence of NE plus GTP. The addition of NE plus GTP caused a marked increase in the tonic contraction but only transiently elevated the level of MLC phosphorylation over that observed in the presence of Ca2+ alone. NE plus GTP induced similar increases in force and stiffness, but shortening velocity depended solely on the [Ca2+]. A regulated MLC phosphatase could explain the initial increase in force and MLC phosphorylation, but not the maintenance of enhanced force while MLC phosphorylation levels fell to values similar to those in response to Ca2+ alone. Therefore, additional elements must be involved in the maintenance of the receptor and G protein-dependent increase in myofilament Ca2+ sensitivity.

scholarly journals FFAR1 activation attenuates histamine-induced myosin light chain phosphorylation and cortical tension development in human airway smooth muscle cells

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Shengjie Xu ◽  
Anthony Schwab ◽  
Nikhil Karmacharya ◽  
Gaoyuan Cao ◽  
Joanna Woo ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Airway Hyperreactivity ◽  
Human Airway Smooth Muscle ◽  
Tension Development ◽  
Cortical Tension ◽  
Human Airway ◽  
Mlc Phosphorylation

Abstract Background Activation of free fatty acid receptors (FFAR1 and FFAR4) which are G protein-coupled receptors (GPCRs) with established (patho)physiological roles in a variety of obesity-related disorders, induce human airway smooth muscle (HASM) cell proliferation and shortening. We reported amplified agonist-induced cell shortening in HASM cells obtained from obese lung donors. We hypothesized that FFAR1 modulate excitation–contraction (EC) coupling in HASM cells and play a role in obesity-associated airway hyperresponsiveness. Methods In HASM cells pre-treated (30 min) with FFAR1 agonists TAK875 and GW9508, we measured histamine-induced Ca2+ mobilization, myosin light chain (MLC) phosphorylation, and cortical tension development with magnetic twisting cytometry (MTC). Phosphorylation of MLC phosphatase and Akt also were determined in the presence of the FFAR1 agonists or vehicle. In addition, the effects of TAK875 on MLC phosphorylation were measured in HASM cells desensitized to β2AR agonists by overnight salmeterol treatment. The inhibitory effect of TAK875 on MLC phosphorylation was compared between HASM cells from age and sex-matched non-obese and obese human lung donors. The mean measurements were compared using One-Way ANOVA with Dunnett’s test for multiple group comparisons or Student’s t-test two-group comparison. For cortical tension measurements by magnetic twisted cytometry, mixed effect model using SAS V.9.2 was applied. Means were considered significant when p ≤ 0.05. Results Unexpectedly, we found that TAK875, a synthetic FFAR1 agonist, attenuated histamine-induced MLC phosphorylation and cortical tension development in HASM cells. These physiological outcomes were unassociated with changes in histamine-evoked Ca2+ flux, protein kinase B (AKT) activation, or MLC phosphatase inhibition. Of note, TAK875-mediated inhibition of MLC phosphorylation was maintained in β2AR-desensitized HASM cells and across obese and non-obese donor-derived HASM cells. Conclusions Taken together, our findings identified the FFAR1 agonist TAK875 as a novel bronchoprotective agent that warrants further investigation to treat difficult-to-control asthma and/or airway hyperreactivity in obesity.

scholarly journals Blockade of ROCK inhibits migration of human primary keratinocytes and malignant epithelial skin cells by regulating actomyosin contractility

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Srisathya Srinivasan ◽  
Sreya Das ◽  
Vishakha Surve ◽  
Ankita Srivastava ◽  
Sushant Kumar ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Focal Adhesions ◽  
Primary Keratinocytes ◽  
Skin Cells ◽  
Physiological Processes ◽  
Actomyosin Contractility ◽  
Malignant Skin ◽  
And Migration ◽  
Mlc Phosphorylation

AbstractActomyosin contractility, crucial for several physiological processes including migration, is controlled by the phosphorylation of myosin light chain (MLC). Rho-associated protein kinase (ROCK) and Myosin light chain kinase (MLCK) are predominant kinases that phosphorylate MLC. However, the distinct roles of these kinases in regulating actomyosin contractility and their subsequent impact on the migration of healthy and malignant skin cells is poorly understood. We observed that blockade of ROCK in healthy primary keratinocytes (HPKs) and epidermal carcinoma cell line (A-431 cells) resulted in loss of migration, contractility, focal adhesions, stress fibres, and changes in morphology due to reduction in phosphorylated MLC levels. In contrast, blockade of MLCK reduced migration, contractile dynamics, focal adhesions and phosphorylated MLC levels of HPKs alone and had no effect on A-431 cells due to the negligible MLCK expression. Using genetically modified A-431 cells expressing phosphomimetic mutant of p-MLC, we show that ROCK dependent phosphorylated MLC controls the migration, focal adhesion, stress fibre organization and the morphology of the cells. In conclusion, our data indicate that ROCK is the major kinase of MLC phosphorylation in both HPKs and A-431 cells, and regulates the contractility and migration of healthy as well as malignant skin epithelial cells.

FK506 BINDING PROTEIN 8 (FKBP8) INTERACTIONS WITH MYOSIN LIGHT CHAIN KINASE 1 ARE REQUIRED FOR TNF-INDUCED TIGHT JUNCTION BARRIER LOSS

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S25-S25
Author(s):  
Li Zuo ◽  
Feng Cao ◽  
Wei-Ting Kuo ◽  
Jerrold Turner
Keyword(s):  
Tight Junction ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Intestinal Barrier ◽  
Intestinal Epithelial ◽  
Binding Interaction ◽  
Chaperone Protein ◽  
Tight Junction Permeability ◽  
Mlc Phosphorylation

Abstract Background Tumor necrosis factor (TNF) regulates intestinal epithelial tight junction permeability by activating myosin light chain kinase 1 (MLCK1) expression and enzymatic activity. MLCK1 recruitment to the apical perijunctional actomyosin ring (PAMR) is, however, required for barrier regulation; Divertin, a small molecule drug that blocks this recruitment, prevents barrier loss and attenuates both acute and chronic experimental diarrheal disease. We therefore hypothesized that MLCK1 recruitment to the PAMR requires interactions with as yet unidentified chaperone protein(s). Objective To identify binding partners and define the mechanisms by which they activate MLCK1 recruitment to the PAMR. Results We performed a yeast-2-hybrid (Y2H) screen using the MLCK1 domains required for PAMR recruitment as bait. FKBP8, which encodes a peptidyl-prolyl cis-trans isomerase (PPI), was recovered, and direct binding to the MLCK1 domains (Kd=~5mM) was confirmed using microscale thermophoresis (MST). This binding interaction required the FK506-binding PPI domain and was specifically inhibited by FK506 (tacrolimus). Immunofluorescent microscopy demonstrated partial colocalization of MLCK1 and FKBP8 within intestinal epithelial monolayers; TNF caused both to concentrate around the PAMR. To further characterize this interaction, we performed proximity ligation assays (PLA) and found that TNF increased interaction between MLCK1 and FKBP8 > 2-fold. FK506 prevented TNF-induced increases in PLA signal. FK506 was also able to reverse TNF-induced increases in myosin light chain (MLC) phosphorylation and tight junction permeability. In Caco-2 monolayers, FKBP8 knockout blocked TNF-induced MLCK1 recruitment, MLC phosphorylation, and tight junction barrier loss; all of which were restored by FKBP8 re-expression. In mice, MLC phosphorylation and intestinal barrier loss triggered by acute, anti-CD3-induced, T cell activation were blocked by luminal FK506. Importantly, this local FK506 treatment did not prevent anti-CD3-induced increases in mucosal TNF, IL-1b, and IFNg. Immunostains of biopsies from IBD patients documented increased PAMR MLC phosphorylation, MLCK1 recruitment, FKBP8 recruitment, and MLCK1-FKBP8 PLA signal relative to control subjects. Conclusions FKBP8 is a chaperone protein required for TNF-induced MLCK1 recruitment and barrier loss. This requires direct interaction between MLCK1 and the PPI domain of FKBP8. FK506 binding to the PPI domain displaces MLCK1 thereby preventing recruitment to the PAMR and barrier loss. These activities are separate from the immunosuppressive effects of FK506. We speculate that molecular blockade of the FKBP8-MLCK1 interaction may be a novel approach to barrier restoration and therapy of diseases associated with intestinal barrier dysfunction. Support NIH (DK068271, DK061931) and the NNSF of China (81800464, 82070548).

Encoding of amplitude and rate of forces applied to the teeth by human periodontal mechanoreceptive afferents

1994 ◽  
Vol 72 (4) ◽  
pp. 1734-1744 ◽  
Author(s):  
M. Trulsson ◽  
R. S. Johansson
Keyword(s):  
Steady State ◽  
Discharge Rate ◽  
Inferior Alveolar Nerve ◽  
Steady State Response ◽  
Force Amplitude ◽  
Stimulus Response ◽  
State Response ◽  
Dynamic Sensitivity ◽  
Linear Relationships ◽  
State Force

1. The encoding of force amplitude and force rate by human periodontal mechanoreceptive afferents was studied. Recordings were obtained from 19 single periodontal afferents in the inferior alveolar nerve with the use of tungsten microelectrodes. Loads consisting of a force increase (loading ramp), a phase of maintained force (static phase), and a force decrease (unloading ramp) were applied to the receptor bearing tooth, which was most often an incisor. The static forces applied ranged between 0.05 and 5 N, and the rate of force applied during the loading ramps ranged between 0.4 and 70 N/s. The forces were primarily applied in one of six directions (lingual, labial, mesial, distal, upward, or downward) that evoked the greatest discharge activity. 2. For each force application, the steady-state response was defined as the mean discharge rate during a 1-s period starting 0.5 s after the end of the loading ramp. Most afferents (15/19) exhibited a “hyperbolic” (viz., negatively accelerating) relationship between the amplitude of the stimulation force and the steady-state response, featuring a pronounced saturation tendency: the highest sensitivity to changes in static force was observed at force levels below 1 N. At higher force levels the sensitivity gradually diminished. Moreover, the dynamic sensitivity similarly decreased with increasing amplitude of static background force. For a subsample of afferents studied, comparable stimulus-response relationships were obtained in directions other than the most responsive one, but the discharge rates were lower. 3. In contrast to the response of most afferents, four (4/19) differed in that they consistently exhibited a nearly linear relationship between force amplitude and the steady-state response. Moreover, these afferents maintained their dynamic sensitivity as the amplitude of the background force was increased. 4. The steady-state response of all afferents was well described as a constant times F/ (F + c), where F represents the steady-state force, and c the force generating one-half the estimated maximum discharge rate that could be evoked by steady-state force stimulation. The c-parameter was on average 0.42 N (range 0.05–1.1 N) for the afferents exhibiting hyperbolic stimulus-response relationships. In contrast it ranged between 5 and 22 N for those exhibiting “nearly linear” relationships. A hypothetical model of the mechanics of the periodontal ligament supporting the F/(F + c) transform is proposed. 5. A general transfer function was developed to predict the instantaneous discharge rate of an individual afferent to arbitrary force profiles applied to the receptor bearing tooth.(ABSTRACT TRUNCATED AT 400 WORDS)

Modelling Age Differences in Isometric Elbow Flexion Using Hill's Three-Element Visco-Elastic Model

1993 ◽  
Vol 37 (2) ◽  
pp. 202-205
Author(s):  
R. Darin Ellis ◽  
Kentaro Kotani
Keyword(s):  
Mechanical Properties ◽  
Steady State ◽  
Age Differences ◽  
Elbow Flexion ◽  
Model Parameters ◽  
Elastic Model ◽  
Tissue Elasticity ◽  
Age Related ◽  
Complex Models ◽  
State Force

A visco-elastic model of the mechanical properties of muscle was used to describe age-differences in the buildup of force in isometric elbow flexion. Given information from the literature on age-related physiological changes, such as decreasing connective-tissue elasticity, one would expect changes in the mechanical properties of skeletal muscle and their related model parameters. Force vs. time curves were obtained for 7 young (aged 21–27) and 7 old (aged 69–83) female subject. There were significant age group differences in steady-state force level and the best fitting model parameters. In particular, the viscous damping element of the model plays a large role in describing the increased time to reach steady-state force levels in the older subject group. Implications of this research include incorporating parameter differences into more complex models, such as crash impact models.

Tearing Resistance of Advanced Double Hulls

1997 ◽  
Vol 41 (01) ◽  
pp. 69-80
Author(s):  
Mark D. Bracco ◽  
Tomasz Wierzbicki
Keyword(s):  
Steady State ◽  
Cutting Force ◽  
Average Error ◽  
Small Scale ◽  
Complex Deformation ◽  
Scale Models ◽  
State Force ◽  
Deformation Patterns ◽  
Double Hull ◽  
Tearing Resistance

This paper studies the cutting by a wedge of advanced double hull (ADH) small-scale models. A total of six cutting experiments were performed with six different wedge geometries. Complex deformation patterns observed in the damaged specimens were simplified to obtain a closed-form upper bound for the steady-state cutting force. The ADH steady-state cutting force solution varied from 6% above to 12% below the experimental mean steady-state force. The absolute average error is 5%.

Transient responses and continuous behavior of active smooth muscle during controlled stretches

1982 ◽  
Vol 242 (3) ◽  
pp. C146-C158 ◽  
Author(s):  
R. A. Meiss
Keyword(s):  
Steady State ◽  
Smooth Muscles ◽  
Muscle Length ◽  
Initial Slope ◽  
Stretch Rate ◽  
Rate Dependent ◽  
Upward Slope ◽  
Length Changes ◽  
State Force ◽  
The Relationship

Controlled length changes were imposed on mesotubarium superius and ovarian ligament smooth muscles from the reproductive tracts of female rabbits in constant estrus. Stretches of up to 35% of the muscle length were applied during isometric contraction, relaxation, and steady-state force levels. Force was continuously monitored and was plotted as a function of length. During constant velocity stretches there was an initial steep rise in force, a rapid downward deviation from the initial slope, and a long region with a constant upward slope. Stretches made during contraction showed smaller initial rises in force and steeper linear portions than did identical comparison stretches made during relaxation. The value of the slope was independent of the prior developed force, but it did depend on whether the muscle was contracting or relaxing. During contraction and steady-state force levels, the slope was independent of the stretch rate, but it was strongly rate dependent during relaxation. Changes in the stretch rate during stretch caused associated changes in muscle force; the relationship was curvilinear and was exaggerated during relaxation. The findings are placed in the context of a sliding-filament--cross-bridge hypothesis.

Sign in / Sign up

Export Citation Format

Share Document