scholarly journals Androgens Induce Nongenomic Stimulation of Colonic Contractile Activity through Induction of Calcium Sensitization and Phosphorylation of LC20 and CPI-17

2010 ◽  
Vol 24 (5) ◽  
pp. 1007-1023 ◽  
Author(s):  
María C. González-Montelongo ◽  
Raquel Marín ◽  
Tomás Gómez ◽  
Jorge Marrero-Alonso ◽  
Mario Díaz
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Contractile Activity ◽  
Rho Kinase ◽  
Regulatory Subunit ◽  
Calcium Sensitization ◽  
Regulatory Myosin Light Chain ◽  
Rapid Activation ◽  
Stimulation Of

Abstract We show that androgens, testosterone and 5α-dihydrotestosterone (DHT), acutely (∼40 min) provoke the mechanical potentiation of spontaneous and agonist-induced contractile activity in mouse colonic longitudinal smooth muscle. The results using flutamide, finasteride, cycloheximide, and actinomycin D indicate that androgen-induced potentiation is dependent on androgen receptors, requires reduction of testosterone to DHT, and occurs independently of transcriptional and translational events. Using permeabilized colonic smooth muscle preparations, we could demonstrate that mechanical potentiation is entirely due to calcium sensitization of contractile machinery. In addition, DHT (10 nm) increased phosphorylation of both 20-kDa myosin light chain (LC20) [regulatory myosin light chain, (MLC)] and CPI-17 (an endogenous inhibitor of MLC phosphatase). Paralleling these findings, inhibition of Rho-associated Rho kinase (ROK) and/or protein kinase C (PKC) with, respectively, Y27632 and chelerythrine, prevented LC20 phosphorylation and abolished calcium sensitization. In addition, inhibition of ROK prevents CPI-17 phosphorylation, indicating that ROK is located upstream PKC-mediated CPI-17 modulation in the signalling cascade. Additionally, androgens induce a rapid activation of RhoA and its translocation to the plasma membrane to activate ROK. The results demonstrate that androgens induce sensitization of colonic smooth muscle to calcium through activation of ROK, which in turn, activates PKC to induce CPI-17 phosphorylation. Activation of this pathway induces a potent steady stimulation of LC20 by inhibiting MLC phosphatase and displacing the equilibrium of the regulatory subunit towards its phosphorylated state. This is the first demonstration that colonic smooth muscle is a physiological target for androgen hormones, and that androgens modulate force generation of smooth muscle contractile machinery through nongenomic calcium sensitization pathways.

Relationship between force and regulatory myosin light chain phosphorylation in airway smooth muscle

2000 ◽  
Vol 279 (1) ◽  
pp. L52-L58 ◽  
Author(s):  
Tetsuya Kai ◽  
Hayashi Yoshimura ◽  
Keith A. Jones ◽  
David O. Warner
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Myosin Light Chain Phosphorylation ◽  
Mean Values ◽  
Regulatory Myosin Light Chain ◽  
Muscarinic Stimulation ◽  
The Relationship ◽  
Force Relationship ◽  
Stimulation Of

We tested the hypothesis that increases in force at a given cytosolic Ca2+ concentration (i.e., Ca2+ sensitization) produced by muscarinic stimulation of canine tracheal smooth muscle (CTSM) are produced in part by mechanisms independent of changes in regulatory myosin light chain (rMLC) phosphorylation. This was accomplished by comparing the relationship between rMLC phosphorylation and force in α-toxin-permeabilized CTSM in the absence and presence of acetylcholine (ACh). Forces were normalized to the contraction induced by 10 μM Ca2+ in each strip, and rMLC phosphorylation is expressed as a percentage of total rMLC. ACh (100 μM) plus GTP (1 μM) significantly shifted the Ca2+-force relationship curve to the left (EC50: 0.39 ± 0.06 to 0.078 ± 0.006 μM Ca2+) and significantly increased the maximum force (104.4 ± 4.8 to 120.2 ± 2.8%; n = 6 observations). The Ca2+-rMLC phosphorylation relationship curve was also shifted to the left (EC50: 1.26 ± 0.57 to 0.13 ± 0.04 μM Ca2+) and upward (maximum rMLC phosphorylation: 70.9 ± 7.9 to 88.5 ± 5.1%; n = 6 observations). The relationships between rMLC phosphorylation and force constructed from mean values at corresponding Ca2+concentrations were not different in the presence and absence of ACh. We find no evidence that muscarinic stimulation increases Ca2+ sensitivity in CTSM by mechanisms other than increases in rMLC phosphorylation.

scholarly journals Maturation and the role of PKC-mediated contractility in ovine cerebral arteries

2009 ◽  
Vol 297 (6) ◽  
pp. H2242-H2252 ◽  
Author(s):  
Ravi Goyal ◽  
Ashwani Mittal ◽  
Nina Chu ◽  
Lijun Shi ◽  
Lubo Zhang ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Contractile Response ◽  
Cerebral Arteries ◽  
Age Groups ◽  
Rho Kinase ◽  
Immunoblot Analysis ◽  
Mek Inhibitor ◽  
Inhibitory Protein ◽  
Regulatory Myosin Light Chain

Ca2+-independent pathways such as protein kinase C (PKC), extracellular-regulated kinases 1 and 2 (ERK1/2), and Rho kinase 1 and 2 (ROCK1/2) play important roles in modulating cerebral vascular tone. Because the roles of these kinases vary with maturational age, we tested the hypothesis that PKC differentially regulates the Ca2+-independent pathways and their effects on cerebral arterial contractility with development. We simultaneously examined the responses of arterial tension and intracellular Ca2+ concentration and used Western immunoblot analysis to measure ERK1/2, RhoA, 20 kDa regulatory myosin light chain (MLC20), PKC-potentiated inhibitory protein of 17 kDa (CPI-17), and caldesmon. Phorbol 12,13-dibutyrate (PDBu)-mediated PKC activation produced a robust contractile response, which was increased a further 20 to 30% by U-0126 (MEK inhibitor) in cerebral arteries of both age groups. Of interest, in the fetal cerebral arteries, PDBu leads to an increased phosphorylation of ERK2 compared with ERK1, whereas in adult arteries, we observed an increased phosphorylation of ERK1 compared with ERK2. Also, in the present study, RhoA/ROCK played a significant role in the PDBu-mediated contractility of fetal cerebral arteries, whereas in adult cerebral arteries, CPI-17 and caldesmon had a significantly greater role compared with the fetus. PDBu also led to an increased MLC20 phosphorylation, a response blunted by the inhibition of myosin light chain kinase only in the fetus. Overall, the present study demonstrates an important maturational shift from RhoA/ROCK-mediated to CPI-17/caldesmon-mediated PKC-induced contractile response in ovine cerebral arteries.

Calcium sensitization produced by G protein activation in airway smooth muscle

2001 ◽  
Vol 281 (3) ◽  
pp. L631-L638 ◽  
Author(s):  
Hayashi Yoshimura ◽  
Keith A. Jones ◽  
William J. Perkins ◽  
Tetsuya Kai ◽  
David O. Warner
Keyword(s):  
Smooth Muscle ◽  
G Proteins ◽  
Myosin Light Chain ◽  
Calcium Sensitization ◽  
Protein Activation ◽  
Maximal Force ◽  
Acute Response ◽  
Regulatory Myosin Light Chain ◽  
G Protein Activation ◽  
Intracellular Ca

We determined whether activation of G proteins can affect the force developed for a given intracellular Ca2+ concentration ([Ca2+]; i.e., the Ca2+ sensitivity) by mechanisms in addition to changes in regulatory myosin light chain (rMLC) phosphorylation. Responses in α-toxin-permeabilized canine tracheal smooth muscle were determined with Ca2+ alone or in the presence of ACh, endothelin-1 (ET-1), or aluminum fluoride (AlF[Formula: see text]; acute or 1-h exposure). Acute exposure to each compound increased Ca2+sensitivity without changing the response to high [Ca2+] (maximal force). However, chronic exposure to AlF[Formula: see text], but not to chronic ACh or ET-1, increased maximal force by increasing the force produced for a given rMLC phosphorylation. Studies employing thiophosphorylation of rMLC showed that the increase in force produced by chronic AlF[Formula: see text] exposure required Ca2+during activation to be manifest. Unlike the acute response to receptor agonists, which is mediated solely by increases in rMLC phosphorylation, chronic direct activation of G proteins further increases Ca2+ sensitivity in airways by additional mechanisms that are independent of rMLC phosphorylation.

Rat pulmonary arterial smooth muscle myosin light chain kinase and phosphatase activities decrease with age

2006 ◽  
Vol 290 (3) ◽  
pp. L509-L516 ◽  
Author(s):  
J. Belik ◽  
Ewa Kerc ◽  
Mary D. Pato
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Rho Kinase ◽  
Adult Tissue ◽  
Adult Rat ◽  
Pulmonary Arterial ◽  
Pulmonary Arterial Smooth Muscle ◽  
Specific Activities

We and others have shown that the fetal pulmonary arterial smooth muscle potential for contraction and relaxation is significantly reduced compared with the adult. Whether these developmental changes relate to age differences in the expression and/or activity of key enzymes regulating the smooth muscle mechanical properties has not been previously evaluated. Therefore, we studied the catalytic activities and expression of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) catalytic (PP1cδ) and regulatory (MYPT) subunits in late fetal, early newborn, and adult rat intrapulmonary arterial tissues. In keeping with the greater force development and relaxation of adult pulmonary artery, Western blot analysis showed that the MLCK, MYPT, and PP1cδ contents increased significantly with age and were highest in the adult rat. In contrast, their specific activities (activity/enzyme content) were significantly higher in the fetal compared with the adult tissue. The fetal and newborn pulmonary arterial muscle relaxant response to the Rho-kinase inhibitor Y-27632 was greater than the adult tissue. In addition to the 130-kDa isoform of MLCK, we documented the presence of minor higher-molecular-weight embryonic isoforms in the fetus and newborn. During fetal life, the lung pulmonary arterial MLCK- and MLCP-specific activities are highest and appear to be related to Rho-kinase activation during lung morphogenesis.

373 Role of myosin light chain kinase and Rho kinase in carbachol-induced contractions of human detrusor smooth muscle

2012 ◽  
Vol 11 (1) ◽  
pp. e373
Author(s):  
C. Protzel ◽  
T. Kirschstein ◽  
K. Porath ◽  
T. Sellmann ◽  
R. Koehling ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Rho Kinase ◽  
Detrusor Smooth Muscle ◽  
Human Detrusor

α1-Adrenoceptor-Gq-RhoA signaling is upregulated to increase myofibrillar Ca2+ sensitivity in failing hearts

2001 ◽  
Vol 281 (2) ◽  
pp. H637-H646 ◽  
Author(s):  
Nobuhiro Suematsu ◽  
Shinji Satoh ◽  
Shintaro Kinugawa ◽  
Hiroyuki Tsutsui ◽  
Shunji Hayashidani ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Expression ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Adrenergic Stimulation ◽  
Regulatory Myosin Light Chain ◽  
Kinase C

α1-Adrenergic stimulation, coupled to Gq, has been shown to promote heart failure. However, the role of α1-adrenergic signaling in the regulation of myocardial contractility in failing myocardium is still poorly understood. To investigate this, we observed 1) the effect of phenylephrine on myofibrillar Ca2+ sensitivity in α-toxin-skinned cardiomyocytes, and 2) protein expression of Gq, RhoA, and myosin light chain phosphorylation using tachypacing-induced canine failing hearts. Phenylephrine significantly increased myofibrillar Ca2+ sensitivity in failing but not in normal cardiomyocytes. Whereas Y-27632 (Rho kinase inhibitor) blocked the phenylephrine-induced Ca2+ sensitization in the failing myocytes, calphostin C (protein kinase C inhibitor) had no effect on Ca2+ sensitization. The protein expression of Gαq and RhoA and the phosphorylation level of regulatory myosin light chain significantly increased in the failing myocardium. Our results suggest that α1-adrenoceptor-Gq signaling is upregulated in the failing myocardium to increase the myofibrillar Ca2+sensitivity mainly through the RhoA-Rho kinase pathway rather than through the protein kinase C pathway.

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