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.

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.

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.

Postnatal development alters functional compartmentalization of myosin light chain kinase in ovine carotid arteries

2021 ◽  
Author(s):  
Dane W. Sorensen ◽  
Elisha R. Injeti ◽  
Luisa Mejia-Aguilar ◽  
James M. Williams ◽  
William J. Pearce
Keyword(s):  
Electrical Stimulation ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
High Speed ◽  
Time Course ◽  
Myosin Light Chain ◽  
Age Groups ◽  
Regulatory Myosin Light Chain ◽  
Near Term

The rate-limiting enzyme for vascular contraction, Myosin Light Chain Kinase (MLCK), phosphorylates regulatory myosin light chain (MLC20) at rates that appear faster despite lower MLCK abundance in fetal compared to adult arteries. This study explores the hypothesis that greater apparent tissue activity of MLCK in fetal arteries is due to age-dependent differences in intracellular distribution of MLCK in relation to MLC20. Under optimal conditions, common carotid artery homogenates from non-pregnant adult female sheep and near-term fetuses exhibited similar values of Vmax and Km for MLCK. A custom-designed, computer-controlled apparatus enabled electrical stimulation and high-speed freezing of arterial segments at exactly 0, 1, 2, and 3 seconds, calculation of in situ rates of MLC20 phosphorylation, and measurement of time-dependent colocalization between MLCK and MLC20. The in situ rate of MLC20 phosphorylation divided by total MLCK abundance averaged to values more than 147% greater in fetal (1.06 ± 0.28) than adult (0.43 ± 0.08) arteries, which corresponded respectively to 43±10% and 31±3% of the Vmax values measured in homogenates. Confocal colocalization analysis revealed in fetal and adult arteries that 33 ± 6% and 20 ± 5% of total MLCK colocalized with pMLC20, and that MLCK activation was greater in peri-luminal than peri-adventitial regions over the time-course of electrical stimulation in both age groups. Together, these results demonstrate that the catalytic activity of MLCK is similar in fetal and adult arteries, but that the fraction of total MLCK in the functional compartment involved in contraction is significantly greater in fetal than adult arteries.

CPI-17 Contributes to Calcium-Independent ADP-Induced Platelet Shape Change through P2Y1-Gq-PKC Pathways.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3644-3644
Author(s):  
Todd M. Getz ◽  
Kamala Bhavaraju ◽  
Satya P. Kunapuli
Keyword(s):  
Platelet Activation ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Shape Change ◽  
Rho Kinase ◽  
Human Platelets ◽  
Inhibitory Protein ◽  
Pkc Inhibitors ◽  
Pkc Activation ◽  
Platelet Shape

Abstract The initial event in platelet activation is the reorganization of the cytoskeleton causing the platelets to change from a discoid to a spiculated spheroid shape. Platelet shape change is primarily regulated by the phosphorylation of myosin light chain kinase. We have shown that this process is mediated through both calcium-dependent and calcium-independent Rho kinase pathways. CPI-17, a Protein kinase C (PKC) phosphorylated inhibitory protein of myosin light chain phosphatase, has been shown to have a role in platelet shape change downstream of thrombin-induced platelet activation. CPI-17 is a 17 kDa protein expressed in human platelets shown to inhibit myosin light chain phosphotase activity via PKCs. In this study we examined the role of CPI-17 in ADP-induced shape change and phosphorylation of CPI-17, downstream of the Gq coupled, P2Y1, and the Gi coupled, P2Y12 receptors. CPI-17 phosphorylation occurred upon activation of platelets with 2MeSADP. This phosphorylation was abolished in the presence of the P2Y1 receptor antagonist, MRS-2179. These results indicated that Gq signaling is important for platelet shape change and phosphorylation of CPI-17. In the presence of the calcium chelator, BAPTA, platelets changed shape in response to 2MeSADP; CPI-17 phosphorylation, however, was unaffected by BAPTA treatment under these conditions. However, CPI-17 phosphorylation was inhibited in the presence of the pan PKC inhibitors. These results indicate that CPI-17 phosphorylation occurs downstream of PKC activation. In the presence of BAPTA, treatment with PKC inhibitors decreased platelet shape change possibly due to reduced CPI-17 phosphorylation. The shape change caused by p160ROCK downstream of G12/13 pathways was unaffected by pan PKC inhibitors, but abolished by p160ROCK inhibitors H1152 or Y27632. Platelets incubated with BAPTA, pan PKC inhibitors, and p160ROCK inhibitor H1152, abolished ADP-induced platelet shape change and CPI-17 phosphorylation. In conclusion, ADP-induced platelet shape change occurs through a Gq-mediated, calcium-independent signaling pathway regulated by CPI-17 phosphorylation via PKC activation.

scholarly journals PKC-induced ERK1/2 interactions and downstream effectors in ovine cerebral arteries

2005 ◽  
Vol 289 (1) ◽  
pp. R164-R171 ◽  
Author(s):  
Yu Zhao ◽  
Lubo Zhang ◽  
Lawrence D. Longo
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Cerebral Arteries ◽  
Rho Kinase ◽  
Thick Filament ◽  
Smooth Muscle Contraction ◽  
Inhibitory Effect ◽  
Isometric Tension ◽  
Free Calcium ◽  
Pkc Activation

Both protein kinase C (PKC) and extracellular signal-regulated kinases (ERK1/2) are involved in mediating vascular smooth muscle contraction. We tested the hypotheses that in addition to PKC activation of ERK1/2, by negative feedback ERKs modulate PKC-induced contraction, and that their interactions modulate both thick and thin myofilament pathways. In ovine middle cerebral arteries (MCA), we measured isometric tension and intracellular free calcium concentration ([Ca2+]i) responses to PKC stimulation [phorbol 12,13-dibutyrate (PDBu), 3 × 10−6 M] in the absence or presence of ERK1/2 inhibition (U-0126, 10−5 M). After PDBu ± ERK1/2 inhibition, we also examined by Western immunoblot the levels of total and phosphorylated ERK1/2, caldesmonSer789, myosin light chain20 (MLC20), and CPI-17. PDBu induced significant increase in tension in the absence of increased [Ca2+]i. PDBu also increased phosphorylated ERK1/2 levels, a response blocked by U-0126. In turn, U-0126 augmented PDBu-induced contractions. PDBu also was associated with significant increases in phosphorylated caldesmonSer789 and MLC20 levels, each of which peaked at 5 to 10 min. PDBu also increased phosphorylated CPI-17 levels, which peaked at 2 to 3 min. Rho kinase inhibition (Y-27632, 3 × 10−7 M) did not alter PDBu-induced contraction. These results support the idea that PKC activation can increase CPI-17 phosphorylation to decrease myosin light chain phosphatase activity. In turn, this increases MLC20 phosphorylation in the thick filament pathway and increases Ca2+ sensitivity. In addition, ERK1/2-dependent phosphorylation of caldesmonSer789 was not necessary for PDBu-induced contraction and appears not to be involved in the reversal of caldesmon's inhibitory effect on actin-myosin ATPase.

scholarly journals Praziquantel Affects the Regulatory Myosin Light Chain of Schistosoma mansoni

2008 ◽  
Vol 53 (3) ◽  
pp. 1054-1060 ◽  
Author(s):  
Munirathinam Gnanasekar ◽  
Ashok M. Salunkhe ◽  
A. Krishna Mallia ◽  
Yi Xun He ◽  
Ramaswamy Kalyanasundaram
Keyword(s):  
Schistosoma Mansoni ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Dna Analysis ◽  
Peptide Mapping ◽  
Immunoblot Analysis ◽  
Drug Of Choice ◽  
Regulatory Myosin Light Chain

ABSTRACT Praziquantel (PZQ) is the drug of choice for schistosomiasis and probably is the only highly effective drug currently available for treating schistosomiasis-infected individuals. The mode of action of PZQ involves increasing the calcium uptake of the parasite, resulting in tegumental damage and death of the parasite. Despite its remarkable function, the target of PZQ has not been identified yet. To begin to understand where PZQ acts, in this study we expressed the cDNA library of Schistosoma mansoni on the surface of T7 bacteriophages and screened this library with labeled PZQ. This procedure identified a clone that strongly bound to PZQ. Subsequent DNA analysis of inserts showed that the clone coded for regulatory myosin light chain protein. The gene was then cloned, and recombinant S. mansoni myosin light chain (SmMLC) was expressed. Immunoblot analysis using antibodies raised to recombinant SmMLC (rSmMLC) showed that SmMLC is abundantly expressed in schistosomula and adult stages compared to the amount in cercarial stages. In vitro analyses also confirmed that PZQ strongly binds to rSmMLC. Further, peptide mapping studies showed that PZQ binds to amino acids 46 to 76 of SmMLC. Immunoprecipitation analysis confirmed that SmMLC is phosphorylated in vivo upon exposure to PZQ. Interestingly, significant levels of anti-SmMLC antibodies were present in vaccinated mice compared to the amount in infected mice, suggesting that SmMLC may be a potential target for protective immunity in schistosomiasis. These findings suggest that PZQ affects SmMLC function, and this may have a role in PZQ action.

scholarly journals A Novel Regulatory Mechanism of Myosin Light Chain Phosphorylation via Binding of 14-3-3 to Myosin Phosphatase

2008 ◽  
Vol 19 (3) ◽  
pp. 1062-1071 ◽  
Author(s):  
Yasuhiko Koga ◽  
Mitsuo Ikebe
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Regulatory Mechanism ◽  
Kinase Inhibitor ◽  
Myosin Ii ◽  
Critical Role ◽  
Rho Kinase ◽  
Myosin Phosphatase ◽  
Dependent Cell ◽  
Direct Binding

Myosin II phosphorylation–dependent cell motile events are regulated by myosin light-chain (MLC) kinase and MLC phosphatase (MLCP). Recent studies have revealed myosin phosphatase targeting subunit (MYPT1), a myosin-binding subunit of MLCP, plays a critical role in MLCP regulation. Here we report the new regulatory mechanism of MLCP via the interaction between 14-3-3 and MYPT1. The binding of 14-3-3β to MYPT1 diminished the direct binding between MYPT1 and myosin II, and 14-3-3β overexpression abolished MYPT1 localization at stress fiber. Furthermore, 14-3-3β inhibited MLCP holoenzyme activity via the interaction with MYPT1. Consistently, 14-3-3β overexpression increased myosin II phosphorylation in cells. We found that MYPT1 phosphorylation at Ser472 was critical for the binding to 14-3-3. Epidermal growth factor (EGF) stimulation increased both Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase inhibitor inhibited the EGF-induced Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase specific siRNA also decreased EGF-induced Ser472 phosphorylation correlated with the decrease in MLC phosphorylation. The present study revealed a new RhoA/Rho-kinase–dependent regulatory mechanism of myosin II phosphorylation by 14-3-3 that dissociates MLCP from myosin II and attenuates MLCP activity.

scholarly journals Rho kinase inhibitors stimulate the migration of human cultured osteoblastic cells by regulating actomyosin activity

2011 ◽  
Vol 16 (2) ◽  
Author(s):  
Xuejiao Zhang ◽  
Cheng Li ◽  
Huiling Gao ◽  
Hiroaki Nabeka ◽  
Tetsuya Shimokawa ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Kinase Inhibitors ◽  
Rho Kinase ◽  
Cell Types ◽  
Osteosarcoma Cells ◽  
Human Osteoblasts ◽  
Protein Levels ◽  
Human Osteosarcoma Cells ◽  
Joint Prostheses

AbstractWe investigated the effects of Rho-associated kinase (ROCK) on migration and cytoskeletal organization in primary human osteoblasts and Saos-2 human osteosarcoma cells. Both cell types were exposed to two different ROCK inhibitors, Y-27632 and HA-1077. In the improved motility assay used in the present study, Y-27632 and HA-1077 significantly increased the migration of both osteoblasts and osteosarcoma cells on plastic in a dose-dependent and reversible manner. Fluorescent images showed that cells of both types cultured with Y-27632 or HA-1077 exhibited a stellate appearance, with poor assembly of stress fibers and focal contacts. Western blotting showed that ROCK inhibitors reduced myosin light chain (MLC) phosphorylation within 5 min without affecting overall myosin light-chain protein levels. Inhibition of ROCK activity is thought to enhance the migration of human osteoblasts through reorganization of the actin cytoskeleton and regulation of myosin activity. ROCK inhibitors may be potentially useful as anabolic agents to enhance the biocompatibility of bone and joint prostheses.

scholarly journals Involvement of Rho-Kinase–Mediated Phosphorylation of Myosin Light Chain in Enhancement of Cerebral Vasospasm

2000 ◽  
Vol 87 (3) ◽  
pp. 195-200 ◽  
Author(s):  
Motohiko Sato ◽  
Eiichi Tani ◽  
Hirokazu Fujikawa ◽  
Kozo Kaibuchi
Keyword(s):  
Cerebral Vasospasm ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Rho Kinase

scholarly journals H2O2 mediates Ca2+- and MLC20phosphorylation-independent contraction in intact and permeabilized vascular muscle

2000 ◽  
Vol 279 (3) ◽  
pp. H1185-H1193 ◽  
Author(s):  
Nancy J. Pelaez ◽  
Tracey R. Braun ◽  
Richard J. Paul ◽  
Richard A. Meiss ◽  
C. Subah Packer
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Kinase Inhibitor ◽  
Isometric Force ◽  
Mesenteric Arteries ◽  
Regulatory Myosin Light Chain ◽  
Pulmonary Arterial ◽  
Dose Dependent ◽  
Arteries And Veins ◽  
Permeabilized Muscle

One purpose of the current study was to establish whether vasoconstriction occurs in all vessel types in response to H2O2. Isometric force was measured in pulmonary venous and arterial rings, and isobaric contractions were measured in mesenteric arteries and veins in response to H2O2. A second purpose was to determine whether H2O2-induced contraction is calcium independent. The addition of H2O2 to calcium-depleted (using the Ca2+ ionophore ionomycin in zero calcium EGTA buffer) muscle caused contraction. Furthermore, permeabilized muscle contracted in response to H2O2 even in zero Ca2+. The final purpose was to determine whether the 20-kDa regulatory myosin light chain (MLC20) phosphorylation plays a role in H2O2-induced contraction. Pulmonary arterial strips were freeze-clamped at various time points during H2O2-induced contractions, and the relative amounts of phosphorylated MLC20 were measured. H2O2 caused dose-dependent contractions that were independent of MLC20 phosphorylation. ML-9, a myosin light chain kinase inhibitor, had no effect on the H2O2 contractile response. In conclusion, H2O2 induces Ca2+- and MLC20 phosphorylation-independent contraction in pulmonary and systemic arterial and venous smooth muscle.

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