Absence of force suppression in rabbit bladder correlates with low expression of heat shock protein 20

Background  

Nitroglycerin can induce relaxation of swine carotid artery without sustained reductions in [Ca²⁺]_i or myosin regulatory light chain (MRLC) phosphorylation. This has been termed force suppression and been found to correlate with ser¹⁶-phosphorylation of heat shock protein 20 (HSP20). We tested for the existence of this mechanism in a smooth muscle that is not responsive to nitric oxide.     

Insulin and GH signaling in human skeletal muscle in vivo following exogenous GH exposure: impact of an oral glucose load

Introduction

GH induces acute insulin resistance in skeletal muscle in vivo, which in rodent models has been attributed to crosstalk between GH and insulin signaling pathways. Our objective was to characterize time course changes in signaling pathways for GH and insulin in human skeletal muscle in vivo following GH exposure in the presence and absence of an oral glucose load.

Methods

Eight young men were studied in a single-blinded randomized crossover design on 3 occasions: 1) after an intravenous GH bolus 2) after an intravenous GH bolus plus an oral glucose load (OGTT), and 3) after intravenous saline plus OGTT. Muscle biopsies were taken at t = 0, 30, 60, and 120. Blood was sampled at frequent intervals for assessment of GH, insulin, glucose, and free fatty acids (FFA).

Results

GH increased AUC_glucose after an OGTT (p<0.05) without significant changes in serum insulin levels. GH induced phosphorylation of STAT5 independently of the OGTT. Conversely, the OGTT induced acute phosphorylation of the insulin signaling proteins Akt (ser⁴⁷³ and thr³⁰⁸), and AS160.The combination of OGTT and GH suppressed Akt activation, whereas the downstream expression of AS160 was amplified by GH.

We Concluded the Following

1) A physiological GH bolus activates STAT5 signaling pathways in skeletal muscle irrespective of ambient glucose and insulin levels 2) Insulin resistance induced by GH occurs without a distinct suppression of insulin signaling proteins 3) The accentuation of the glucose-stimulated activation of AS 160 by GH does however indicate a potential crosstalk between insulin and GH.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00477997","term_id":"NCT00477997"}}NCT00477997     

Pennogenin Tetraglycoside Induces Rat Myometrial Contraction and MLC20 Phosphorylation via PLC-IP3 and RhoA/Rho Kinase Signaling Pathways

Background

Total steroidal saponins extracted from the rhizome of Paris polyphylla Sm. var. yunnanensis (TSSPs) have been widely used in China for the treatment of abnormal uterine bleeding. We previously studied the main active constituents of TSSPs and their structure-activity relationships with respect to rat myometrial contractions. Tg (pennogenin tetraglycoside) was identified as one of the active ingredients in TSSPs able to induce rat myometrial contractions. However, the mechanisms underlying the pharmacological actions on uterine activity have not been described clearly.

Methods

Here Tg was screened for effects on contractile activity in isolated uterine strips from estrogen-primed rats and on MLC20 phosphorylation and related signaling pathways in cultured rat myometrial cells as determined by Western blot. Intracellular calcium ([Ca²⁺]_i) was monitored under a confocal microscope using Fluo-4 AM-loaded myometrial cells.

Results

Tg dose-dependently stimulated rat myometrial contractions as well as MLC20 phosphorylation in vitro, which could be completely suppressed by an inhibitor of myosin light chain kinase (MLCK). Use of Ca²⁺ channel blockers and kinase inhibitors demonstrated that Tg-induced myometrial contractions are mediated by activation of the phospholipase C (PLC)-inositol triphosphate (IP3) signaling pathway, resulting in increased MLC20 phosphorylation. Furthermore, Y27632, a specific inhibitor of Rho kinase (ROK), notably suppressed Tg-stimulated myometrial contractions and decreased MLC20 phosphorylation.

Conclusions

These data provide evidence that rat myometrial contractility induced by Tg results from enhanced MLC20 phosphorylation, while both PLC-IP3 and RhoA/ROK signaling pathways mediate the process. These mechanisms may be responsible for the therapeutic effects of TSSPs on abnormal uterine bleeding.     

Effect of Testosterone on Insulin Stimulated IRS1 Ser Phosphorylation in Primary Rat Myotubes—A Potential Model for PCOS-Related Insulin Resistance

Background

Polycystic ovary syndrome (PCOS) is characterized by a hyperandrogenic state and frequently develops skeletal muscle insulin resistance. We determined whether testosterone adversely affects insulin action by increasing serine phosphorylation of IRS-1^636/639 in differentiated rat skeletal muscle myotubes. The phosphorylation of Akt, mTOR, and S6K, downstream targets of the PI3-kinase-IRS-1 complex were also studied.

Methods

Primary differentiated rat skeletal muscle myotubes were subjected to insulin for 30 min after 16-hour pre-exposure to either low (20 ng/ml) or high (200 ng/ml) doses of testosterone. Protein phosphorylation of IRS-1 Ser^636/639, Akt Ser⁴⁷³, mTOR-Ser²⁴⁴⁸, and S6K-Thr³⁸⁹ were measured by Western blot with signal intensity measured by immunofluorescence.

Results

Cells exposed to 100 nM of insulin had increased IRS-1 Ser^636/639 and Akt Ser⁴⁷³ phosphorylation. Cells pre-exposed to low-dose testosterone had significantly increased insulin-induced mTOR-Ser²⁴⁴⁸ and S6K-Thr³⁸⁹ phosphorylation (p<0.05), and further increased insulin-induced IRS-1 Ser^636/639 phosphorylation (p = 0.042) compared to control cells. High-dose testosterone pre-exposure attenuated the insulin-induced mTOR-Ser²⁴⁴⁸ and S6K-Thr³⁸⁹ phosphorylation.

Conclusions

The data demonstrated an interaction between testosterone and insulin on phosphorylation of intracellular signaling proteins, and suggests a link between a hyperandrogenic, hyperinsulinemic environment and the development of insulin resistance involving serine phosphorylation of IRS-1 Ser^636/639. These results may guide further investigations of potential mechanisms of PCOS-related insulin resistance.     

Computational studies of the effects of myocardial blood flow reductions on cardiac metabolism

Background  

A computational model of myocardial energy metabolism was used to assess the metabolic responses to normal and reduced myocardial blood flow. The goal was to examine to what extent glycolysis and lactate formation are controlled by the supply of glycolytic substrate and/or the cellular redox (NADH/NAD⁺) and phosphorylation (ATP/ADP) states.     

Mapping phosphoproteins in <Emphasis Type="Italic">Mycoplasma genitalium</Emphasis> and <Emphasis Type="Italic">Mycoplasma pneumoniae</Emphasis>

Background  

Little is known regarding the extent or targets of phosphorylation in mycoplasmas, yet in many other bacterial species phosphorylation is known to play an important role in signaling and regulation of cellular processes. To determine the prevalence of phosphorylation in mycoplasmas, we examined the CHAPS-soluble protein fractions of Mycoplasma genitalium and Mycoplasma pneumoniae by two-dimensional gel electrophoresis (2-DE), using a combination of Pro-Q Diamond phosphoprotein stain and ³³P labeling. Protein spots that were positive for phosphorylation were identified by peptide mass fingerprinting using MALDI-TOF-TOF mass spectrometry.     

[HCO<Subscript>3</Subscript><Superscript>-</Superscript>]-regulated expression and activity of soluble adenylyl cyclase in corneal endothelial and Calu-3 cells

Background  

Bicarbonate activated Soluble Adenylyl Cyclase (sAC) is a unique cytoplasmic and nuclear signaling mechanism for the generation of cAMP. HCO₃ ^- activates sAC in bovine corneal endothelial cells (BCECs), increasing [cAMP] and stimulating PKA, leading to phosphorylation of the cystic fibrosis transmembrane-conductance regulator (CFTR) and increased apical Cl^- permeability. Here, we examined whether HCO₃ ^- may also regulate the expression of sAC and thereby affect the production of cAMP upon activation by HCO₃ ^- and the stimulation of CFTR in BCECs.     

The role of sphingosine kinase 1/sphingosine-1-phosphate pathway in the myogenic tone of posterior cerebral arteries

Aims

The goal of the current study was to determine whether the sphingosine kinase 1 (SK1)/sphingosine-1-phosphate (S1P) pathway is involved in myogenic vasoconstriction under normal physiological conditions. In the present study, we assessed whether endogenous S1P generated by pressure participates in myogenic vasoconstriction and which signaling pathways are involved in SK1/S1P-induced myogenic response under normal physiological conditions.

Methods and Results

We measured pressure-induced myogenic response, Ca²⁺ concentration, and 20 kDa myosin light chain phosphorylation (MLC₂₀) in rabbit posterior cerebral arteries (PCAs). SK1 was expressed and activated by elevated transmural pressure in rabbit PCAs. Translocation of SK1 by pressure elevation was blocked in the absence of external Ca²⁺ and in the presence of mechanosensitive ion channel and voltage-sensitive Ca²⁺ channel blockers. Pressure-induced myogenic tone was inhibited in rabbit PCAs treated with sphingosine kinase inhibitor (SKI), but was augmented by treatment with NaF, which is an inhibitor of sphingosine-1-phosphate phosphohydrolase. Exogenous S1P further augmented pressure-induced myogenic responses. Pressure induced an increase in Ca²⁺ concentration leading to the development of myogenic tone, which was inhibited by SKI. Exogenous S1P further increased the pressure-induced increased Ca²⁺ concentration and myogenic tone, but SKI had no effect. Pressure- and exogenous S1P-induced myogenic tone was inhibited by pre-treatment with the Rho kinase inhibitor and NADPH oxidase inhibitors. Pressure- and exogenous S1P-induced myogenic tone were inhibited by pre-treatment with S1P receptor blockers, W146 (S1P1), JTE013 (S1P2), and CAY10444 (S1P3). MLC₂₀ phosphorylation was increased when the transmural pressure was raised from 40 to 80 mmHg and exogenous S1P further increased MLC₂₀ phosphorylation. The pressure-induced increase of MLC₂₀ phosphorylation was inhibited by pre-treatment of arteries with SKI.

Conclusions

Our results suggest that the SK1/S1P pathway may play an important role in pressure-induced myogenic responses in rabbit PCAs under normal physiological conditions.     

PKCα-Specific Phosphorylation of the Troponin Complex in Human Myocardium: A Functional and Proteomics Analysis

Aims

Protein kinase Cα (PKCα) is one of the predominant PKC isoforms that phosphorylate cardiac troponin. PKCα is implicated in heart failure and serves as a potential therapeutic target, however, the exact consequences for contractile function in human myocardium are unclear. This study aimed to investigate the effects of PKCα phosphorylation of cardiac troponin (cTn) on myofilament function in human failing cardiomyocytes and to resolve the potential targets involved.

Methods and Results

Endogenous cTn from permeabilized cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy was exchanged (∼69%) with PKCα-treated recombinant human cTn (cTn (DD+PKCα)). This complex has Ser23/24 on cTnI mutated into aspartic acids (D) to rule out in vitro cross-phosphorylation of the PKA sites by PKCα. Isometric force was measured at various [Ca²⁺] after exchange. The maximal force (F_max) in the cTn (DD+PKCα) group (17.1±1.9 kN/m²) was significantly reduced compared to the cTn (DD) group (26.1±1.9 kN/m²). Exchange of endogenous cTn with cTn (DD+PKCα) increased Ca²⁺-sensitivity of force (pCa₅₀ = 5.59±0.02) compared to cTn (DD) (pCa₅₀ = 5.51±0.02). In contrast, subsequent PKCα treatment of the cells exchanged with cTn (DD+PKCα) reduced pCa₅₀ to 5.45±0.02. Two PKCα-phosphorylated residues were identified with mass spectrometry: Ser198 on cTnI and Ser179 on cTnT, although phosphorylation of Ser198 is very low. Using mass spectrometry based-multiple reaction monitoring, the extent of phosphorylation of the cTnI sites was quantified before and after treatment with PKCα and showed the highest phosphorylation increase on Thr143.

Conclusion

PKCα-mediated phosphorylation of the cTn complex decreases F_max and increases myofilament Ca²⁺-sensitivity, while subsequent treatment with PKCα in situ decreased myofilament Ca²⁺-sensitivity. The known PKC sites as well as two sites which have not been previously linked to PKCα are phosphorylated in human cTn complex treated with PKCα with a high degree of specificity for Thr143.     

ACE as a mechanosensor to shear stress influences the control of its own regulation via phosphorylation of cytoplasmic Ser(1270)

Objectives

We tested whether angiotensin converting enzyme (ACE) and phosphorylation of Ser¹²⁷⁰ are involved in shear-stress (SS)-induced downregulation of the enzyme.

Methods and Results

Western blotting analysis showed that SS (18 h, 15 dyn/cm²) decreases ACE expression and phosphorylation as well as p-JNK inhibition in human primary endothelial cells (EC). CHO cells expressing wild-type ACE (wt-ACE) also displayed SS-induced decrease in ACE and p-JNK. Moreover, SS decreased ACE promoter activity in wt-ACE, but had no effect in wild type CHO or CHO expressing ACE without either the extra- or the intracellular domains, and decreased less in CHO expressing a mutated ACE at Ser¹²⁷⁰ compared to wt-ACE (13 vs. 40%, respectively). The JNK inhibitor (SP600125, 18 h), in absence of SS, also decreased ACE promoter activity in wt-ACE. Finally, SS-induced inhibition of ACE expression and phosphorylation in EC was counteracted by simultaneous exposure to an ACE inhibitor.

Conclusions

ACE displays a key role on its own downregulation in response to SS. This response requires both the extra- and the intracellular domains and ACE Ser¹²⁷⁰, consistent with the idea that the extracellular domain behaves as a mechanosensor while the cytoplasmic domain elicits the downstream intracellular signaling by phosphorylation on Ser¹²⁷⁰.     

Phosphorylation of Mycobacterium tuberculosis Ser/Thr phosphatase by PknA and PknB

Background

The integrated functions of 11 Ser/Thr protein kinases (STPKs) and one phosphatase manipulate the phosphorylation levels of critical proteins in Mycobacterium tuberculosis. In this study, we show that the lone Ser/Thr phosphatase (PstP) is regulated through phosphorylation by STPKs.

Principal Findings

PstP is phosphorylated by PknA and PknB and phosphorylation is influenced by the presence of Zn²⁺-ions and inorganic phosphate (Pi). PstP is differentially phosphorylated on the cytosolic domain with Thr¹³⁷, Thr¹⁴¹, Thr¹⁷⁴ and Thr²⁹⁰ being the target residues of PknB while Thr¹³⁷ and Thr¹⁷⁴ are phosphorylated by PknA. The Mn²⁺-ion binding residues Asp³⁸ and Asp²²⁹ are critical for the optimal activity of PstP and substitution of these residues affects its phosphorylation status. Native PstP and its phosphatase deficient mutant PstP_c ^D38G are phosphorylated by PknA and PknB in E. coli and addition of Zn²⁺/Pi in the culture conditions affect the phosphorylation level of PstP. Interestingly, the phosphorylated phosphatase is more active than its unphosphorylated equivalent.

Conclusions and Significance

This study establishes the novel mechanisms for regulation of mycobacterial Ser/Thr phosphatase. The results indicate that STPKs and PstP may regulate the signaling through mutually dependent mechanisms. Consequently, PstP phosphorylation may play a critical role in regulating its own activity. Since, the equilibrium between phosphorylated and non-phosphorylated states of mycobacterial proteins is still unexplained, understanding the regulation of PstP may help in deciphering the signal transduction pathways mediated by STPKs and the reversibility of the phenomena.     

Tyrosine phosphorylation of myosin heavy chain during skeletal muscle differentiation: an integrated bioinformatics approach

Background  

Previously it has been shown that insulin-mediated tyrosine phosphorylation of myosin heavy chain is concomitant with enhanced association of C-terminal SRC kinase during skeletal muscle differentiation. We sought to identify putative site(s) for this phosphorylation event.     

Robust MS quantification method for phospho-peptides using 18O/16O labeling

Background  

Quantitative measurements of specific protein phosphorylation sites, as presented here, can be used to investigate signal transduction pathways, which is an important aspect of cell dynamics. The presented method quantitatively compares peptide abundances from experiments using ¹⁸O/¹⁶O labeling starting from elaborated MS spectra. It was originally developed to study signaling cascades activated by amyloid-β treatment of neurons used as a cellular model system with relevance to Alzheimer's disease, but is generally applicable.     

Two isoaccepting seryl tRNAs coded by separate mitochondrial genes in yeast

Summary In S. cerevisiae four isoacceptor mitochondrial tRNAs for serine have been separated by reversed phase chromatography. At least two of these species are products of different genes. In this work the deletion mapping technique has been used to locate two genes for tRNA^ser. The gene for tRNA^ser previously localized in the oli I region of the mitochondrial genome has been found to code for tRNA _ser ² , and another gene coding for tRNA _ser ¹ has been detected in the region where most of other tRNA genes are found. Results of fine mapping experiments allowed to localize this gene in the proximity of the gene for tRNA^arg.     

Regulation of Fibre Contraction in a Rat Model of Myocardial Ischemia

Background

The changes in the actomyosin crossbridge cycle underlying altered contractility of the heart are not well described, despite their importance to devising rational treatment approaches.

Methodology/Principal Findings

A rat ischemia–reperfusion model was used to determine the transitions of the crossbridge cycle impacted during ischemia. Compared to perfused hearts, the maximum force per cross-sectional area and Ca²⁺ sensitivity of fibers from ischemic hearts were both reduced. Muscle activation by photolytic release of Ca²⁺ and ATP suggested that the altered contractility was best described as a reduction in the rate of activation of noncycling actomyosin crossbridges to activated, cycling states. More specifically, the apparent forward rate constant of the transition between the nonforce bearing A-M.ADP.Pi state and the bound, force bearing AM*.ADP.Pi state was reduced in ischemic fibers, suggesting that this transition is commensurate with initial crossbridge activation. These results suggested an alteration in the relationship between the activation of thin filament regulatory units and initial crossbridge attachment, prompting an examination of the post-translational state of troponin (Tn) T and I. These analyses indicated a reduction in the diphosphorylated form of TnT during ischemia, along with lower Ser23/24 phosphorylation of TnI. Treatment of perfused fibers by 8-Br-cAMP increased Ser23/24 phosphorylation of TnI, altering the reverse rate constant of the Pi isomerization in a manner consistent with the lusitropic effect of β-adrenergic stimulation. However, similar treatment of ischemic fibers did not change TnI phosphorylation or the kinetics of the Pi isomerization.

Conclusions

Ischemia reduces the isomerization from A-M.ADP.Pi to AM*.ADP.Pi, altering the kinetics of crossbridge activation through a mechanism that may be mediated by altered TnT and TnI phosphorylation.     

Cancer stem cells in solid tumors: elusive or illusive?

Background

The fibroblast growth factor receptor (FGFR) interprets concentration gradients of FGF ligands and structural changes in the heparan sulfate (HS) co-receptor to generate different cellular responses. However, whether the FGFR generates different signals is not known.

Results

We have previously shown in rat mammary fibroblasts that in cells deficient in sulfation, and so in HS co-receptor, FGF-2 can only stimulate a transient phosphorylation of p42/44^MAPK and so cannot stimulate DNA synthesis. Here we demonstrate that this is because in the absence of HS, FGF-2 fails to stimulate the phosphorylation of the adaptor FGFR substrate 2 (FRS2). In cells possessing the HS co-receptor, FGF-2 elicits a bell-shaped dose response: optimal concentrations stimulate DNA synthesis, but supramaximal concentrations (≥ 100 ng/mL) have little effect. At optimal concentrations (300 pg/mL) FGF-2 stimulates a sustained dual phosphorylation of p42/44^MAPK and tyrosine phosphorylation of FRS2. In contrast, 100 ng/mL FGF-2 only stimulates a transient early peak of p42/44^MAPK phosphorylation and fails to stimulate appreciably the phosphorylation of FRS2 on tyrosine.

Conclusions

These results suggest that the nature of the FGFR signal produced is determined by a combination of the HS co-receptor and the concentration of FGF ligand. Both the phosphorylation of the adaptor FRS2, the kinetics (sustained or transient) of phosphorylation of p42/44(MAPK) are varied, and so differing cellular responses are produced.     

Myomegalin is a novel A-kinase anchoring protein involved in the phosphorylation of cardiac myosin binding protein C

Background  

Cardiac contractility is regulated by dynamic phosphorylation of sarcomeric proteins by kinases such as cAMP-activated protein kinase A (PKA). Efficient phosphorylation requires that PKA be anchored close to its targets by A-kinase anchoring proteins (AKAPs). Cardiac Myosin Binding Protein-C (cMyBPC) and cardiac troponin I (cTNI) are hypertrophic cardiomyopathy (HCM)-causing sarcomeric proteins which regulate contractility in response to PKA phosphorylation.     

Reversible Blockade of Complex I or Inhibition of PKCβ Reduces Activation and Mitochondria Translocation of p66Shc to Preserve Cardiac Function after Ischemia

Aim

Excess mitochondrial reactive oxygen species (mROS) play a vital role in cardiac ischemia reperfusion (IR) injury. P66^Shc, a splice variant of the ShcA adaptor protein family, enhances mROS production by oxidizing reduced cytochrome c to yield H₂O₂. Ablation of p66^Shc protects against IR injury, but it is unknown if and when p66^Shc is activated during cardiac ischemia and/or reperfusion and if attenuating complex I electron transfer or deactivating PKCβ alters p66^Shc activation during IR is associated with cardioprotection.

Methods

Isolated guinea pig hearts were perfused and subjected to increasing periods of ischemia and reperfusion with or without amobarbital, a complex I blocker, or hispidin, a PKCβ inhibitor. Phosphorylation of p66^Shc at serine 36 and levels of p66^Shc in mitochondria and cytosol were measured. Cardiac functional variables and redox states were monitored online before, during and after ischemia. Infarct size was assessed in some hearts after 120 min reperfusion.

Results

Phosphorylation of p66^Shc and its translocation into mitochondria increased during reperfusion after 20 and 30 min ischemia, but not during ischemia only, or during 5 or 10 min ischemia followed by 20 min reperfusion. Correspondingly, cytosolic p66^Shc levels decreased during these ischemia and reperfusion periods. Amobarbital or hispidin reduced phosphorylation of p66^Shc and its mitochondrial translocation induced by 30 min ischemia and 20 min reperfusion. Decreased phosphorylation of p66^Shc by amobarbital or hispidin led to better functional recovery and less infarction during reperfusion.

Conclusion

Our results show that IR activates p66^Shc and that reversible blockade of electron transfer from complex I, or inhibition of PKCβ activation, decreases p66^Shc activation and translocation and reduces IR damage. These observations support a novel potential therapeutic intervention against cardiac IR injury.