Dataset Information

ITRAQ-Based Proteomics Analysis Reveals the Effect of Neoliensinine on KCl-Induced Vascular Smooth Muscle Contraction by Inhibiting Regulatory Light Chain Phosphorylation.

ABSTRACT: Smooth muscle (SM) contraction is one of the important physiological functions of the human body, and SM abnormal contraction will induce many diseases. The phosphorylated regulatory light chains (p-RLC) play a decisive role in SM contraction, and dephosphorylation of p-RLC is an effective way to relax SM. Our previous study showed that the novel benzylisoquinoline alkaloid, neoliensinine (Neo), could relax microvascular SM contracted by KCl hyperpolarization. In this study, mesenteric capillaries isolated from 45 mice were divided into normal tension group (Control), 124?mM KCl induced contraction model group (Model), and KCl and Neo-treatment group (Drug). The dephosphorylation levels of RLC in the three groups were measured. Compared with the model group, the phosphorylation of RLC in the drug group was decreased dramatically as expected, suggesting that the relaxation effect of Neo was caused by downregulating p-RLC of microvessel SM. In order to fully understand its fundamental mechanism, our research focused on the identification of target proteins in mice with KCl-induced contractile mesenteric capillary. Isobaric tags for relative and absolute quantification (ITRAQ) tagging was carried out by nanospray liquid chromatography-tandem mass spectrometry. The results allowed the upregulation of 164 differential abundance proteins (DAPs) among the 3,474 protein abundance disturbances identified from the model/control samples. Further comparison showed that there were 16 DAP convergences associated with vascular SM contraction between the drug/model and the drug/control samples. Among them, two proteins with known function, PLC? and RhoGEF12, were selected as target proteins of the relaxation effect of Neo. The two selective target DAPs were verified by Western blot at protein level. The results suggested that changes of the two proteins were consistent with that of the iTRAQ results. Our present work reveals that Neo relaxes vascular smooth muscle via inhibition of RLC phosphorylation, and PLC? and RhoGEF12 may be potential biomarkers for evaluating the effects mediated by Neo.

SUBMITTER: Yang GM

PROVIDER: S-EPMC6749048 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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ITRAQ-Based Proteomics Analysis Reveals the Effect of Neoliensinine on KCl-Induced Vascular Smooth Muscle Contraction by Inhibiting Regulatory Light Chain Phosphorylation.

Yang Guang-Ming GM Yan Ke K Wang Peng P Zhang Jun-Li JL Pan Zi-Hao ZH Pan Yang Y

Frontiers in pharmacology 20190911

Smooth muscle (SM) contraction is one of the important physiological functions of the human body, and SM abnormal contraction will induce many diseases. The phosphorylated regulatory light chains (p-RLC) play a decisive role in SM contraction, and dephosphorylation of p-RLC is an effective way to relax SM. Our previous study showed that the novel benzylisoquinoline alkaloid, neoliensinine (Neo), could relax microvascular SM contracted by KCl hyperpolarization. In this study, mesenteric capillari ...[more]

PMID: 31572175

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Project description:Berberine is a natural isoquinoline alkaloid possessing various pharmacological effects, particularly apparent in the treatment of diarrhea, but the underlying mechanism remains unclear. Smooth muscle myosin light-chain kinase (MLCK) plays a crucial role in the smooth muscle relaxation-contraction events, and it is well known that berberine can effectively depress the contraction of smooth muscle. Hence, whether berberine could inhibit MLCK and then depress the smooth muscle contractility might be researched.The purpose of this study is to investigate the effects of berberine on MLCK. Based on this, the contractility of gastro-intestine, catalysis activity of MLCK, and molecular docking are going to be evaluated.The experiment of smooth muscle contraction was directly monitored the contractions of the isolated gastrointestine by frequency and amplitude at different concentration of berberine. The effects of berberine on MLCK were measured in the presence of Ca2+-calmodulin, using the activities of 20 kDa myosin light chain (MLC20) phosphorylation, and myosin Mg2+-ATPase induced by MLCK. The docking study was conducted with expert software in the meantime.The phosphorylation of myosin and the Mg2+-ATPase activity is reduced in the presence of berberine. Moreover, berberine could inhibit the contractibility of isolated gastric intestine smooth muscle. Berberine could bind to the ATP binding site of MLCK through hydrophobic effect and hydrogen bonding according to the docking study.The present work gives a deep insight into the molecular mechanism for the treatment of diarrhea with berberine, i.e., berberine could suppress the contractility of smooth muscle through binding to MLCK and depressing the catalysis activity of MLCK.Berberine significantly reduced the amplitude of contraction in isolated duodenum and gastric strips in ratsBerberine inhibited the phosphorylated extents of MLC20 and Mg2+-ATPase activity of phosphorylated myosin induced by MLCKBerberine binds to the ATP binding site of MLCK by hydrophobic effect and hydrogen bondingBerberine may modulate contraction of smooth muscle by inhibiting MLCK. Abbreviations used: MLCK: Myosin light chain kinase; MLC20: 20 KDa regulating myosin light chain; CaM: Calmodulin.

Project description:Efficient mechanotransduction in vascular smooth muscle cells (VSMCs) is intimately coupled to physical coupling of the cell to extracellular matrix proteins (ECM) by integrins. Integrin adhesion receptors are essential for normal vascular function and defective integrin signaling is associated with cardiovascular disease. However, less is known about the mechanism of integrin activation in VSMCs in relation to vasoregulation. Our laboratory previously demonstrated that the vasoconstrictor Angiotensin II increases VSMC stiffness in concert with enhanced adhesion to fibronectin (FN), indicating an important role for adhesion in contraction. However, the mechanism of this coordination remains to be clarified. In this study, intracellular Ca2+ ([Ca2+]i) was hypothesized to link integrin activation through inside-out signaling pathways leading to enhanced adhesion in response to AII. By using atomic force microscopy (AFM) with an anti-?5 antibody coated AFM probe, we confirmed that cell stiffness was increased by AII, while we observed no change in adhesion to an ?5 integrin antibody. This indicated that increases in cell adhesion to FN induced by AII were occurring through an integrin activation process, as increased membrane integrin expression/receptor density would have been accompanied by increased adhesion to the anti-?5 antibody. Further studies were performed using either KCl or BAPTA-AM to modulate the level of [Ca2+]i. After KCl, VSMCs showed a rapid transient increase in cell stiffness as well as cell adhesion to FN, and these two events were synchronized with superimposed transient increases in the level of [Ca2+]i, which was measured using the Ca2+ indicator, fluo-4. These relationships were unaffected in VSMCs pretreated with the myosin light chain kinase inhibitor, ML-7. In contrast, unstimulated VSMCs incubated with an intracellular calcium chelator, BAPTA-AM, showed reduced cell adhesion to FN as well the expected decrease in [Ca2+]i. These data suggest that in VSMCs, integrin activation is linked to signaling events tied to levels of [Ca2+]i while being less dependent on events at the level of contractile protein activation. These findings provide additional evidence to support a role for adhesion in VSMC contraction and suggest that following cell contractile activation, that adhesion may be regulated in tandem with the contractile event.

Dataset Information

ITRAQ-Based Proteomics Analysis Reveals the Effect of Neoliensinine on KCl-Induced Vascular Smooth Muscle Contraction by Inhibiting Regulatory Light Chain Phosphorylation.

Publications

ITRAQ-Based Proteomics Analysis Reveals the Effect of Neoliensinine on KCl-Induced Vascular Smooth Muscle Contraction by Inhibiting Regulatory Light Chain Phosphorylation.

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