Browsing by Subject "Smooth Muscle"

Now showing 1 - 4 of 4
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    Endothelium-independent dilation in children and adolescents
    (2011-08) Marlatt, Kara Lynn
    Peak brachial artery dilation post-nitroglycerin (NTG) administration occurs between 3 and 5-min in adults. The purpose of this study was to identify the time to peak dilation response to sublingual NTG (0.3 mg) in youth. Endothelium-independent dilation (EID) was measured in 198 healthy (113 males, 85 females) youth (6-18 yrs) via ultrasound imaging of the brachial artery following NTG administration up to 5-min. Time to peak EID was 4-min, 28-sec, following NTG administration. There was a significant (p<0.001) difference post-NTG at the 3 vs. 4-min, 4 vs. 5-min, and 3 vs. 5-min time points. Peak EID (males: 24.8 + 0.5 vs. females: 25.3 + 0.6 %, p=0.6) was not significantly different between genders after adjusting for baseline brachial diameter. Endothelium-independent dilation was measured up to 8-min in a small sub-group of 20 youth (12 males, 8 females), resulting in a time to peak dilation of 4-min, 24-sec, and an average change in peak dilation of 23.8 + 1.2 %. No significant difference existed beyond the 4-min time point within the small sub-group. In conclusion, peak response to NTG administration occurred between 4 and 5-min. The results demonstrate the importance of measuring EID up to 5-min post-nitroglycerin administration in youth.
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    Phosphorylation-induced structural changes in smooth muscle myosin.
    (2010-07) Kast, David John Edward
    Activation of smooth muscle myosin (SMM) requires phosphorylation of the myosin's regulatory light chain (RLC) to relieve autoinhibitory head-head interactions, but the structural basis of this mechanism in unknown. There are no crystal structures of any fragment of SMM, and there are no crystal structures of any RLC that contains the N-terminal 24 amino acids required for phosphorylation. Site-directed spin labeling of this N-terminal segment, referred to as the phosphorylation domain (PD), showed that phosphorylation increases α-helicity, mobility and solvent accessibility of the PD. A model emerged, where the unphosphorylated RLC is compact with a disordered PD, and phosphorylation causes the PD extend away from the RLC while inducing helical ordering. The goal of this research is to test the hypothesis that the PD functions as a structural switch that changes the structure of RLC upon phosphorylation, and to define these structural changes in atomic detail. Complementary fluorescence resonance energy transfer (FRET) experiments and molecular dynamics (MD) simulations were performed to elucidate structural changes in the phosphorylation domain (PD) of smooth muscle regulatory light chain (RLC). MD simulations on the isolated PD reveal disorder-to-order transition, where residues K11-Q15 are disordered in the unphosphorylated PD but completely α-helical in the phosphorylated PD. A salt bridge formed between R16 and the phosphorylated S19 promotes ordering by stabilizing α-helicity and reducing conformational fluctuations. Consequently, this disorder-to-order transition is regulated by delicate balance between enthalpy and entropy. To elucidate the structural changes of the PD in context with the RLC bound to smooth muscle myosin, donor-acceptor pairs of probes were attached to three site-directed di-Cys mutants of RLC, each having one Cys at position 129 in the C-terminal lobe and the other at position 2, 3, or 7 in the N-terminal PD. Labeled RLC was reconstituted onto myosin S1. Time-resolved FRET demonstrated two simultaneously resolved structural states of the RLC, closed and open, which are present in both unphosphorylated and phosphorylated biochemical states. All three FRET pairs show that phosphorylation shifts the equilibrium toward the open state, increasing its mole fraction by 23%. Molecular dynamics simulations agree with FRET data in remarkable detail, supporting the coexistence of two structural states, with phosphorylation shifting the system toward a more open and mobile structure. This agreement between experiment and simulation validates the additional structural details provided by the MD simulations: In the closed state, the PD is bent onto the surface of the C-terminal lobe, stabilized by two specific interdomain salt bridges. In the open state, the PD is more helical and straight, resides farther from the C-terminal lobe, and is stabilized by a specific intradomain salt bridge. The closed and open states are also present in phosphorylated HMM, while unphosphorylated HMM possess the closed state an intermediate distance distribution. Phosphorylation forces the PD to adopt S1-like states without increasing the mean separation of the two myosin heads. The result is a vivid atomic-resolution model of the molecular mechanism by which phosphorylation activates smooth muscle.
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    Regulation of CD38 expression in human airway smooth muscle (HASM) cells.
    (2009-05) Jude, Joseph Antony
    CD38 is a multifunctional enzyme-cum-receptor expressed in a variety of mammalian tissues including airway smooth muscles (ASM). The ADP-ribosyl cyclase activity of CD38 generates cyclic ADP-ribose (cADPR), a Ca2+ mobilizing agent in ASM cells. In vivo studies in mice showed that CD38 plays an important role in the development of airway hyperresponsiveness (AHR) following exposure to cytokines. In vitro studies demonstrated that a variety of cytokines, including the inflammatory cytokine TNF-alpha, induce CD38 expression in human ASM (HASM) cells. Studies also showed that the TNF-alpha-induced CD38 expression in HASM cells is mediated through both the transcriptional and post-transcriptional mechanisms and involves activation of mitogen-activated protein kinases (MAPKs) and transcription factors NF-kappaB and AP-1. The role of CD38 in the pathogenesis of AHR in human asthmatics is not known. The current studies demonstrate that HASM cells isolated from asthmatic patients show differentially elevated CD38 expression following TNF-alpha exposure compared to non-asthmatic HASM cells. Basal and TNF-alpha-induced activation of extracellular signal- regulated kinase (ERK) and p38 MAPK are elevated in asthmatic HASM cells, whereas the TNF-alpha-induced activation of c-jun N terminal kinase (JNK) is elevated in non-asthmatic HASM cells compared to asthmatic cells. The TNF-alpha-induced NF-kappaB activation is elevated in asthmatic HASM cells, indicating that the differentially elevated CD38 expression in asthmatic HASM cells is mediated through transcriptional mechanisms. The role of cross-talk between ERK and phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathways in TNF-alpha-induced CD38 expression is investigated in the current studies. Two pharmacological inhibitors of PI3K, LY294002 and wortmannin, show differential effects on the TNF-alpha-induced CD38 expression in HASM cells. Transient expression of PI3K catalytic subunit (p110) elevates CD38 expression while transfection with phosphatase tensin homolog (PTEN) attenuates the TNF-alpha-induced CD38 expression, suggesting that PI3K/Akt pathway mediates CD38 expression in HASM cells in a non-ERK dependant manner. The role of adenylate-uridylate-rich elements (AREs) of the CD38 mRNA 3' untranslated region (3'UTR) in CD38 mRNA stability is currently being investigated. Preliminary findings show that, in HASM cells, the RNA-binding proteins HuR and TIA-1 selectively bind to an ARE of CD38 3'UTR in response to TNF-alpha-exposure, suggesting a potential role for the CD38 mRNA AREs in the post-transcriptional regulation of CD38 expression in HASM cells.