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Self-Immolative Thiocarbamates for Studying COS and H2S Chemical BiologySteiger, Andrea 30 April 2019 (has links)
In recent years, hydrogen sulfide (H2S) has garnered interest as the third addition to the gasotransmitter family. Essential to human physiology, H2S has roles in the cardiovascular, nervous, and respiratory systems and perturbations in physiological H2S levels have been correlated to a variety of diseases. As a result, there has been significant interest in the development of H2S-releasing compounds (H2S donors) that can mimic slow, enzymatic production for research and therapeutic applications. While a large library of H2S donors exists, several common drawbacks persist, such as: lack of spatial and temporal control, poorly understood mechanisms of release, uncontrolled kinetics, and low efficiency. These issues significantly limit the biological applications of many H2S donors.
This dissertation describes recent work to provide biocompatible H2S donors with controllable release kinetics using a robust, novel strategy for H2S delivery that relies on rapid enzymatic hydrolysis of carbonyl sulfide (COS) to H2S by the ubiquitous mammalian enzyme carbonic anhydrase (CA). Self-immolative thiocarbamates can be designed to release COS by a variety of stimuli, and in biological milieu this COS is rapidly converted to H2S by CA. This strategy has enabled the development of the first analyte-replacement fluorescent probe for H2S and has become a popular strategy for H2S delivery in a variety of applications. Additionally, the unexpected cytotoxicity profile of enzyme-activated COS/H2S donors has piqued interest in COS chemical biology, and these donors are being used as tools for studying COS itself. This dissertation includes previously published and unpublished coauthored work. / 2021-04-30
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Novel ways to regulate T-type Ca2+ channelsPeers, C., Elies, Jacobo, Gamper, N. 2015 February 1925 (has links)
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Hydrogen sulfide inhibits Cav3.2 T-type Ca2 channelsElies, Jacobo, Scragg, J.L., Huang, S., Dallas, M.L., Huang, D., MacDougall, D., Boyle, J.P., Gamper, N., Peers, C. 02 September 2014 (has links)
No / The importance of H2S as a physiological signaling molecule continues to develop, and ion channels are emerging as a major family of target proteins through which H2S exerts many actions. The purpose of the present study was to investigate its effects on T-type Ca2+ channels. Using patch-clamp electrophysiology, we demonstrate that the H2S donor, NaHS (10 μM−1 mM) selectively inhibits Cav3.2 T-type channels heterologously expressed in HEK293 cells, whereas Cav3.1 and Cav3.3 channels were unaffected. The sensitivity of Cav3.2 channels to H2S required the presence of the redox-sensitive extracellular residue H191, which is also required for tonic binding of Zn2+ to this channel. Chelation of Zn2+ with N,N,N′,N′-tetra-2-picolylethylenediamine prevented channel inhibition by H2S and also reversed H2S inhibition when applied after H2S exposure, suggesting that H2S may act via increasing the affinity of the channel for extracellular Zn2+ binding. Inhibition of native T-type channels in 3 cell lines correlated with expression of Cav3.2 and not Cav3.1 channels. Notably, H2S also inhibited native T-type (primarily Cav3.2) channels in sensory dorsal root ganglion neurons. Our data demonstrate a novel target for H2S regulation, the T-type Ca2+ channel Cav3.2, and suggest that such modulation cannot account for the pronociceptive effects of this gasotransmitter. / This work was supported by the British Heart Foundation, the Medical Research Council, and the Hebei Medical University
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Characterization of mechanism of action of hydrogen sulfide (H2S) in the regulation of smooth muscle functionNalli, Ancy D 01 January 2015 (has links)
Hydrogen sulfide (H2S) is receiving increasing interest, as much as nitric oxide (NO) and carbon monoxide have received previously, to understand its physiological functions as it meets all the criteria to define as a third gasotransmitter. Endogenous synthesis from L-cysteine via cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS) and the function of H2S as an inhibitor of smooth muscle contraction in gastrointestinal tract are known. However, the loci of generation and action of H2S, and the mechanism of inhibition of contraction are unknown. Hence, my aims in the present study are to: i) identify the expression of enzymes in smooth muscle, ii) determine the effects of endogenously released and exogenously applied H2S on smooth muscle function; and iii) identify the targets and mechanism involved in mediating the effects of H2S using isolated smooth muscle cells from rabbit colon.
I have identified the expression of CSE, but not CBS, in smooth muscle and demonstrated that L-cysteine (an activator of CSE) and NaHS (H2S donor): 1) inhibited carbachol-induced contraction in muscle strips and isolated muscle cells that was independent of KATP channels, a known S-sulfhydration target of H2S; 2) induced S-sulfhydration of small G protein, RhoA leading to inhibition of RhoA and Rho kinase activities, a key pathway in the sustained smooth muscle contraction; and 3) inhibited PDE5 activity leading to augmentation NO-induced cGMP formation and muscle relaxation. Sodium nitroprusside (an NO donor) induced an increase in H2S production via PKG-dependent phosphorylation and activation of CSE.
We conclude that smooth muscle cells selectively express CSE, and endogenous generation of H2S via activation of CSE inhibits muscle contraction and augments muscle relaxation. Inhibition of contraction is mediated via S-sulfhydration of RhoA and suppression of RhoA/Rho kinase pathway. Augmentation of relaxation is mediated via inhibition of PDE5 activity and stimulation of cGMP/PKG pathway, which in addition initiates generation of H2S via PKG-mediated phosphorylation and activation of CSE. The findings are important in providing the underlying mechanisms involved in the regulation of smooth muscle function by H2S and could offer insights for the development of therapeutic agents that may act on smooth muscle in the gut to treat motility disorders.
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H2S does not regulate proliferation via T-type Ca2+ channelsElies, Jacobo, Johnson, E., Boyle, J.P., Scragg, J.L., Peers, C. 24 April 2015 (has links)
No / T-type Ca2+ channels (Cav3.1, 3.2 and 3.3) strongly influence proliferation of various cell types, including vascular smooth muscle cells (VSMCs) and certain cancers. We have recently shown that the gasotransmitter carbon monoxide (CO) inhibits T-type Ca2+ channels and, in so doing, attenuates proliferation of VSMC. We have also shown that the T-type Ca2+ channel Cav3.2 is selectively inhibited by hydrogen sulfide (H2S) whilst the other channel isoforms (Cav3.1 and Cav3.3) are unaffected. Here, we explored whether inhibition of Cav3.2 by H2S could account for the anti-proliferative effects of this gasotransmitter. H2S suppressed proliferation in HEK293 cells expressing Cav3.2, as predicted by our previous observations. However, H2S was similarly effective in suppressing proliferation in wild type (non-transfected) HEK293 cells and those expressing the H2S insensitive channel, Cav3.1. Further studies demonstrated that T-type Ca2+ channels in the smooth muscle cell line A7r5 and in human coronary VSMCs strongly influenced proliferation. In both cell types, H2S caused a concentration-dependent inhibition of proliferation, yet by far the dominant T-type Ca2+ channel isoform was the H2S-insensitive channel, Cav3.1. Our data indicate that inhibition of T-type Ca2+ channel-mediated proliferation by H2S is independent of the channels’ sensitivity to H2S. / This work was supported by the British Heart Foundation (PG/11/84/29146).
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