Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.555856
Title: Molecular basis and pharmacology of Ca2+ homeostasis in vascular disease
Author: Amer, Mohamed Shebl Abd ElAleem
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2010
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Abstract:
The ci+ ion is a crucial signalling molecule that plays an essential role in cardiovascular physiology and pathology. Several proteins are known to control Ca2+ but understanding of the mechanisms involved remains relatively limited. The mammalian transient receptor potential (TRP) proteins can form non-selective cation channels and are implicated in cardiovascular abnormalities such as neointimal hyperplasia. TRPM3, TRPM2 and TRPC5 are examples of TRP channels that are widely expressed and involved in vascular cell functions. Sigma 1 receptor (Sig-IR) is a Ca2+ -regulatory ER protein that can translocate to the plasma membrane and modulate ion channels. Cholesterol is an essential component of cell membranes that has been suggested to modulate TRP channels such as TRPC 1. Translocon, an ER protein conducting channel, has been suggested to act as a Ca2+- leak channel in non-vascular cell types. The aim of this study was to investigate these mechanisms and factors in relation to human vascular cells. Multiple experimental approaches were used including intracellular Ca2+ measurements, RNA interference, quantitative RT-PCR, immunohistochemistry, and protein synthesis measurements. Cell culture, channels over-expressed in cell-line and I clinical samples from patients with coronary artery disease were the source of native cells. Structurally unrelated Sig-I R ligands, SKF 10047, 4- IBP, BD 1063 and BD 1 047 were used to explore the relationship ofTRP channels to Sig-IR. Importantly, all of the ligands inhibited TRPM3 activity but had no effects on TRPM2. However, Sig- lR siRNA studies failed to show a link of TRPM3 to Sig-IR .except for a component of the BDI047 effect. With TRPC5, except for potentiation by BDI063, Sig-l R ligands all produced strong inhibitory effects. Furthermore, so did the putative endogenous Sig-IR ligands; PregS, progesterone and dimethyltryptamine. In addition, Sig-l R ligands inhibited endogenous Ca2+ -entry signals in endothelial cells (ECs) evoked by histamine, vascular endothelial growth factor (VEGF) and H202 where endogenous TRP channels are suggested to be involved. However, again, Sig-lR siRNA revealed no role for Sig-lR. Moreover, immunohistochemistry results suggested that Sig-IR did not eo-localise with ] v TRPM3 or TRPC5. Therefore, the data suggest primarily direct effects of Sig-lR ligands on TRP channels and related endogenous Ca2+ signal of ECs but little or no role of Sig-I R itself. Metaphit, a non-selective sigma receptor antagonist, inhibited TRPM3, TRPC5 and TRPM2 activities and endogenous Ca2+-signals induced by histamine and H202 in ECs and by an oxidized phospholipid in VSMCs. Importantly, metaphit (1 IlM) inhibited the VEGF response in ECs. The data suggest that metaphit is a direct potent blocker ofTRP channels that may be useful as an anti-angiogenic agent. Cholesterol inhibited TRPC5 and potentiated TRPM2. It was shown to dissipate TRPC5 from the cell surface. In ECs, cholesterol regulated the Ca2+ -signal induced by histamine and VEGF. Interestingly, TRPCl siRNA indicated that cholesterol determines the molecular basis of the ECs Ca2+ -signal (i.e. TRPC 1 channel function required cholesterol). In the absence of cholesterol, histamine-induced Ca2+ -entry was mediated by other channels types and probably the CRAC channel based on the demonstrated sensitivity to synta-66 compound. To investigate the role of the tr~slocon as a passive ER Ca2+-leak mechanism in VSMCs, protein synthesis inhibitors, puromycin, anisomycin and emetine were used. The ligands inhibited radioactive methionine incorporation. The translocon opener, puromycin, evoked a transient elevation in cytoplasmic Ca2+ that was abolished by thapsigargin and inhibited by the translocon blockers, anisomycin and emetine, indicating the capacity of the translocon to act as an ER Ca2+ -leak channel. However, the translocon ligands had no effect on thapsigargin, ionomycin, ATP or " endothelin-l-induced Ca2+ -release, indicating that the translocon is normally closed and lacks relevance to calcium leak in VSMCs. In summary, this study showed novel modulation of TRP channels and endogenous Ca2+ -signals by Sig-I R ligands, importance of cholesterol in TRP channel function and capability of the translocon as a Ca2+ -leak pathway in proliferating VSMCs but excluded its physiological role.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.555856  DOI: Not available
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