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Title: Functional role of myristoylated alanine-rich C-kinase substrate in regulating vascular tone
Author: Jahan, Kazi Sultana
ISNI:       0000 0004 8502 8802
Awarding Body: St George's, University of London
Current Institution: St George's, University of London
Date of Award: 2019
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Abstract:
Background: Phosphatidylinositol 4, 5-bisphosphate (PIP2) plays a key role in regulating contraction of vascular smooth muscle cells (VSMCs) through acting as a phospholipase C (PLC) substrate for Gaq/n receptor-mediated signalling and as a direct ligand of ion channels and transporters. But, how does PIP2 have two distinct roles in regulating vascular contractility? A possible explanation is that cells contain two pools of PIP2, a global pool of PIP2 accessible by PLC, and local PIP2 microdomains, produced by PIP2-binding proteins, that act as a gating ligand. To date, there have been no studies on the effect of PIP2-binding proteins on vascular contractility. This thesis investigated the role of the PIP2-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) in such a role. Methods and Results: Western blotting and immunocytochemistry showed that MARCKS is expressed in rat and mice mesenteric artery. Wire myography revealed that disinhibition of MARCKS using MANS peptide (MANS) produced vasoconstrictions which were inhibited by L- and T-type voltage-dependent Ca2+ channel (VDCC) blockers. Co-immunolabelling and proximity ligation assay demonstrated that MARCKS and CaV1.2 proteins co-localise at the plasma membrane (PM), and that MANS and methoxamine (MO) reduced these interactions and cause translocation of MARCKS from the PM to the cytosol. Dot-blotting revealed that PIP2 preferentially binds to MARCKS and not CaV1.2, and that MANS and MO increase interactions between PIP2 and CaV1.2 whilst reducing associations between PIP2and MARCKS. Whole-cell recordings showed that MANS increased peak amplitude of VDCC currents and shifted activation curve to more negative membrane potentials, which was prevented by depleting PIP2 levels. Knockdown of MARCKS using morpholinos significantly reduced contractions induced by MANS and Gaq/n receptor agonists. Conclusions: This thesis shows for the first time that in resting, unstimulated vessels MARCKS has a critical inhibitory effect on vascular contractility through acting as a PIP2 buffer. Disinhibition of MARCKS by MANS or vasoconstrictors induces release of PIP2 into the local environment that binds to, and increases, VDCC activity leading to subsequent contraction.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.795562  DOI: Not available
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