Structural and functional studies of caveolae in the femoral artery



Albrakati, Ashraf
Structural and functional studies of caveolae in the femoral artery. Doctor of Philosophy thesis, University of Liverpool.

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Abstract

Caveolae are important microdomains found in the plasma membrane, which act as signalling hubs in endothelial cells (ECs) and smooth muscle cells (SMCs). Thus, disruption of caveolae by membrane cholesterol depleting agents such as methyl β-cyclodextrin (M-β-CD) has various functional effects on arteries, including impairment of endothelium-dependent relaxation and augmentation of smooth muscle cell contraction independently of the endothelium. The aim of this study was to test the hypotheses that caveolae modulate contractility in rat femoral artery, and more specifically the response to BKCa channel activity. Different methods were used in the study including; transmission electron microscopy (TEM), immunohistochemistry, immunocytochemistry and Western blot, and myography. M-β-CD was used in this work to disrupt caveolae from the plasma membranes of SMCs and ECs of rat femoral artery. TEM examination showed that caveolae were present throughout the plasma membrane of the SMCs and ECs in femoral artery. TEM examination also showed that treatment of the artery with M-β-CD, causes removal of caveolae from most of plasma membrane of the SMCs and ECs. Immunohistochemistry and immunocytochemistry results showed that cav-1, cav-3 and BKCa channels are co-expressed in SMCs, and cav-1 and BKCa channels are present in ECs. The presence of these proteins in femoral artery tissue lysate was confirmed by Western blot. Femoral artery contraction studies using myography showed that caveolar disruption by M-β-CD caused a significant increase in the contraction in endothelium-intact artery rings in response to 20 mM K+ and 100 nM BayK-8644 (20 K/ Bay K). In endothelium-denuded artery rings and/or after incubation of endothelium-intact artery rings with L-NAME (an eNOS inhibitor), there was a non-significant increase in the contraction in response to 20K/ BayK. Incubation of endothelium-intact artery rings with TEA+ or IBTX, both BKCa channel inhibitors, caused a significant increase in the contraction to 20 K/ Bay K. Incubation of endothelium-intact artery rings with L-NAME, reduced the contraction to TEA+ and IBTX. These results provide evidence that contraction of rat femoral artery is inhibited by basal nitric oxide release from the endothelium, and BKCa channels in the SMCs might have a role in this vasorelaxation. Further femoral artery relaxation studies showed that the integrity of caveolae was important for the vasorelaxation of arteries (pre-contracted with 20 K/ Bay K) when BKCa channels were directly activated by NS-1619. In contrast, vasorelaxation by isoproterenol and forskolin in endothelium-denuded artery was not significantly altered after caveolae were disrupted. These data together suggest that caveolae are an important factor in maintaining Ca2+ homeostasis in SMCs in rat artery femoral due to regulation of contractile activation.

Item Type: Thesis (Doctor of Philosophy)
Additional Information: Date: 2013-07 (completed)
Subjects: ?? QP ??
Divisions: Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology
Depositing User: Symplectic Admin
Date Deposited: 08 Jan 2014 16:26
Last Modified: 16 Dec 2022 04:39
DOI: 10.17638/00012775
Supervisors:
  • Quayle, John
  • Dart, Caroline
URI: https://livrepository.liverpool.ac.uk/id/eprint/12775