A study of the function and distribution of alpha1-adrenoceptors in the mouse carotid artery

Methven, Laura (2007) A study of the function and distribution of alpha1-adrenoceptors in the mouse carotid artery. PhD thesis, University of Glasgow.

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Abstract

The primary objective of these experiments was to examine the function and distribution of the alpha1-adrenoceptor (alpha1-AR) subtypes in the mouse carotid artery. The aims were achieved by using wire myography for the functional studies and confocal microscopy for the studies of alpha1-AR distribution. For both types of study, single and double knockouts of the alpha1-AR subtypes were employed, in addition to pharmacological analysis, to provide an insight into the alpha1-ARs in the wild type (WT) mouse. The aim of the first two studies presented in this thesis (Chapters Three and Four) was to establish whether an alpha1A-AR-mediated and/or alpha1e-AR-mediated contractile response exist in the mouse carotid artery, in addition to the predominant alpha1o-AR. It was found that the alpha1A-AR had a contractile role in this vessel based on pharmacological analysis in the WT mouse and knockout mice. Firstly, an alpha1-AR-mediated contractile response was identified in the alpha1B/o-KO. Secondly, the alpha1o-AR and another alpha1-AR subtype were identified as mediating the phenylephrine-induced response by employing subtype selective antagonists. Thirdly, the A-61603-induced response was found to be mediated by the alpha1A-AR and the alpha1D-AR using subtype selective antagonists. This also demonstrated that this alpha1A-AR selective agonist had an action on the alpha1o-AR. In the absence of am-AR selective compounds, comparison of the agonist responses in the WT mouse and knockout mice revealed that a minor alpha1e-AR-mediated response was also present. Thus, the am-AR predominantly mediates the vasoconstriction of the mouse carotid artery but the alpha1A-AR and alpha1B-AR appear to contribute to the contractile response. The aim of Chapter Five was to assess the effect of nitric oxide (NO) on the alpha1-AR- mediated contractile response in the mouse carotid artery. The contractile response to both phenylephrine and A-61603 was augmented by the nitric oxide synthase inhibitor L- NAME. This suggested that the alpha1-AR-mediated contractile response was suppressed by NO. An attempt was made to determine whether NO was released spontaneously in this vessel or in response to alpha1-AR stimulation. There was no evidence of constitutive NO release. The effect of L-NAME was greater with increasing concentrations of alpha1-AR agonist and a non-adrenergic response was shown to be unaffected by L-NAME. From these findings it is evident that in the mouse carotid artery activation of alpha1-ARs triggers NO release, which suppresses the alpha1-AR-mediated contractile response. Chapter Six aimed to examine the distribution of the alpha1-AR subtitles in the media of the mouse carotid artery. A protocol was developed to determine optimum conditions to visualise the binding distribution of the fluorescent alpha1-AR ligand Quinazoline Piperazine Bodipy (QAPB) in the media of this vessel. Comparison of QAPB binding in the WT mouse with the alpha1-AR knockout mice demonstrated that the alpha1o-AR was a major component of the alpha1-AR population. The use of non-fluorescent subtype selective antagonists to compete for QAPB binding sites revealed that the alpha1A-AR and alpha1e-AR also appeared to exist in the media of the WT mouse. Comparison of the QAPB subcellular binding distribution in the WT mouse and knockout mice suggested that the alpha1e-AR may be predominantly located on the cell surface, while, in the absence of the alpha1B-AR, the alpha1A- AR and am-AR may be predominantly located at intracellular sites. The final study in this thesis (Chapter seven) aimed to establish whether alpha1-ARs exist on the endothelium of the mouse carotid artery. Evidence of alpha1-ARs on endothelial cells (EC) was found both in the WT and knockout mice through the use of QAPB and non- fluorescent subtype selective antagonists. The proportion of EC with QAPB binding was reduced in the knockout mice indicative of the loss of an entire alpha1-AR population compared to the WT mouse. Comparing QAPB binding in the WT mouse and knockout mice in the presence of the non-fluorescent antagonists provided evidence that all three alpha1- AR subtypes are present on the endothelium in the carotid artery of the WT mouse. Collectively, the findings of the studies presented in this thesis demonstrate that all three alpha1-ARs are present and are functional in the mouse carotid artery. The alpha1o-AR is confirmed as being the predominant contractile alpha1-AR subtype but both the alpha1A-AR and ttiB-AR do have minor contractile roles in this vessel. furthermore, the activation of alpha1- ARs on the endothelium may result in the release of NO and subsequently cause the suppression of the alpha1-AR-mediated contractile response.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Ian McGrath
Keywords: Pharmacology
Date of Award: 2007
Depositing User: Enlighten Team
Unique ID: glathesis:2007-71113
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 10 May 2019 10:49
URI: http://theses.gla.ac.uk/id/eprint/71113

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