A combined pharmalogical/knockout approach to subtyping alpha1-adrenoceptors in murine tissues

The characterisation of adrenoceptor subtypes has traditionally been achieved using pharmacological methods. However, in recent years molecular biology and biochemical methods have become increasingly popular. Transgenic technology has provided a merging of pharmacology and molecular biology, whereby molecular biology is used to identify and manipulate the gene of interest whilst pharmacology is used to investigate the functional consequences of these genetic manipulations. The process of subtyping a1-adrenoceptor subtypes is often made more complicated by a lack of reliable subtype selective drugs, in particular for the a1B-adrenoceptor, for which there are no such compounds. The a1B-adrenoceptor KO mouse created by Cavalli et al (1997) provides a unique environment in which to study a1-adrenoceptor subtypes. The study of these KO mice may provide a valuable insight into the functional role of individual a1-adrenoceptor subtypes. For this reason my project investigated the functional consequences of deleting the a1B-adrenoceptor using traditional pharmacological methods including radioligand binding and wire myography and the more recently developed method of confocal microscopy. In Chapter 2,1 describe the effects of deletion of the a1B-adrenoceptor from the brain and livers of WT and KO mice. Using five subtype selective ligands (prazosin, RS100329, (R)-A-61603, L765,314 and BMY7378), radioligand binding was used to characterise the a1-adrenoceptor subtypes in WT and KO brain and liver tissue. The WT brain was found to contain a heterogeneous population of a1-adrenoceptors, consisting of a1A- and a1B-adrenoceptors\ whilst the KO brain had a smaller receptor number compared to the WT and a pure population of a1A-adrenoceptors. In the WT liver (4 month old) a pure population of a1B-adrenoceptors was determined to be present, consistent with previous reports (Cavalli et al, 1997; Yang et al, 1998). The KO liver (4 month old) was unexpectedly found to contain a population of a1-adrenoceptors: characterised to be a1A-adrenoceptors. Preliminary results from studies of 3 month old KO livers revealed that there are negligible amounts of a1-adrenoceptors present at this age, whereas in 3 month old WT liver a1-adrenoceptor density is higher compared with 4 month old livers. This study demonstrates the loss (without apparent replacement) of the aiB-adrenoceptor from the KO brain and the substitution of the a1A- adrenoceptor to compensate for the loss of the a1B-adrenoceptor in the KO liver. This upregulation of the a1A-adrenoceptor appears to occur between 3 and 4 months of age. Chapter 3 describes a general pharmacological characterisation of the mouse carotid artery and a detailed investigation into the characterisation of the a1-adrenoceptor subtype mediating contraction in WT and KO carotid arteries. The general pharmacology of the carotid artery was studied using a number of vasoactive compounds. Chapter 4 describes a study investigating the effects which deletion of the a1B-adrenoceptor has on the structure of the adventitia of WT and KO carotid arteries. The number and density of adventitial cells in the KO carotid artery was found to be significantly smaller than in the WT carotid artery. The implications of this are not yet certain, but the a1B-adrenoceptor has been implicated in growth (Chen et al, 1995; Zhang and Faber, 2001) which may account for the observed differences in the KO carotid artery compared with the WT. The results presented in this thesis have gone some way to furthering our knowledge of the a1B-adrenoceptor and demonstrate that the effects of deleting the a1B-adrenoceptor depend not only on the tissue in which it is expressed, but also on the cell type. The merging of transgenic technologies with pharmacological techniques (pharmacogenomics) provides the pharmacologist with information which would not be possible with drugs alone and looks set to be a way forward in clarifying the confusing pharmacology arising from the use of inadequate drugs