Cellular distribution of alpha1-adrenoceptors using visualisation and radioligand binding techniques

Caldwell, Darren (2003) Cellular distribution of alpha1-adrenoceptors using visualisation and radioligand binding techniques. PhD thesis, University of Glasgow.

Abstract

Cellular distributions of alpha1-adrenoceptors (ARs) were assessed using visualisation and radioligand binding techniques in Rat-1 fibroblasts stably expressing the bovine alpha1a -AR. Competitive radioligand binding (RLB) was compared in whole cell and isolated membrane preparations using both classical and updated RLB protocols. A range of agonists and antagonists displayed their known characteristics in membranes. Whole cell competitive RLB for most ligands displayed a high affinity binding site, consistent with a population of -25% of the sites that had affinity ≥ in membranes, whereas the other 75% had affinity variably lower than in membranes according to the competitor. Results suggest the high affinity component represents displacement of 3H-prazosin from cell surface receptors and the low affinity component that follows a slow sustained plateau phase represents a displacement from intracellular binding sites. Displacement by competing ligands occurred in a time dependent, temperature- independent manner. An early hypothesis that ligands penetrated the cell and accessed intracellular compartments via an extraneuronal monoamine transporter (EMT) was dispelled following the inability of corticosterone to block the uptake of ligands entering cells. A more likely method of how ligands access intracellular compartments is by endocytosis via clathrin-coated pits. Concanavalin A is an endocytotic blocker and exerted an inhibiting effect that could be overcome once a certain concentration of antagonist (QAPB) had been applied. It did, however, exert little effect on the ability of an agonist (adrenaline) to displace radiolabelled prazosin from intracellular binding sites. The possibility of an endocytotic pathway for cellular penetration was supported by direct visualisation studies. Following a sustained period of time, the fluorescent alpha1- AR antagonist BODIPY-FL prazosin (QAPB) was detected in high fluorescent intensity "hot spots" throughout the cytosol. The intensity of the fluorescent signal steadily decreased following cumulative applications of the alpha1-AR agonist phenylephrine and was also reversible upon phenylephrine removal. This indicates that not only can antagonists penetrate cells and bind to intracellular compartments, but also agonists can penetrate cells and subsequently displace antagonist from these intracellular binding sites. The ability of antagonists to selectively compete at the cell surface and intracellular regions was validated by results showing the alpha1-AR antagonist phentolamine displacing QAPB from both cell surface and intracellular binding sites. Colocalisation with fluorescent dyes that can identify subcellular compartments (Transferrin Alexa Fluor546) indicated partial localisation with the early and recycling endosomes. This strongly suggests that alpha1a-AR ligand complexes follow the endocytotic recycling pathway, via early and recycling endosomes. Using microspectrofluorimetry techniques, phenylephrine-activated Ca2+ signals were recorded from single Rat-1 fibroblasts stably expressing the bovine alpha1a-AR and used to analyse functional agonist-antagonist interactions. The results demonstrate that the dominant Ca2+ entry pathway evoked by the bovine alpha1a -AR is store-operated, in which the Ca2+ content of agonist-sensitive intracellular Ca2+ stores governs Ca2+ influx. Beyond the concentration of phenylephrine that produced the maximum response (>3xlO-5M), the concentration-response relationships displayed fade, which could be explained by agonist-dependent depletion of Ca2+ stores. Antagonism exhibited by QAPB produced concentration-dependent, nonparallel, rightward displacements in the control concentration-Ca2+ response curve to phenylephrine with a decrease in maximal response. This represents an example of insurmountable antagonism, where the antagonist dissociates very slowly (or not at all) from the receptor. It is demonstrated by a rightward shift as the antagonist concentration increases and also by a decrease in maximal response.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Ian McGrath
• Keywords: Biochemistry.
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: McGrath, Prof. Ian
• Date of Award: 2003
• Depositing User: Enlighten Team
• Unique ID: glathesis:2003-41126
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 15 Apr 2019 15:06
• Last Modified: 07 Jun 2021 15:50
• URI: https://theses.gla.ac.uk/id/eprint/41126