Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

A fluorescent quinazoline derivative, QAPB retains high affinity for alpha-1 adrenoceptors. We have previously described the characterisation and cellular localisation of alpha-1 adrenoceptors in recombinant human alpha-1a, alpha-1b and alpha-1d adrenoceptor subtypes using both radioligand binding and fluorescent ligand binding. The latter method is now used to study dissociated native smooth muscle cells. Briefly, cells were incubated with cumulative doses of QAPB (0.4-10nM) in the presence and absence of 10uM phentolamine. Serial z-section images were acquired at equilibrium for each concentration using a CLSM and 3D-iso-surface modules were constructed using IMARIS software. Fluorescent binding was time and concentration dependent in native smooth muscle cells. In the presence of phentolamine (10mM) QAPB-binding was significantly inhibited and the residue was used to calculate non-specific binding. Specific binding curves were constructed and fluorescence KD (FKD) values calculated which were similar to KD values determined from radioligand binding studies. New quantitative methods for analysis of fluorescence in 3-D were devised so that we could distinguish receptors on the cell surface from those inside the cells. Fluorescence was found to be closely associated with the cell membrane and subcellular compartments around the nuclear membrane.

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Poster Number PAmackenzie0899
Keywords: adrenoceptor, quinazoline, cell, binding, fluorescence