Many irreversible antagonists are known to bind irreversibly to pharmacological
receptors. However, few studies suggest that these irreversible antagonists may also
display irreversible non-specific antagonism by binding irreversibly to non-syntopic
binding sites on the receptor macromolecule, whereby they modulate the signal
transduction of these receptors or reduce the agonist binding affmity.
The aim of this study was to investigate whether the classical irreversible antagonists
phenoxybenzamine, benextramine and 4-DAMP mustard display irreversible nonspecific
antagonism at various G protein-coupled receptor (GPCR) types. In addition,
the subcellular mechanism whereby benextramine displays irreversible non-specific
antagonism was investigated.
Three cell lines were employed to investigate the antagonism by these irreversible
antagonists: Chinese hamster ovary (CHO-K1) cells transfected to express the porcine
a2A-adrenoceptor (a2A-AR) at higher (a2A-H) or lower (a2A-L) numbers, human
neuroblastoma (SH-SY5Y) cells that endogenously express muscarinic acetylcholine
receptors (mACh-Rs), and SH-SY5Y cells transfected (5HT2A-SH-SY5Y)o express
the human 5HT2A-serotonirne ceptor (5HTZA-R).C ells of the appropriate cell line were
pre-treated at the appropriate concentrations and incubation times with an appropriate
irreversible antagonist, with or without an appropriate reversible competitive antagonist
at a sufficient concentration to protect the specific receptors. This was followed by
washing procedures with drug-free media to rinse any unbound or reversibly bound
drugs from the cells. When appropriate, cell membranes were prepared. Receptor
function was evaluated by measuring whole-cell [3H]-cAMP or [3H]-IPx acumulation,
or the binding of [35S]-GTPyS to membraness. Receptor concentrations were
determined from radioligand-binding assays. In addition, the constitutive [35S]-GTPyS binding to Go protein before and after pre-treatment with benextramine was investigated.
Results suggest that phenoxybenzamine (100 uM, 20 minutes) and benextramine (10
uM, 20 minutes) display irreversible non-specific antagonism at a2A-ARs when measuring Gi-mediated effects in a2A-L cells, but the affinity for a2A-ARs in a2A-H cells was not changed. In addition, it was found that the observed irreversible nonspecific antagonism by benextramine appears to be time- and concentration-dependent.
When the mechanism of irreversible antagonism by benextramine was further investigated, benextramine reduced the binding of [35S]-GTPyS to a2A-H membranes with protected a2A-ARs, but did not modulate the constitutive binding of [35S]-GTPyS to Go. In addition, benextramine displays irreversible non-specific antagonism by
inhibiting the G,-mediated effects of a2A-ARs in a2A-H cells and the Gq-mediated
effects of mACh-Rs or 5HT2A-Rs in SH-SY5Y or 5HT2A-SH-SY5Y cells respectively.
4-DAMP mustard (100 uM, 20 minutes) did not display irreversible non-specific
antagonism at mACh-Rs in SH-SY5Y cells, but irreversible non-specific antagonism
was observed when the incubation time was increased (100 uM, 60 minutes).
In conclusion it was found that phenoxybenzamine, benextramine and 4-DAMP
mustard display irreversible non-specific antagonism at typical experimental
conditions. These findings confirm concerns in literature and supports the possibility
that more irreversible antagonists could display irreversible non-specific antagonism,
and that could influence the interpretation of data obtained with such drugs. In
addition, benextramine may prove to be a useful experimental drug in studying GPCR
signalling. / Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2004.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:nwu/oai:dspace.nwu.ac.za:10394/92 |
| Date | January 2003 |
| Creators | Bodenstein, Johannes |
| Publisher | North-West University |
| Source Sets | South African National ETD Portal |
| Detected Language | English |
| Type | Thesis |
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