Geneeskundige Stichting Koningin Elisabeth ... - GSKE - FMRE

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et al., 1992; Panek et al., 1995), coexistence of different receptor subpopulations (Zhang et
al., 1993) and the ability of the antagonist to affect the internalisation of AT 1 receptors within
the cell (Liu et al., 1992).
Molecular action mechanism of AT 1 receptor antagonists.
A similar discrimination between AT 1 receptor antagonists can also be made by investigating
their effect on angiotensin II-mediated responses in intact cell systems but, so far, only limited
attention has been spend to this approach. Yet, there are major technical advantages such
as the ability to measure direct [ 3H]-antagonist binding and inhibition of agonist induced responses
under identical experimental conditions (Fierens et al., 1999a). In this context, Chinese
Hamster Ovary cells have been permanently transfected with the gene coding for the human
AT 1 receptor (CHO-hAT1 cells) (Vanderheyden et al., 1999). These cells express human AT 1
receptors at their surface and their intensive investigation in our laboratory has shed new light
on the molecular action mechanism of nonpeptide AT 1 receptor antagonists. The major findings
can be summarised as follows:
a) When a pre-incubation step with the antagonist is included, surmountable and insurmountable
antagonists can be discriminated from each other by measuring the angiotensin IImediated
IP production in CHO-hAT1 cells. Interestingly, it was already noticed by Criscione et
al. (1993) that valsartan, an insurmountable antagonist in contraction studies, was unable to
depress the maximal angiotensin II- induced aldosterone release in bovine adrenal glomerulosa
cells. These latter experiments were done under co-incubation conditions. Similarly, co-incubation
with candesartan, EXP3174 and irbesartan only produced parallel rightward shifts of the
angiotensin II dose-response curves in CHO-hAT1 cells (Fierens et al., 1999b). Therefore, it
appears that all AT 1 receptor antagonists are competitive with respect to angiotensin II.
Furthermore we have experimental data that indicate that the investigated antagonists bind to
a common or overlapping site on the receptor in a mutually exclusive way (Vanderheyden et
al., 2000a). The ability of certain antagonists to decrease the maximal effect of angiotensin II
is therefore the result of the experimental set-up (i.e. antagonist pre-incubation) and should not
be ascribed to non-competitive behaviour.
b) Radioligand binding experiments reveal that AT 1 receptor antagonists are able to dissociate
from their receptor. Hence, irreversible binding can be excluded as an explanation for
insurmountable antagonism. Instead, insurmountable inhibition is merely related to the slow dissociation
rate of the antagonist- receptor complex: i.e. the blockade is sufficiently long lasting
to prevent the access of the receptors to subsequently added angiotensin II. In contrast, the effect
of surmountable antagonists such as losartan is very brief. Direct (radioligand dissociation binding)
and indirect (restoration of angiotensin II- mediated responses after antagonist preincubation)
estimations of the half-life of antagonist- receptor complexes in CHO-hAT1 cells are 7
min for irbesartan, 17 min for valsartan, 30 min for EXP3174 and 120 min for candesartan
(Fierens et al., 1999a; Vanderheyden et al., 2000a,b; Verheijen et al., 2000).