Geneeskundige Stichting Koningin Elisabeth ... - GSKE - FMRE

46
et al., 1992). At present, no information is available about the factors that might potentially
affect the local accumulation of AT 1 receptor antagonists and, in particular, whether rebinding
requires a special organisation of the AT 1 receptor (e.g. presence in clusters) to take place.
The following experimental approach is proposed. For the receptor-binding characteristics,
we will investigate the antagonistic profiles of candesartan, EXP3174, telmisartan, eprosartan,
irbesartan and losartan by functional studies. Besides measuring the antagonist’s effects on the
angiotensin II- mediated IP production (Table 1a), these cells also offer the possibility of measuring
more distant and relevant functional responses (Table 1b). Provided that the receptor concentration
is sufficiently high, direct binding of [ 3H]candesartan, [ 3H]valsartan and [ 3H]irbesartan
can be investigated as well (Table 1c). If not, indirect information can be obtained by
competition and kinetic (i.e. delay of the association rate in antagonist- pre-treated cells, Hara
et al., 1995; Vanderheyden et al., 2000a) experiments using [ 125I]sarile.
In addition, certain of the above mentioned cell lines may offer the possibility to investigate
the effect of antagonists on angiotensin- mediated cell proliferation and differentiation.
Stimulation of AT 1 receptors will induce the proliferation of vascular smooth muscle cells by a
protein kinase C- dependent pathway (by activation of mitogen-activated protein kinases) as
well as by an independent pathway (by activation of tyrosine kinases such as Scr, Jak en Fak)
(Marrero et al., 1995; Berk, 1999). In contrast, stimulation of AT 2 receptors will inhibit cell differentiation
and cell growth (Schmitz et al., 1997). The L6C5 cell line derived from skeletal muscle
possesses both AT 1 and AT 2 receptors and it has been shown to constitute an excellent
model to investigate the effect of both receptor subtypes on cell differentiation (detected by the
fusion of myoblasts en myotubules and induced by insulin like growth factor 1 (IGF-1)) (Bottari
S.P. et al., personal communication). This cell line offers therefore the possibility to investigate,
on the same experimental system, the ability of AT 1 receptor antagonists to shift a predominantly
AT 1 receptor response to angiotensin II into an AT 2 receptor response.
Table 1
i) Inositol phosphate production
Goal experimental approach
AT 1 receptor activity angiotensin II concentration-response curve
Insurmountable inhibition antagonist inhibition curve (preincubation versus
co-incubation with angiotensin II)
Kinetic parameters delay in response to angiotensin II in washout
experiments (with losartan)
Rebinding same, without losartan