The imidazoline₂ binding sites (I₂BS) have been described in a number of species. I₂BS selective compounds are known to inhibit the monoamine oxidase A and B (MAO) isozymes at micromolar concentrations. A previous study looking at [³H]idazoxan labelling of I₂BS in whole brain homogenates of MAO-A and MAO-B knockout mice concluded that I₂BS are located exclusively on the B isoform (Remaury et al., 2000). However, studies in our laboratory suggest that the population of I₂BS and autoradiographical distribution do not exclusively correlate with MAO-A or B. The aim of this study was to use the highly selective I₂BS ligand [³H]2BFI to investigate I₂BS in mouse kidneys devoid of functional MAO-A. The results have been compared with data from wildtype animals in order to profile the extent of I₂BS associated with MAO-A.

Kidneys from C3H/He mice of both sexes (8 MAO-A knockout (KO) and 8 wildtype (WT); Cases et al., 1995) were obtained, and 20 µm sections cut. Sequential sections were incubated with the radioligands at room temperature as follows, specific binding and wash protocols were as published (Lione et al., 1998; Saura et al., 1994): 3 nM [³H]Ro41-1049 (60 min), 5 nM [ 3H]idazoxan + 5 µM rauwolscine (40 min) and 1.5 nM [ ³H]2BFI (40 min). The labelled sections were apposed to Kodak Biomax film with tritium standards. The regional binding (cortex, inner and outer medulla) was quantified by computer aided densitometry (MCID 5, Imaging Associates, UK).

The regional labelling of MAO-A ([³H]Ro41-1049) in the wild-type kidneys concurred with previously published data (Saura et al., 1994). In the knockout kidneys, MAO-A binding was reduced to background levels (two way ANOVA, P<0.0001). I₂ binding as shown by [ ³H]idazoxan (+ rauwolscine) was also significantly lower (P<0.0001) in the knockouts, and this finding was replicated using the selective I₂ radioligand [³H]2BFI (Table 1). In conclusion, these data indicate that in kidney at least, I₂ binding is in part associated with MAO-A.

Table 1 – MAO-A ([³H]Ro41-1049) and I₂ ([³H]idazoxan and [³H]2BFI) binding to the mouse kidney sections. Data are expressed in fmol.mg -1 wet tissue ± the standard deviation.

Cases, O, et al. (1995) Science, 268, 1763-6
Lione, LA, et al. (1998) Eur. J. Pharmacol., 353, 123-35
Remaury, A, et al. (2000) Mol. Pharmacol., 58, 1085-90
Saura, J, et al. (1994) Neurobiol. Aging, 15, 399-408

[³H]Ro41-1049 was kindly donated by Dr. Borroni at Hoffmann La Roche