[3H]Rilmenidine-labelled imidazoline-receptor binding sites co-localize with [3H]2-(benzofuranyl)-2-imidazoline-labelled imidazoline-receptor binding sites and monoamine oxidase-B in rabbit, but not rat, kidney

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Abstract

The distribution and relative densities of imidazoline-receptor binding sites (I-RBS) and monoamine oxidase (MAO)-A and -B enzyme(s) in rat and rabbit kidney were compared autoradiographically using fixed nanomolar concentrations of [3H]rilmenidine and [3H]2-(benzofuranyl)-2-imidazoline ([3H]2-BFI) to label I-RBS, and [3H]RO41-1049 and [3H]RO19-6327 to label MAO-A and -B isoenzymes, respectively. In rat kidney, high densities of I-RBS labelled by [3H]rilmenidine were observed in the cortex and outer stripe (120–280 fmol/mg tissue), in contrast to low I-RBS densities labelled by [3H]2-BFI (<4 fmol/mg). A relatively high density of [3H]RO41-1049 binding to MAO-A enzyme was present in all regions of the rat kidney (160–210 fmol/mg) compared with a low density of [3H]RO19-6327 binding to MAO-B (<25 fmol/mg). Comparison of MAO-A and -B distributions with that of [3H]rilmenidine-labelled I-RBS strongly suggests a lack of association in rat kidney. Similarly, the extremely low densities of [3H]2-BFI-labelled I2-RBS in rat kidney contrasts with the density of MAO-A, but is consistent with the low density of MAO-B. Rabbit kidney cortex and outer stripe contained high relative densities of [3H]rilmenidine-labelled I-RBS (200–215 fmol/mg) and [3H]2-BFI-labelled I2-RBS (45–60 fmol/mg) with lower densities in the inner stripe and inner medulla (⩽100 and 30 fmol/mg respectively). A high density of MAO-A binding was observed in the inner stripe (515 fmol/mg) with lower levels in the cortex and outer stripe (100–240 fmol/mg), while high densities of MAO-B binding were observed in the cortex and outer stripe (290–450 fmol/mg) with lower levels in the inner stripe (65 fmol/mg). The correlation between the localization of [3H]rilmenidine-labelled I-RBS and [3H]RO19-6327-labelled MAO-B in rabbit kidney (r=0.87, P=0.057) suggest that [3H]rilmenidine may label a binding site co-existent with MAO-B, but not MAO-A (n.s.), in this tissue, but rilmenidine did not inhibit [3H]RO41-1049 or [3H]RO19-6327 binding. The distribution of [3H]2-BFI-labelled I2-RBS overlapped the combined distributions of both MAO-A and -B isoenzymes, suggesting that [3H]2-BFI may label sites on both enzymes in the rabbit, but [3H]2-BFI binding only correlated with [3H]RO19-6327 (r=0.84, P=0.07), not [3H]RO41-1049 binding (n.s.). Moreover, 2-BFI only inhibited [3H]RO19-6327, not [3H]RO41-1049 binding. These data are consistent with reports that I2-RBS are located on MAO-B and allosterically influence the catalytic site. The relationship of [3H]rilmenidine- and [3H]2-BFI-labelled I-RBS and the identity of non-MAO-associated [3H]rilmenidine-labelled I-RBS requires further investigation.

Introduction

Antihypertensive agents such as rilmenidine and clonidine are reported to act via α2-adrenoceptors and/or imidazoline receptor binding sites (I-RBS). [3H]Rilmenidine has been reported to label both α2A-adrenoceptors and novel I2B like-RBS throughout the rat central nervous system (King et al., 1992, King et al., 1995a). High densities of [3H]rilmenidine-labelled I2B like-RBS were located in the arcuate nucleus, ependymal cell layer of the third ventricle, interpeduncular nucleus and pineal gland (King et al., 1995a), regions shown to also contain high relative densities of [3H]idazoxan-labelled I2-RBS (Mallard et al., 1992), as well as MAO-B enzymes (Willoughby et al., 1988; Saura et al., 1992). In addition, in situ hybridization histochemistry studies reveal a high density of MAO-B mRNA localized in these regions of the rat brain (Luque et al., 1995).

Monoamine oxidase (MAO) enzymes are located on the outer mitochondrial membrane and are responsible for the deamination of biogenic and trace amines. Substrate selectivity and inhibitor sensitivity form the basis of classification of two isoforms of the enzyme. MAO-A is selectively inhibited by nanomolar concentrations of clorgyline, and MAO-B by l-deprenyl. The strong association of I2-RBS and MAO enzymes is well documented in that they share common subcellular localization in human and rabbit kidney and liver (Lachaud-Pettiti et al., 1991; Tesson et al., 1991); chronic treatment of rats with MAO inhibitors decreases I2-RBS densities in rat brain and liver (Olmos et al., 1993; Alemany et al., 1995, Alemany et al., 1997); I2-RBS and MAO-B densities positively correlate with aging in human brain (Sastre and Garcı́a-Sevilla, 1993); elevated levels of MAO enzyme activity and I2-RBS occur in Alzheimic brain (Ruiz et al., 1993; Saura et al., 1994); MAO and I2-RBS have high (micro)sequence homology; and expression of MAO-A and -B in yeast results in a co-expression of I2-RBS (Tesson et al., 1995). However, biochemical studies have revealed photoaffinity/radioligand labelling of I2-RBS was unaffected by MAO inhibitors such as pargyline (Lanier et al., 1995) and that MAO activity was inhibited by some imidazoline derivatives, but not idazoxan itself (Carpéné et al., 1995), suggesting the location of I2-RBS at a site distinct from the catalytic site on MAO enzyme molecules.

In light of these findings and previous studies in our laboratory on the distribution and characterization of [3H]rilmenidine-labelled I-RBS in rat kidney (King et al., 1995b) and I2-RBS labelled by the selective ligand [3H]2-BFI in rabbit kidney membranes (Hosseini et al., 1997), the aim of this study was to directly compare the regional distribution of [3H]rilmenidine- and [3H]2-BFI-labelled I-RBS with that of [3H]RO41-1049- and [3H]RO19-6327-labelled MAO-A and MAO-B enzymes in rat and rabbit kidney to determine whether [3H]rilmenidine- and/or [3H]2-BFI label I-RBS that are independent of MAO isoenzymes.

Section snippets

Animals

Ethical approval was granted for all procedures associated with these studies by the Austin and Repatriation Medical Centre Animal Welfare Committee and experiments were carried out according to guidelines issued by the National Health and Medical Research Council of Australia. Dutch rabbits and Wistar–Kyoto rats were obtained from the Biological Research Laboratories at the Austin and Repatriation Medical Centre. Male Wistar–Kyoto rats (250 g) were stunned and killed by decapitation. Rabbits

Rat

In rat kidney, [3H]rilmenidine labelled a high density of non-adrenergic binding sites in the cortex and outer stripe of rat kidney (Fig. 1A, B) that were not inhibited by the imidazole derivatives, imidazole-4-acetic acid and cimetidine, or the imidazoline, idazoxan (Table 1). In contrast, very low levels of [3H]2-BFI binding were present in the rat kidney (Fig. 1D, E; Table 2). [3H]RO41-1049 labelled a high density of presumed MAO-A in all regions of the rat kidney (Fig. 1G; Table 3), while

Discussion

The present study reports the comparative localization and relative densities of I-RBS labelled by [3H]rilmenidine and [3H]2-BFI, with MAO-A and -B enzymes labelled by the highly selective radioligands, [3H]RO41-1049 and [3H]RO19-6327, in rat and rabbit kidney. Results demonstrate that species-dependent differences exist in the distribution and densities of both I-RBS and MAO isoenzymes and provide further evidence that I2-RBS are closely associated with MAO enzymes and that [3H]rilmenidine

Conclusion

In summary, the current findings suggest that [3H]rilmenidine labels a high density of novel I-RBS in rat kidney and multiple I-RBS in rabbit kidney that are presumably localized to the proximal tubules in the outer stripe and medullary rays in the cortex. The pattern of [3H]rilmenidine binding is unlike the distribution pattern of MAO-A and -B in rat kidney, demonstrating [3H]rilmenidine-labelled sites are predominantly not associated with either enzyme in this species. Therefore, the rat

Acknowledgements

This work was supported by grants from the National Health and Medical Research Council of Australia, and the Austin Hospital Medical Research Foundation. S.S. was the recipient of an Aichi Medical University Research Scholarship and A.R.H. was the recipient of a scholarship from the Ministry of Health and Medical Education, Iran.

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  • Cited by (5)

    1

    Present address: Owari Asahi Clinic, 2505-1 Harada, Higashi Daidoucho, Owari Asahi, Aichi 488, Japan.

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    Present address: PO Box 358, Malek Ashtar Post Office, Tehran 13475, Iran.

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