[3H]RS79948-197 binding to human, rat, guinea pig and pig α2A-, α2B- and α2C-adrenoceptors. Comparison with MK912, RX821002, rauwolscine and yohimbine

https://doi.org/10.1016/S0014-2999(97)01521-5Get rights and content

Abstract

The Kd values of the recently introduced radioligand [3H]RS79948-197 ((8aR,12aS,13aS)-5,8,8a,9,10,11,12,12a,13,13a-decahydro-3-methoxy-12-(ethylsulphonyl)-6H-isoquino[2,1-g][1,6]naphthyridine) were determined for the recombinant human and rat α2A-, α2B- and α2C- as well as guinea pig α2B- and α2C-adrenoceptors expressed in COS (CV-1 Origin, SV40) cells. In addition, the Kd values were also determined for [3H]RS79948-197 for the guinea pig spleen α2A-adrenoceptor and for pig α2A-, α2B- and α2C-adrenoceptors in membranes obtained from kidney and striatum. Available radioligands for α2-adrenoceptors, besides [3H]RS79948-197 are the tritiated forms of MK912 ((2S,12bS)1′,3′-dimethylspiro(1,3,4,5′,6,6′,7,12b-octahydro-2H-benzo[b]furo[2,3-a]quinazoline)-2,4′-pyrimidin-2′-one), RX821002 (2-methoxy-idazoxan), rauwolscine and yohimbine. In the present article the binding constants of all these substances for the α2A-, α2B- and α2C-adrenoceptor subtypes in human, pig, rat and guinea pig are reviewed. In all species tested MK912 was α2C-selective, RX821002 showed a minor α2A-selectivity, whereas [3H]RS79948-197 was non-selective among the α2-adrenoceptor subtypes, showing high affinity for all three subtypes. Rauwolscine and yohimbine showed relatively low affinities for most of the α2-adrenoceptor subtypes investigated, the exception being rauwolscine having high affinity for the human and porcine α2C-adrenoceptors.

Introduction

Studies of the recombinant α2-adrenoceptor subtypes have shown that they correlate pharmacologically well with the corresponding subtypes of native tissues (Uhlén et al., 1992; Bylund et al., 1992). In a report from the `IUPHAR Nomenclature of Adrenoceptors Committee' these α2-adrenoceptor subtypes were designated α2A-, α2B- and α2C (Bylund et al., 1994). The rat and the human orthologs of the α2A-adrenoceptor show discrepant pharmacology and the rat ortholog has been designated as the α2D-adrenoceptor, but there is now the general consensus that there are only three α2-adrenoceptor subtypes within one single species (Hieble and Bond, 1994).

In the present study, the Kd values of the recently introduced radioligand [3H]RS79948-197 ((8aR,12aS,13aS)-5,8,8a,9,10,11,12,12a,13,13a-decahydro-3-methoxy-12-(ethylsulphonyl)-6H-isoquino[2,1-g][1,6]naphthyridine; Hume et al., 1996; Milligan et al., 1997) were determined for the expressed human and rat recombinant α2A-, α2B- and α2C-, for guinea pig recombinant α2B- and α2C-adrenoceptors and for guinea pig native α2A-adrenoceptors in spleen membranes. In addition, we determined the affinity of [3H]RS79948-197 for the pig α2A-, α2B- and α2C-adrenoceptors in striatal and kidney membranes.

Available α2-adrenoceptor radioligands, besides [3H]RS79948-197, are [3H]MK912 ((2S,12bS)1′,3′-dimethylspiro(1,3,4,5′,6,6′,7,12b-octahydro-2H-benzo[b]furo[2,3-a]quinazoline)-2,4′-pyrimidin-2′-one), [3H]RX821002 (2-methoxy-idazoxan), [3H]rauwolscine and [3H]yohimbine. When radioligand binding is applied in tissues containing more than one α2-adrenoceptor subtype, the less abundant population could often be detected only by the use of a subtype-selective radioligand. For example, the use of the α2C-selective radioligand [3H]MK912 (Pettibone et al., 1989) enabled the detection of both α2A- and α2C-adrenoceptors in the rat spinal cord (Uhlén et al., 1992), even though the α2C-adrenoceptors constituted only 5% of the total α2-adrenoceptor population in this particular tissue.

In some recent studies from our laboratory, we determined the Kd values of MK912, RX821002, rauwolscine and yohimbine for α2A-, α2B- and α2C-adrenoceptors in different species. From the present and our previous studies, where exactly the same assay conditions were used, we are now able to compile a complete table of the Kd values and thereby the subtype selectivities, of [3H]RS79948-197, MK912, RX821002, rauwolscine and yohimbine for all of the human, rat, guinea pig and pig α2-adrenoceptor subtypes. The data should enable researchers to choose the most appropriate α2-adrenoceptor radioligands for their particular studies.

Section snippets

Isotopes and drugs

The following isotopes and drugs were used: [3H]MK912 (83 Ci/mmol) was purchased from New England Nuclear (NEN) through DuMedical AB, Stockholm, Sweden. [Ethyl-3H]RS79948-197 (76 Ci/mmol) was purchased from Amersham through Amersham Sweden AB, Solna, Sweden. For the structure of RS79948-197 see Hume et al. (1996). BDF8933 (4-fluoro-2-(imidazoline-2-ylamino)-isoindoline; Armah, 1988) was a gift from Beiersdorf AG, Hamburg, Germany; BRL44408

Determination of the Kd values of [3H]RS79948-197 for the recombinant human α2A-, α2B- and α2C-adrenoceptors

In order to determine the Kd values of [3H]RS79948-197 for the human α2A-, α2B- and α2C-adrenoceptors saturation experiments were performed. In these experiments one plain curve, representing total binding and one curve where binding was blocked by 2 μM BDF8933 (representing non-specific binding), were obtained. As can be seen in Table 1 the Kd values of [3H]RS79948-197 for the human α2-adrenoceptors ranged between 0.46 and 0.77 nM.

Determination of the Kd values of [3H]RS79948-197 for the recombinant rat α2A-, α2B- and α2C-adrenoceptors

The Kd values of [3H]RS79948-197 for the rat α2A-, α2B- and α2C

Discussion

In the present paper the binding properties of the recently introduced α2-adrenoceptor antagonist radioligand [3H]RS79948-197 were investigated. It was found that [3H]RS79948-197 had high affinity for each of the human, rat, pig and guinea pig α2A-, α2B- and α2C-adrenoceptors. Combined with its low non-specific binding, this makes [3H]RS79948-197 a good general radioligand for α2-adrenoceptors.

For the human and rat α2A-, α2B- and α2C-adrenoceptors, and for the guinea pig α2B- and α2C

Acknowledgements

Supported by the Swedish MRC (04X-05957) and the Berth von Kantzow, the Magnus Bergwall, the Clas Groschinsky and the Åke Wiberg foundations.

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