Identification of alpha 1L-adrenoceptor in mice and its abolition by alpha 1A-adrenoceptor gene knockout

Abstract

Background and purpose: The alpha(1L)-adrenoceptor has pharmacological properties that distinguish it from three classical alpha(1)-adrenoceptors (alpha(1A), alpha(1B) and alpha(1D)). The purpose of this was to identify alpha(1L)-adrenoceptors in mice and to examine their relationship to classical alpha(1)-adrenoceptors.

Experimental approach: Radioligand binding and functional bioassay experiments were performed on the cerebral cortex, vas deferens and prostate of wild-type (WT) and alpha(1A)-, alpha(1B)- and alpha(1D)-adrenoceptor gene knockout (AKO, BKO and DKO) mice.

Key results: The radioligand [(3)H]-silodosin bound to intact segments of the cerebral cortex, vas deferens and prostate of WT, BKO and DKO but not of AKO mice. The binding sites were composed of two components with high and low affinities for prazosin or RS-17053, indicating the pharmacological profiles of alpha(1A)-adrenoceptors and alpha(1L)-adrenoceptors. In membrane preparations of WT mouse cortex, however, [(3)H]-silodosin bound to a single population of prazosin high-affinity sites, suggesting the presence of alpha(1A)-adrenoceptors alone. In contrast, [(3)H]-prazosin bound to two components having alpha(1A)-adrenoceptor and alpha(1B)-adrenoceptor profiles in intact segments of WT and DKO mouse cortices, but AKO mice lacked alpha(1A)-adrenoceptor profiles and BKO mice lacked alpha(1B)-adrenoceptor profiles. Noradrenaline produced contractions through alpha(1L)-adrenoceptors with low affinity for prazosin in the vas deferens and prostate of WT, BKO and DKO mice. However, the contractions were abolished or markedly attenuated in AKO mice.

Conclusions and implications: alpha(1L)-Adrenoceptors were identified as binding and functional entities in WT, BKO and DKO mice but not in AKO mice, suggesting that the alpha(1L)-adrenoceptor is one phenotype derived from the alpha(1A)-adrenoceptor gene.

Figure 1

Binding of [³H]-silodosin and [³H]-prazosin to mouse cerebral cortex. (a) Saturation curves for specific binding of [³H]-silodosin in tissue segments and membrane preparations of WT mouse cortex and in tissue segments of AKO mouse cortex. (b) Binding density of [³H]-silodosin estimated from saturation experiments with WT, AKO, BKO and DKO mouse cortices. The saturation experiments were conducted with intact tissue segments (S) and membranes (M) of cerebral cortex. High- and low-affinity sites for prazosin at [³H]-silodosin binding sites were represented as α_1A and α_1L, respectively. (c) Binding density of [³H]-prazosin estimated from saturation experiments with intact segments of WT, AKO, BKO and DKO mouse cortices. High- and low-affinity sites for silodosin at [³H]-prazosin-binding sites were represented as α_1A and α_1B, respectively. The number of experiments was six in WT, five in AKO, four in BKO and three in DKO. ^*Significantly different from WT mouse (P<0.01). AKO, α_1A-adrenoceptor gene knockout; BKO, α_1B-adrenoceptor gene knockout; DKO, α_1D-adrenoceptor gene knockout; WT, wild type.

Figure 2

Competition curves for various antagonists in mouse cerebral cortex. (a and b) Competition curves for prazosin and RS-17053 at 500 pM [³H]-silodosin-binding sites in the intact segments and membranes of the WT mouse cortex. (c) Competition curves for silodosin and BMY 7378 at 500 pM [³H]-prazosin-binding sites in the intact segments of the WT mouse cortex. (d) Competition of silodosin in the intact segments of AKO, BKO, and DKO mouse cortices at 500 pM [³H]-prazosin-binding sites. Each curve is representative of similar results obtained in 2–5 experiments. AKO, α_1A-adrenoceptor gene knockout; BKO, α_1B-adrenoceptor gene knockout; DKO, α_1D-adrenoceptor gene knockout; WT, wild type.

Figure 3

Binding of [³H]-silodosin to mouse vas deferens and prostate. (a) Representative saturation curves for specific binding of [³H]-silodosin in tissue segments of vas deferens isolated from WT and AKO mice. (b) Representative competition curve for prazosin at 500 pM [³H]-silodosin-binding sites in tissue segments of the WT mouse vas deferens. (c and d) Binding capacities of 500 pM [³H]-silodosin in the intact segments of WT, AKO, BKO and DKO mouse vas deferentia (c) and prostate (d). In panel c, high- and low-affinity sites for prazosin at [³H]-silodosin-binding sites were represented as α_1A and α_1L, respectively. ^*Significantly different from WT mice (P<0.01). Mean±s.e.mean of 3–6 experiments.

Figure 4

Contractile responses to noradrenaline in mouse vas deferens (epididymal portion). (a) Effects of prazosin and silodosin on the contractile responses to 10 μM noradrenaline (NA) in the vas deferens isolated from WT mice. In contrast to silodosin (3 nM), prazosin (10 nM) failed to inhibit the contractile response to noradrenaline. (b) Effects of prazosin, silodosin, 5-methylurapidil (5-MU) and BMY 7378 on the concentration–response curves for noradrenaline in the WT mouse vas deferens. Mean±s.e.mean of 4–6 experiments. WT, wild type.

Figure 5

Effects of α₁-adrenoceptor gene knockout on the contractile responses in mouse vas deferens (epididymal portion). (a) Representative responses to 100 μM noradrenaline (NA) in WT, AKO, BKO and DKO mice. (b) Contractile responses induced by 100 μM noradrenaline and 1 μM α, β-methylene ATP (α,β-Me ATP). Mean±s.e.mean of 3–6 experiments. ^*Significantly different from other columns (P<0.01). AKO, α_1A-adrenoceptor gene knockout; BKO, α_1B-adrenoceptor gene knockout; DKO, α_1D-adrenoceptor gene knockout; WT, wild type.

Figure 6

Effects of prazosin (a), silodosin (b), 5-methylurapidil (c; 5-MU) and BMY 7378 (d) on the concentration–response curves for noradrenaline in WT mouse prostate. Mean±s.e.mean of 4–6 experiments. WT, wild type.

Figure 7

Contractile responses to noradrenaline in mouse prostate. (a) Concentration–response curves for noradrenaline in WT and AKO mouse prostate. Mean±s.e.mean of 4–6 experiments. (b) Effects of silodosin, BMY 7378 and prazosin on the contractile response to noradrenaline in the AKO mouse prostate. This is a representative result obtained from three AKO mice. The response induced by 100 μM noradrenaline in the control was equivalent to 18.5 mg, which was taken as 100%. Prazosin (1 nM) produced insurmountable inhibition. (c) Contractile responses induced by 100 μM noradrenaline and 10 μM α,β-methylene ATP (α,β-Me ATP). Mean±s.e.mean of 3–6 experiments. ^*Significantly different from other columns (P<0.01). AKO, α_1A-adrenoceptor gene knockout; WT, wild type.