Lack of effect of the alpha2C-adrenoceptor Del322-325 polymorphism on inhibition of cyclic AMP production in HEK293 cells

Abstract

Background and purpose: The alpha(2C)-adrenoceptor has multiple functions, including inhibiting release of noradrenaline from presynaptic nerve terminals. A human alpha(2C) polymorphism, Del322-325, a potential risk factor for heart failure, has been reported to exhibit reduced signalling in CHO cells. To further understand the role of the Del322-325 polymorphism on receptor signalling, we attempted to replicate and further study the reduced signalling in HEK293 cells.

Experimental approach: Human alpha(2C) wild-type (WT) and Del322-325 adrenoceptors were stably transfected into HEK293 cells. Radioligand binding was performed to determine affinities for both receptors. In intact cells, inhibition of forskolin-stimulated cyclic AMP production by WT and Del322-325 clones with a range of receptor densities (200-2320 fmol.mg(-1) protein) was measured following agonist treatment.

Key results: Noradrenaline, brimonidine and clonidine exhibited similar binding affinities for WT and Del322-325. Brimonidine and clonidine also had similar efficacies and potencies for both receptors for the inhibition of cyclic AMP production at all receptor densities tested. A linear regression analysis comparing efficacy and potency with receptor expression levels showed no differences in slopes between WT and Del322-325.

Conclusions and implications: The alpha(2C) WT and Del322-325 adrenoceptors exhibited similar binding properties. Additionally, inhibition of cyclic AMP production by Del322-325 was similar to that of WT over a range of receptor densities. Therefore, in intact HEK293 cells, the alpha(2C)-Del322-325 polymorphism does not exhibit reduced signalling to adenylyl cyclase and may not represent a clinically important phenotype.

Figure 1

High- and low-affinity competition binding of brimonidine at α_2C WT (A) and Del322-325 (B) receptors in the absence or presence of Gpp(NH)p. These curves are from single experiments and are representative of the results from the global fit analysis. Experiments were performed in duplicate and data are plotted as means ± SEMs.

Figure 2

Inhibition of cyclic AMP production by the α₂ agonists brimonidine (A) and clonidine (B) acting at α_2C WT and Del322-325 adrenoceptors stably expressed in HEK293 cells. The data are normalized to the maximal forskolin stimulation and the values plotted are the means ± SEM from three to four experiments. WT B_max= 1460 fmol·mg⁻¹ protein; Del322-325 B_max= 1100 fmol·mg⁻¹ protein.

Figure 3

Comparison of the functional responses of α_2C WT and Del322-325 adrenoceptors at various receptor expression levels in HEK293 cells. Efficacy and potency values for the inhibition of cyclic AMP production by brimonidine (A and C, respectively) and clonidine (B and D, respectively) are plotted against the log B_max values of multiple WT and Del322-325 clones. Plotted values are the means ± SEM. The slopes of the lines for WT and Del322-325 were determined by linear regression analysis and did not differ between the two receptors for either agonist.