Agonist trafficking of G(i/o)-mediated alpha(2A)-adrenoceptor responses in HEL 92.1.7 cells

Abstract

1. The ability of 19 agonists to elevate Ca(2+) and inhibit forskolin-induced cyclic AMP elevation through alpha(2A)-adrenoceptors in HEL 92.1.7 cells was investigated. Ligands of catecholamine-like- (five), imidazoline- (nine) and non-catecholamine-non-imidazoline-type (five) were included. 2. The relative maximum responses were similar in both assays. Five ligands were full or nearly full agonists, six produced 20 - 70% of the response to a full agonist and the remaining eight gave lower responses (< 20%) so that their potencies were difficult to evaluate. 3. Marked differences in the potencies of the agonists with respect to the two measured responses were seen. The catecholamines were several times less potent in decreasing cyclic AMP than in increasing Ca(2+), whereas the other, both imidazoline and ox-/thiazoloazepine ligands, were several times more potent with respect to the former than the latter response. For instance, UK14,304 was more potent than adrenaline with respect to the cyclic AMP response but less potent than adrenaline with respect to the Ca(2+) response. 4. All the responses were sensitive to pertussis toxin-pretreatment. Also the possible role of PLA(2), beta-adrenoceptors or ligand transport or metabolism as a source of error could be excluded. The results suggest that the active receptor states produced by catecholamines and the other agonists are markedly different and therefore have different abilities to activate different signalling pathways.

Figure 1

Ca²⁺ and cyclic AMP responses to two ligands, moxonidine (an imidazoline) and B-HT 920 (an oxazoloazepine), as mean±s.e.mean of three determinations in triplicate.

Figure 2

Correlation of the abilities of α₂-adrenoceptor agonists to induce cyclic AMP decrease and Ca²⁺ elevation. (A) The maximum responses; (B) the EC₅₀ values. Also drawn are the best linear fit (solid line) and the best linear fit forced to origo (dotted line) in (A) and the relationships 1 : 3.7 and 59 : 1 in (B). The drawn relationships of indicated value are based on the average of logarithms of EC_50-Ca²⁺/EC_{50-cyclic AMP} for the corresponding ligand groups. The overall r (calculated for the logarithmic data) for (A) is 0.903 and for (B) 0.437. The overall EC_50-Ca²⁺/EC_{50-cyclic AMP} in (B) is 2.84±0.79. Catecholamines alone have EC_50-Ca²⁺/EC_{50-cyclic AMP} of 0.0219± 0.0095 and the imidazolines and ox-/thiazoloazepines 4.05±0.72 (significance for difference between groups: P<0.01).

Figure 3

A schematic representation of the hypothesis with three somewhat different models, which are all possible based on the results. R is the inactive and R^* and R^** the two active receptor conformations, T1 and T2 are two signal transducers (e.g. G proteins) and E1 and E2 are the separate signal cascades which finally lead to a generation of a second messenger response (Ca²⁺ and cyclic AMP, respectively). The solid arrows indicate the direction of the response (the thicker the arrow, the stronger the coupling) and the dotted arrows in the top scheme the interconvertibility of the receptor conformations. In (A) a single receptor conformation only couples to a single transducer, which then couples to a single second messenger cascade. Catecholamines bind more strongly to R^* whereas the other ligands bind more strongly to R^** leading to a stronger activation of E1 (Ca²⁺) or E2 (cyclic AMP) response, respectively. In (B) the agonist trafficking of the receptor signals is accomplished at the level of receptor-transducer-coupling: the catecholamine-activated receptor (R^*) is more effective in coupling to T1 (and thus to E1) whereas the non-catecholamine-activated receptor (R^*) is more effective in its coupling to T2 (and thus to E2). In the third scheme, (C), a single receptor conformation only couples to a single transducer (as in A), which then couples to both second messenger cascades. The agonist trafficking of the receptor signals is brought about by the different ability of transducers to activate different cascades: the catecholamine-activated transducer T1 is more effective in coupling to E1 whereas the non-catecholamine-activated transducer T2 is more effective in its coupling to E2. The schemes (B) and (C) are essentially similar to the scheme presented in Berg et al. (1998), though somewhat more complex.