Anne Marjamäki

Molecular Pharmacology of α2-adrenoceptor Subtypes

α2adrenergic receptors mediate many of the physiological actions of the endogenous catecholamines adrenaline and noradrenaline, and are targets of several therapeutic agents, α2-adrenoceptor agonists are currently used as antihypertensives and as veterinary sedative anaesthetics. They are also used experimentally in humans as adjuncts to anaesthesia, as spinal analgesics, and to treat opioid, nicotine and alcohol dependence and withdrawal. Three human α2-adrenoceptor subtype genes have been cloned and designated α2-C10, α2-C4 and α2-C2, according to their location on human chromosomes 10, 4 and 2. They correspond to the previously identified pharmacological receptor subtypes α2A, α2C and α2B. The receptor proteins share only about 50% identity in their amino acid sequence, but some structurally and functionally important domains are very well conserved. The most obvious functionally important differences between the receptor subtypes are based on their different tissue distributions; e.g. the α2A subtype ...

α2-Adrenoceptor regulation of adenylyl cyclase in CHO cells: Dependence on receptor density, receptor subtype and current activity of adenylyl cyclase

Chinese hamster ovary (CHO) cells stably transfected to express different densities of the human alpha2A-, alpha2B- and alpha2C-adrenoceptor subtypes, were used to characterize the regulation of adenylyl cyclase activity by alpha2-adrenoceptor agonists. In isolated cell membranes, activation of alpha2A- and alpha2C-adrenoceptors did not affect basal enzyme activity, but activation of alpha2B-adrenoceptors stimulated adenylyl cyclase activity. The extent of stimulation was dependent on the receptor density and was insensitive to pertussis toxin treatment. In the presence of 10 microM forskolin all three receptor subtypes mediated inhibition of adenylyl cyclase activity in a pertussis toxin-sensitive manner. In experiments performed with intact cells the same pattern could be seen: the basal production of cAMP was not affected when alpha2C-adrenoceptors were activated, but activated alpha2B-adrenoceptors mediated stimulation of cAMP production. In the presence of forskolin, both receptor subtypes mediated inhibition of cAMP production. Our results suggest that alpha2B-adrenoceptors are coupled to both Gi and Gs proteins. The signal transduction pathway to which the receptor is coupled is not dependent on receptor density, but its effect on adenylyl cyclase regulation is dependent on the current activity of adenylyl cyclase. The results also suggest that the alpha2A- and alpha2C-subtypes are preferentially coupled to Gi and transduce only inhibition of adenylyl cyclase activity in transfected CHO cells. At low densities of alpha2C-adrenoceptors, clonidine was a partial agonist, but in clones expressing high levels of alpha2C-adrenoceptors, clonidine acted as a full agonist by inhibiting cAMP accumulation with the same efficacy as (-)-noradrenaline. This demonstrates that receptor reserve can mask partial agonist activity.

Coupling of human α2-adrenoceptor subtypes to regulation of cAMP production in transfected S115 cells

Stable S115 mouse mammary tumour cell lines, expressing separately alpha 2A-C10, alpha 2B-C2 and alpha 2C-C4 adrenoceptors were used to compare the receptor binding properties of alpha 2-adrenoceptor agonists with their potency in inhibiting cAMP production. All tested agonists detected high and low affinity binding sites in all three receptor subtypes. In the presence of the GTP analogue Gpp(NH)p (10 microM), all displacement curves were shifted to the right and were best modelled by one-site fits, suggesting that the receptor subtypes are coupled to G-proteins. The extent of the Gpp(NH)p-induced shift was greatest in the alpha 2A-C10 subtype, smaller in alpha 2C-C4, and minimal in alpha 2B-C2. All three receptor subtypes were also coupled to inhibition of forskolin-stimulated cAMP production through pertussis toxin-sensitive G-proteins. For the full agonists noradrenaline, UK 14,304, and dexmedetomidine, the maximal inhibitory effect on cAMP production was smaller in the alpha 2B-C2 subtype (35%) than in the alpha 2A-C10 and alpha 2C-C4 subtypes (50-70%). After treatment of cells expressing alpha 2B-C2 receptors with pertussis toxin, cAMP production was increased by up to 58% by alpha 2-adrenoceptor agonists. Similar stimulation of adenylyl cyclase activity could not be demonstrated at the other two receptor subtypes. In conclusion, these results demonstrate that (1) alpha 2-adrenoceptor agonists may be characterized by an agonist-type binding pattern in homogenates of transfected S115 cells, (2) all three alpha 2-adrenoceptor subtypes are coupled to inhibition of adenylyl cyclase in S115 cells through pertussis toxin-sensitive G-proteins, (3) the receptor-effector coupling in S115 cells is different among the subtypes so that the alpha 2A-C10 subtype is coupled with high efficacy but with low sensitivity, the alpha 2B-C2 subtype with low efficacy but high sensitivity, and the alpha 2C-C4 subtype with both high efficacy and high sensitivity, and (4) at least alpha 2B-C2 receptors may also be coupled to stimulation of adenylyl cyclase activity, presumably through Gs.

Use of recombinant human α2-adrenoceptors to characterize subtype selectivity of antagonist binding

Abstract Cloning of the genes encoding three subtypes of human α 2 -adrenoceptors allows the separate heterologous expression of each subtype. We have generated stably transfected Shionogi S115 mouse mammary tumour cell lines expressing the human α 2 -adrenoceptor subtypes α 2 -C10, α 2 -C 2 , and α 2 -C4 at densities of 0.2–7 pmol/mg total cellular protein. Binding of [ 3 H]rauwolscine was inhibited by co-incubation of S115 cell homogenates with ten α 2 -adrenoceptor antagonists and oxymetazoline, a partial agonist known to discriminate the receptor subtypes. Other useful agents for discrimination of subtypes were prazosin, chlorpromazine, phentolamine, and yohimbine. The most sensitive indices for differences between the three subtypes were the binding inhibition coefficient (K i ) ratios chlorpromazie/oxymetazoline ( α 2 -C10 202; α 2 -C 0.004; α 2 -C4: 0.8), prazosin/oxymetazoline (430; 0.03; 0.5) and chlorpromazine/atipamezole (1612; 5.8; 77). Correlation analysis between our results for human-type receptors and published data for their rat α 2 -adrenoceptor homologues demonstrated excellent general agreement, with some interspecies differences in the affinity of rauwolscine, phentolamine and oxymetazoline. The use of recombinant human receptors produced in stably transfected cell lines should facilitate the development of new, subtype-selective α 2 -adrenoceptor ligands.

Conserved structural, pharmacological and functional properties among the three human and five zebrafish α2‐adrenoceptors

1 Zebrafish has five distinct α2‐adrenoceptors. Two of these, α2Da and α2Db, represent a duplicated, fourth α2‐adrenoceptor subtype, while the others are orthologue of the human α2A‐, α2B‐ and α2C‐adrenoceptors. Here, we have compared the pharmacological properties of these receptors to infer structural determinants of ligand interactions. 2 The zebrafish α2‐adrenoceptors were expressed in Chinese hamster ovary cells and tested in competitive ligand binding assays and in a functional assay (agonist‐stimulated [35S]GTPγS binding). The affinity results were used to cluster the receptors and, separately, the ligands using both principal component analysis and binary trees. 3 The overall ligand binding characteristics, the order of potency and efficacy of the tested agonists and the G‐protein coupling of the zebrafish and human α2‐adrenoceptors, separated by ∼350 million years of evolution, were found to be highly conserved. The binding affinities of the 20 tested ligands towards the zebrafish α2‐adrenoceptors are generally comparable to those of their human counterparts, with a few compounds showing up to 40‐fold affinity differences. 4 The α2A orthologues and the zebrafish α2D duplicates clustered as close pairs, but the relationships between the orthologues of α2B and α2C were not clearly defined. Applied to the ligands, our clustering methods segregated the ligands based on their chemical structures and functional properties. As the ligand binding pockets formed by the transmembrane helices show only minor differences among the α2‐adrenoceptors, we suggest that the second extracellular loop – where significant sequence variability is located – might contribute significantly to the observed affinity differences.

Stable expression of recombinant human α2-adrenoceptor subtypes in two mammalian cell lines: characterization with [3H]rauwolscine binding, inhibition of adenylate cyclase and RNase protection assay

Cloning of the genes encoding distinct subtypes of human alpha 2-adrenergic receptors (alpha 2-AR) allows the separate recombinant expression of each individual subtype in heterologous systems. We report here the transfection, selection and preliminary pharmacological characterization of two mammalian cell lines, adherent Shionogi S115 mouse mammary tumour cells and human B-lymphoblastoid IBW4 cells growing in suspension, expressing the human alpha 2-AR subtypes alpha 2-C4 and alpha 2-C10 at densities of approx. 2 x 10(5) receptors/cell. Transfection of the subtype genes was verified using a specific RNase protection assay. Pharmacological characterization was carried out with [3H]rauwolscine binding, which was inhibited by oxymetazoline and prazosin in a subtype-selective manner. The sensitivity of (-)-noradrenaline binding to the GTP-analogue 5-guanylylimidodiphosphate suggested that the receptors are coupled to G-proteins. This was verified in S115 cells by efficient inhibition of forskolin-stimulated cAMP production by the alpha 2-AR agonists, (-)-noradrenaline and clonidine. These cell lines thus appear to be suitable for pharmacological studies on receptor function and ligand binding.

Molecular mechanisms of ligand–receptor interactions in transmembrane domain V of the α2A-adrenoceptor

The structural determinants of catechol hydroxyl interactions with adrenergic receptors were examined using 12 α2‐adrenergic agonists and a panel of mutated human α2A‐adrenoceptors. The α2ASer201 mutant had a Cys → Ser201 (position 5.43) amino‐acid substitution, and α2ASer201Cys200 and α2ASer201Cys204 had Ser → Cys200 (5.42) and Ser → Cys204 (5.46) substitutions, respectively, in addition to the Cys → Ser201 substitution. Automated docking methods were used to predict the receptor interactions of the ligands. Radioligand‐binding assays and functional [35S]GTPγS‐binding assays were performed using transfected Chinese hamster ovary cells to experimentally corroborate the predicted binding modes. The hydroxyl groups of phenethylamines were found to have different effects on ligand affinity towards the activated and resting forms of the wild‐type α2A‐adrenoceptor. Substitution of Ser200 or Ser204 with cysteine caused a deterioration in the capability of catecholamines to activate the α2A‐adrenoceptor. The findings indicate that (i) Cys201 plays a significant role in the binding of catecholamine ligands and UK14,304 (for the latter, by a hydrophobic interaction), but Cys201 is not essential for receptor activation; (ii) Ser200 interacts with the meta‐hydroxyl group of phenethylamine ligands, affecting both catecholamine binding and receptor activation; while (iii) substituting Ser204 with a cysteine interferes both with the binding of catecholamine ligands and with receptor activation, due to an interaction between Ser204 and the para‐hydroxyl group of the catecholic ring.

Use of a hollow fiber bioreactor for large-scale production of α2-adrenoceptors in mammalian cells

Gene cloning has revealed the existence of receptors, which are structurally similar but pharmacologically distinct. One recent example is the alpha 2-adrenergic receptor (alpha 2AR) family with three members. Preparation of membrane-embedded G-protein coupled receptor subtypes in pure form is practically impossible from natural sources and only recombinant techniques have provided possibilities to study these receptors in great detail. In this respect, both yeast and insect cell hosts have been applied successfully but no good mammalian alternative has been described for large-scale production. We describe in this report the use of S115 mouse mammary tumor cells as an effective host for large-scale production of alpha 2-adrenoceptors. These cells can be easily adapted to grow in a hollow fiber bioreactor, with up to 2.8 g of total cellular protein produced in one 0.8 m2 casette. We also show that each recombinant alpha 2-subtype exhibits their expected ligand binding properties, and suggest therefore that this system could be generally applicable to other eukaryotic plasma membrane proteins.

Similar ligand binding in recombinant human α2C2-adrenoceptors produced in mammalian, insect and yeast cells

Abstract Ligand binding properties were investigated in recombinant human α2C2-adrenoceptors expressed in three different host systems: Shionogi S115 mouse mammary tumour cells, Spodoptera frugiperda Sf9 insect cells and Saccharomyces cerevisae yeast cells. The expected 43 kDa α2C2 protein was visualized with immunoblotting using a polyclonal α2C2-receptor antibody. [3H]Rauwolscine binding in cell homogenates or membranes (Bmax 3–11 pmol/mg protein; Kd approximately 5.5 nM) was inhibited by prazosin, oxymetazoline, RX821002, chlorpromazine and (−)-noradrenaline with and without the GTP-analogue Gpp(NH)p with similar Ki values in the different host systems. This indicates that α2C2-adrenoceptors retain their binding characteristics irrespective of the host environment.

Sulfonamide inhibitors of α2β1 integrin reveal the essential role of collagen receptors in in vivo models of inflammation

Small molecule inhibitors of α2β1 integrin, a major cellular collagen receptor, have been reported to inhibit platelet function, kidney injury, and angiogenesis. Since α2β1 integrin is abundantly expressed on various inflammation‐associated cells, we tested whether recently developed α2β1 blocking sulfonamides have anti‐inflammatory properties. Integrin α2β1 inhibitors were shown to reduce the signs of inflammation in arachidonic acid‐induced ear edema, PAF stimulated air pouch, ovalbumin‐induced skin hypersensitivity, adjuvant arthritis, and collagen‐induced arthritis. Thus, these sulfonamides are potential drugs for acute and allergic inflammation, hypersensitivity, and arthritis. One sulfonamide with potent anti‐inflammatory activity has previously been reported to be selective for activated integrins, but not to inhibit platelet function. Thus, the experiments also revealed fundamental differences in the action of nonactivated and activated α2β1 integrins in inflammation when compared to thrombosis.