Adrenaline (ADR) and noradrenaline (NA) can simultaneously activate inhibitory α2‐ and stimulatory β‐adrenoceptors (AR). However, ADR and NA differ significantly in that ADR is a potent β2‐AR agonist while NA is not. Only recently has the interaction resulting from the simultaneous activation of α2‐ and β2‐AR been examined at the cellular level to determine the mechanisms of α2‐AR regulation following concomitant activation of both α2‐ and β2‐ARs by chronic ADR. This study evaluates β2‐AR regulation of α2A‐AR signalling following chronic ADR (300 nM) and NA (1 and 30 μM) treatments of BE(2)‐C human neuroblastoma cells that natively express both β2‐ and α2A‐ARs. Chronic (24 h) treatment with ADR (300 nM) desensitized the response to the α2A‐AR agonist, brimonidine, in BE(2)‐C cells. Addition of the β‐AR antagonist, propranolol, blocked the ADR‐induced α2A‐AR desensitization. Unlike ADR, chronic NA (1 μM) treatment had no effect on the α2A‐AR response. However if NA was increased to 30 μM for 24 h, α2A‐AR desensitization was observed; this desensitization was partially reversed by propranolol. Chronic ADR (300 nM) treatment reduced α2A‐AR binding levels, contributing to the α2A‐AR desensitization. This decrease was prevented by addition of propranolol during ADR treatment. Chronic NA (30 μM), like ADR, treatment lowered specific binding, whereas 1 μM NA treatment was without effect. Chronic ADR treatment produced a significant increase in GRK3 levels and this was blocked by propranolol or GRK2/3 antisense DNA treatment. This antisense DNA, common to both GRK2 and GRK3, also blocked chronic ADR‐induced α2A‐AR desensitization and down‐regulation. Acute (1 h) ADR (300 nM) or NA treatment (1 μM) produced α2A‐AR desensitization. The desensitization produced by acute treatment was β‐AR independent, as it was not blocked by propranolol. We conclude that chronic treatment with modest levels of ADR produces α2A‐AR desensitization by mechanisms that involve up‐regulation of GRK3 and down‐regulation of α2A‐AR levels through interactions with the β2‐AR. British Journal of Pharmacology (2003) 138, 921–931. doi:

Serum prolactin concentrations and dopamine turnover in the striatum and median eminence were studied in male rats after the administration of estradiol benzoate. The alpha-methyltyrosine-induced reduction of dopamine concentrations in these brain regions was used to evaluate relative rates of turnover. Steady state dopamine concentrations in the median eminence and striatum were not altered by 1, 3 or 5 days of estradiol treatment. However, 3 or 5 days of estradiol administration enhanced dopamine turnover in the median eminence but not in the striatum. Estradiol treatment failed to alter dopamine turnover in the median eminence of hypophysectomized rats. Estradiol increased serum prolactin concentrations at all of the times examined. Although alpha-methyltyrosine also increased serum prolactin, this increase was further enhanced in estradiol-treated rats. The increased prolactin response to alpha-methyltyrosine and increased dopamine turnover in the median eminence of estradiol-treated rats suggests that tuberoinfundibular dopaminergic neurons may be part of a hormonal-neuronal negative feedback loop which functions to regulate prolactin secretion.

We recently reported that ␣ 2A -adrenoceptor (AR) desensitization and down-regulation occurs after 24-h treatment with epinephrine (EPI) (0.3 M) in BE(2)-C cells that express both ␣ 2 -and ␤ 2 -ARs. The same concentration of norepinephrine (NE) has no effect. The effect of EPI is prevented by ␤ 2 -AR blockade and is associated with an increase in G protein-coupled receptor kinase 3 (GRK3) expression. Because differences in agonist-induced down-regulation of the ␣ 2A -versus ␣ 2B -ARs have been reported, the present study examines the effects of simultaneous activation of ␣ 2B -and ␤ 2 -ARs on ␣ 2B -AR number and signaling. We studied NG108 cells that naturally express ␣ 2B -ARs, and BN17 cells, NG108 cells transfected to express the human ␤ 2 -AR. In NG108 cells, ␣ 2B -AR desensitization and down-regulation require treatment with 20 M EPI or NE; GRK expression was not changed. In BN17 cells expressing ␤ 2 -ARs, the threshold EPI concentration for ␣ 2B -AR desensitization and down-regulation was reduced to 0.3 M; 10 M NE was required for the same effect. Furthermore, 24-h EPI or NE treatments that produced desensitization also resulted in a selective 2-fold up-regulation of GRK3; GRK2 was unchanged. The ␤-AR antagonist alprenolol (1 M) and GRK3 antisense (but not sense) DNA blocked 0.3 M EPI-and 10 M NE-induced desensitization and down-regulation of the ␣ 2B -AR as well as GRK3 up-regulation. In conclusion, simultaneous activation of ␣ 2B -and ␤ 2 -ARs results in a 67-fold decrease in the threshold concentration of EPI required for ␣ 2B -AR down-regulation. This lower threshold for down-regulation is associated with ␣ 2B -and ␤ 2 -AR dependent up-regulation of GRK3 expression.Over the last decade, considerable attention has been given to the short-and long-term regulation of ␣ 2 -adrenoceptor signaling. Primarily using transfected receptors in cell lines that do not endogenously express the ␣ 2 -adrenoceptor, Liggett and coworkers have conducted a series of experiments examining the short-term and long-term desensitization of the three ␣ 2 -adrenoceptor subtypes (␣ 2A/D,2B and 2C ) (Liggett, 1998). Through these studies, structural components of the ␣ 2 -adrenoceptor have been identified as sites for GRK phosphorylation that are important for the short-term desensitization of these receptors. For example, four serines in the third intracellular loop are reported to be critical for shortterm desensitization of the ␣ 2A -adrenoceptor (Eason et al., 1995). However, there are structural differences within the ␣ 2 -adrenoceptor family that may lead to heterogeneity in the regulation of the three ␣ 2 -subtypes (Eason and Liggett, 1992). For example, significantly higher agonist concentrations are reportedly required for desensitization of ␣ 2A compared with ␣ 2B -adrenoceptors (Heck and Bylund, 1997). Moreover, studies have suggested that phosphorylation sites critical to short-term ␣ 2 -adrenoceptor desensitization are not important for long-term desensitization and down-regulation (Jewell-Motz and Liggett, 19...

Exposure of rats to unpredictable, inescapable stress results in two distinct behaviors during subsequent escape testing. One behavior, suggestive of lack of stress resilience, is prolonged escape latency compared to non-stressed rats and is labeled learned helplessness (LH). The other behavior suggestive of stress resilience is normal escape latency and is labeled non-helpless (NH). This study examines the effects of unpredictable, inescapable tail-shock stress (TSS) on alpha2-adrenoceptor (α2-AR) and corticotropin-releasing factor 1 receptor (CRF1) regulation as well as protein levels of G protein-coupled receptor kinase 3 (GRK3), GRK2, tyrosine hydroxylase (TH) plus carbonylated protein levels in locus coeruleus (LC), amygdala (AMG), cortex (COR) and striatum (STR). In NH rats, α2-AR and CRF1 receptors were significantly down-regulated in LC after TSS. No changes in these receptor levels were observed in the LC of LH rats. GRK3, which phosphorylates receptors and thereby contributes to α2-AR and CRF1 receptor down-regulation, was reduced in the LC of LH but not NH rats. GRK2 levels were unchanged. In AMG, GRK3 but not GRK2 levels were reduced in LH but not NH rats, and receptor regulation was impaired in LH rats. In STR, no changes in GRK3 or GRK2 levels were observed. Finally, protein carbonylation, an index of oxidative stress, was increased in the LC and AMG of LH but not NH rats. We suggest that reduced stress resilience after TSS may be related to oxidative stress, depletion of GRK3 and impaired regulation of α2-AR and CRF1 receptor in LC.

Chronic coactivation of ␣ 2B -and ␤ 2 -adrenoceptors (AR) was recently reported to down-regulate the ␣ 2B -AR at a lower threshold epinephrine (EPI) concentration compared with the activation of ␣ 2B -AR alone. This is the result of a modest ␤ 2 -AR-dependent up-regulation of G protein-coupled receptor kinase 3 (GRK3). In the present study, we determined that increasing GRK2 or GRK3 levels, independent of ␤ 2 -AR activation, decreases the EC 50 concentration for agonist-induced down-regulation of the ␣ 2B -AR using NG108 cells with or without overexpression (2-to 10-fold) of GRK2 or GRK3. In parental NG108 cells, the EC 50 concentration of EPI required for downregulation of the ␣ 2B -AR is 30 M. A 2-to 3-fold overexpression of GRK3 in NG108 cells, however, reduces the EC 50 to 0.2 M (a 150-fold decrease), whereas a comparable overexpression of GRK2 reduces it to 1 M (a 30-fold decrease). However, when GRK3 or GRK2 in NG108 cells are overexpressed 8-to 10-fold, the EC 50 concentration (0.02 M EPI) for ␣ 2B -AR down-regulation is reduced 1000-fold. These data clearly suggest that a modest (2-to 3-fold) up-regulation of GRK3 is more effective at enhancing the sensitivity of ␣ 2B -AR to down-regulation after exposure to EPI than a modest up-regulation of GRK2, but that both GRK2 and GRK3 are equally effective at inducing ␣ 2B -AR down-regulation when up-regulated 8-to 10-fold. To our knowledge, this is the first report to systematically demonstrate that GRKs, particularly GRK3, play a pivotal role in modulating the agonist EC 50 concentration that down-regulates the ␣ 2B -AR and thus adds a new dimension to an already intricate signaling network.