Effects of ß-blockers on house dust mite-driven murine models pre- and post-development of an asthma phenotype.

BACKGROUND: Our previous studies suggested certain ß-adrenoceptor blockers (ß-blockers) attenuate the asthma phenotype in ovalbumin driven murine models of asthma. However, the ovalbumin model has been criticized for lack of clinical relevance. METHODS: We tested the non-selective ß-blockers, carvedilol and nadolol, in house dust mite (HDM) driven murine asthma models where drugs were administered both pre- and post-development of the asthma phenotype. We measured inflammation, mucous metaplasia, and airway hyper-responsiveness (AHR). We also measured the effects of the ß-blockers on extracellular-signal regulated kinase (ERK 1/2) phosphorylation in lung homogenates. RESULTS: We show that nadolol, but not carvedilol, attenuated inflammation and mucous metaplasia, and had a moderate effect attenuating AHR. Following HDM exposure, ERK1/2 phosphorylation was elevated, but the level of phosphorylation was unaffected by ß-blockers, suggesting ERK1/2 phosphorylation becomes dissociated from the asthma phenotype. CONCLUSION: Our findings in HDM models administering drugs both pre- and post-development of the asthma phenotype are consistent with previous results using ovalbumin models and show differential effects for nadolol and carvedilol on the asthma phenotype. Lastly, our data suggest that ERK1/2 phosphorylation may be involved in development of the asthma phenotype, but may have a limited role in maintaining the phenotype.

Differential effects of inescapable stress on locus coeruleus GRK3, alpha2-adrenoceptor and CRF1 receptor levels in learned helpless and non-helpless rats: a potential link to stress resilience.

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 alpha(2)-adrenoceptor (α(2A)-AR) and corticotropin-releasing factor 1 receptor (CRF(1)-R) 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, α(2A)-AR and CRF(1)-R 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 α(2A)-AR and CRF(1)-R 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 α(2A)-AR and CRF(1)-R in LC.

The presence of beta2-adrenoceptors sensitizes alpha2A-adrenoceptors to desensitization after chronic epinephrine treatment.

BACKGROUND: In addition to the regulation of blood pressure, alpha2- and beta-adrenoceptor (AR) subtypes play an important role in the modulation of noradrenergic neurotransmission in the human CNS and PNS. Several studies suggest that the alpha2-AR responsiveness in cells and tissues after chronic epinephrine (EPI) or norepinephrine (NE) exposure may vary, depending on the beta-AR activity present there. Recently, we reported that in BE(2)-C human neuroblastoma cells (endogenously expressing alpha2A- and beta2-AR), chronic EPI treatment (300 nM) produced a dramatic beta-adrenoceptor-dependent desensitization of the alpha2A-AR response. The aim of this study is to determine if stable addition of a beta2-AR to a second neuroblastoma cell line (SH-SY5Y), that normally expresses only alpha2A-ARs that are not sensitive to 300 nM EPI exposure, would suddenly render alpha2A-ARs in that cell line sensitive to treatment with the same EPI concentration. METHODS: These studies employed RT-PCR, receptor binding and inhibition of cAMP accumulation to confirm alpha2-AR subtype expression. Stable clones of SH-SY5Y cells transfected to stably express functional beta2-ARs (SHbeta2AR4) were selected to compare sensitivity of alpha2-AR to EPI in the presence or absence of beta2-ARs. RESULTS: A series of molecular, biochemical and pharmacological studies indicated that the difference between the cell lines could not be attributed to alpha2-AR heterogeneity. We now report that after transfection of functional beta2-AR into SH-SY5Y cells (SHbeta2AR4), chronic treatment with modest levels of EPI desensitizes the alpha2A-AR. This effect results from a beta2-AR dependent down-regulation of native alpha2A-ARs by EPI accompanied by enhanced translocation of GRK2 and GRK3 to the membrane (required for GRK-mediated phosphorylation of agonist-occupied receptors). CONCLUSION: This study further supports the hypothesis that the presence of the beta-AR renders the alpha2A-AR more susceptible to desensitization with physiological levels of EPI.

Activation of the CRF(1) receptor causes ERK1/2 mediated increase in GRK3 expression in CATH.a cells.

G-protein coupled receptor kinase 3 (GRK3) mediates desensitization of alpha(2)-adrenergic (alpha(2)-AR) and CRF(1) receptors. CRF(1) receptors, alpha(2)-AR and GRK3, are localized to the primary source of noradrenergic inputs to higher brain centers critical in both the response to stress and the development of depression, namely, locus coeruleus (LC). This study utilizing CATH.a cells (derived from the LC), demonstrates for the first time, that the stress hormone, CRF selectively up-regulates GRK3 expression via an ERK1/2-mediated mechanism accompanied by the activation of Sp-1 and Ap-2 transcription factors. This observation has important implications for the regulation of stress signaling in the brain.

Extracellular signal-regulated kinase 1/2-mediated transcriptional regulation of G-protein-coupled receptor kinase 3 expression in neuronal cells.

Relatively small changes in G-protein-coupled receptor kinase (GRK) 3 expression (approximately 2-fold) profoundly affect alpha2-adrenergic receptor (AR) function and preferentially regulate neuronal alpha2A- and alpha2B-AR signaling. In the present study, we provide evidence that epinephrine (EPI)-induced up-regulation of GRK3 protein expression in two neuronal cell lines, BE(2)-C cells (endogenously express alpha2A- and beta2AR) and BN17 cells [endogenously express alpha2B (NG108) and transfected to express beta2-AR] is due in part to increased GRK3 gene expression. In both cell lines, the increase in GRK3 transcription occurred via an extracellular signal-regulated kinase (ERK) 1/2-dependent mechanism because the increase in GRK3 mRNA is eliminated in the presence of the mitogen-activated protein kinase/ERK kinase 1/2 inhibitor, U0126 [1,4-diamino-2,3-dicyano-1,4-bis (2-amino phenylthiobutadiene)]. EPI-induced GRK3 mRNA up-regulation also is prevented in the presence of propranolol or phentolamine. Moreover, GRK3 mRNA did not increase in response to EPI treatment in NG108 cells (endogenously express alpha2B-AR with no beta2-AR). Both these results suggest that simultaneous activation of alpha2- and beta2-AR by EPI is required for the ERK1/2-dependent increase in GRK3 mRNA. The EPI-induced increase in GRK3 mRNA was unaffected in the presence of the protein kinase C inhibitor, chelerythrine chloride. Finally, EPI treatment resulted in increased nuclear translocation and accumulation of the transcription factors, Sp-1 and Ap-2, in BE(2)-C cells. Taken together, our results demonstrate the involvement of the ERK1/2 pathway in selective up-regulation of GRK3 mRNA expression, possibly via activation of Sp-1 and Ap-2 transcription factors in neuronal cells.

Role of 90-kDa heat shock protein (Hsp 90) and protein degradation in regulating neuronal levels of G protein-coupled receptor kinase 3.

Cellular levels of G protein-coupled receptor kinase (GRK)3 determine the sensitivity of the alpha(2A/B)-adrenoceptor (alpha(2)-AR) to agonist-induced down-regulation. Using human neuroblastoma BE(2)-C cells, this study examines how cellular GRK3 levels are affected by several mechanisms reported to influence stability and degradation of other GRKs. We first examined the interaction between the 90-kDa heat shock protein (Hsp90) and GRK3; Hsp90 reportedly affects the maturation and stability of GRK2. In unstimulated cells, GRK3 coimmunoprecipitates with Hsp90, suggesting a physical interaction. Moreover, when GRK3 protein expression was increased by 24-h epinephrine (EPI) treatment, Hsp90 protein expression increased with a similar but slightly delayed time course. To investigate the influence of Hsp90 on GRK3 protein stability, we determined the effect of the Hsp90 inhibitor geldanamycin (GA) on cellular GRK3 levels. GA eliminated the interaction between Hsp90 with GRK3 and produced a rapid, proteasome-mediated, 70% decrease in GRK3 levels within 24 h. To investigate the influence of Hsp90 on up-regulation of GRK3 expression, we examined the effect of GA on EPI-induced up-regulation. GA reduced the absolute increase in GRK3; however, the percentage of increase in GRK3 by EPI was not significantly different in the absence versus presence of GA (141 +/- 41 versus 94 +/- 12%). Finally, we examined the influence of Ca(2+)-activated proteases on cellular GRK3. Treatment with the calcium ionophore ionomycin produced a rapid decrease in GRK3 levels that was inhibited by the calpain inhibitor calpeptin. In conclusion, several mechanisms influence the degradation of GRK3 and therefore have the potential to affect GPCR signaling by regulating GRK3 levels in neurons.

Involvement of G protein-coupled receptor kinase (GRK) 3 and GRK2 in down-regulation of the alpha2B-adrenoceptor.

Increasing the cellular levels of G protein-coupled receptor kinase (GRK) 2 or GRK3 renders the alpha2B-adrenoceptor (AR) more sensitive to agonist-induced down-regulation (J Pharmacol Exp Ther 312:767-773, 2005). However, an absolute requirement of GRK3 and GRK2 for alpha2B-AR down-regulation is controversial. In this study, using NG108 cells (endogenous alpha2B-AR), we provide strong evidence for a critical role of both GRK3 and GRK2 in down-regulation of the alpha2B-AR. Pretreatment of NG108 cells with 20 microM epinephrine (EPI) begins down-regulating the alpha2B-AR by 2 h. The translocation of GRK3 and GRK2 to the membrane peaks at 30 min, decreasing by 1 h. Although these results may implicate GRK3 and GRK2 in alpha2B-AR down-regulation, significant receptor down-regulation is not observed until 2 h, after GRK3 and GRK2 translocation has peaked and is declining. To more directly establish a role for GRK3 and GRK2 in alpha2B-AR down-regulation, NG108 cells were transfected to express GRK3ct, which binds to liberated Gbetagamma subunits, preventing GRK3 and GRK2 translocation to the membrane. Overexpression of GRK3ct prevented not only the translocation of GRK3 and GRK2 but also the down-regulation of the alpha2B-AR caused by 24-h pretreatment with 20 microM EPI. Taken together, these data provide direct evidence for a role of GRK3 and GRK2 in the down-regulation of the alpha2B-AR and contribute significantly to the increasing evidence in the literature for a pivotal role of GRKs in modulating the agonist-induced down-regulation of the alpha2-AR.

Cellular G protein-coupled receptor kinase levels regulate sensitivity of the {alpha}2b-adrenergic receptor to undergo agonist-induced down-regulation.

Chronic coactivation of alpha(2B)- and beta(2)-adrenoceptors (AR) was recently reported to down-regulate the alpha(2B)-AR at a lower threshold epinephrine (EPI) concentration compared with the activation of alpha(2B)-AR alone. This is the result of a modest beta(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 beta(2)-AR activation, decreases the EC(50) concentration for agonist-induced down-regulation of the alpha(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 down-regulation of the alpha(2B)-AR is 30 microM. A 2- to 3-fold overexpression of GRK3 in NG108 cells, however, reduces the EC(50) to 0.2 microM (a 150-fold decrease), whereas a comparable overexpression of GRK2 reduces it to 1 microM (a 30-fold decrease). However, when GRK3 or GRK2 in NG108 cells are overexpressed 8- to 10-fold, the EC(50) concentration (0.02 microM EPI) for alpha(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 alpha(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 alpha(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 alpha(2B)-AR and thus adds a new dimension to an already intricate signaling network.

Simultaneous alpha2B- and beta2-adrenoceptor activation sensitizes the alpha2B-adrenoceptor for agonist-induced down-regulation.

We recently reported that alpha(2A)-adrenoceptor (AR) desensitization and down-regulation occurs after 24-h treatment with epinephrine (EPI) (0.3 microM) in BE(2)-C cells that express both alpha(2)- and beta(2)-ARs. The same concentration of norepinephrine (NE) has no effect. The effect of EPI is prevented by beta(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 alpha(2A)-versus alpha(2B)-ARs have been reported, the present study examines the effects of simultaneous activation of alpha(2B)- and beta(2)-ARs on alpha(2B)-AR number and signaling. We studied NG108 cells that naturally express alpha(2B)-ARs, and BN17 cells, NG108 cells transfected to express the human beta(2)-AR. In NG108 cells, alpha(2B)-AR desensitization and down-regulation require treatment with 20 microM EPI or NE; GRK expression was not changed. In BN17 cells expressing beta(2)-ARs, the threshold EPI concentration for alpha(2B)-AR desensitization and down-regulation was reduced to 0.3 microM; 10 microM 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 beta-AR antagonist alprenolol (1 microM) and GRK3 antisense (but not sense) DNA blocked 0.3 microM EPI- and 10 microM NE-induced desensitization and down-regulation of the alpha(2B)-AR as well as GRK3 up-regulation. In conclusion, simultaneous activation of alpha(2B)- and beta(2)-ARs results in a 67-fold decrease in the threshold concentration of EPI required for alpha(2B)-AR down-regulation. This lower threshold for down-regulation is associated with alpha(2B)- and beta(2)-AR dependent up-regulation of GRK3 expression.

Desensitization of alpha 2A-adrenoceptor signalling by modest levels of adrenaline is facilitated by beta 2-adrenoceptor-dependent GRK3 up-regulation.

(1) Adrenaline (ADR) and noradrenaline (NA) can simultaneously activate inhibitory alpha(2)- and stimulatory beta-adrenoceptors (AR). However, ADR and NA differ significantly in that ADR is a potent beta(2)-AR agonist while NA is not. Only recently has the interaction resulting from the simultaneous activation of alpha(2)- and beta(2)-AR been examined at the cellular level to determine the mechanisms of alpha(2)-AR regulation following concomitant activation of both alpha(2)- and beta(2)-ARs by chronic ADR. (2) This study evaluates beta(2)-AR regulation of alpha(2A)-AR signalling following chronic ADR (300 nM) and NA (1 and 30 micro M) treatments of BE(2)-C human neuroblastoma cells that natively express both beta(2)- and alpha(2A)-ARs. (3) Chronic (24 h) treatment with ADR (300 nM) desensitized the response to the alpha(2A)-AR agonist, brimonidine, in BE(2)-C cells. Addition of the beta-AR antagonist, propranolol, blocked the ADR-induced alpha(2A)-AR desensitization. Unlike ADR, chronic NA (1 micro M) treatment had no effect on the alpha(2A)-AR response. However if NA was increased to 30 micro M for 24 h, alpha(2A)-AR desensitization was observed; this desensitization was partially reversed by propranolol. (4) Chronic ADR (300 nM) treatment reduced alpha(2A)-AR binding levels, contributing to the alpha(2A)-AR desensitization. This decrease was prevented by addition of propranolol during ADR treatment. Chronic NA (30 micro M), like ADR, treatment lowered specific binding, whereas 1 micro M NA treatment was without effect. (5) 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 alpha(2A)-AR desensitization and down-regulation. (6) Acute (1 h) ADR (300 nM) or NA treatment (1 micro M) produced alpha(2A)-AR desensitization. The desensitization produced by acute treatment was beta-AR independent, as it was not blocked by propranolol. (7) We conclude that chronic treatment with modest levels of ADR produces alpha(2A)-AR desensitization by mechanisms that involve up-regulation of GRK3 and down-regulation of alpha(2A)-AR levels through interactions with the beta(2)-AR.