Benzo(a)pyrene triggers desensitization of β2-adrenergic pathway

Abstract

Exposure to environmental polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene (B(a)P), has been linked to several health-threatening risks. PAHs were also shown to hinder adrenergic receptor (ADR) responses. As we previously demonstrated that B(a)P can directly interact with the β2ADR, we investigated here whether B(a)P could decrease β2ADR responsiveness by triggering receptor desensitization phenomena. We firstly showed that exposure to B(a)P reduced β2ADR-mediated epinephrine-induced induction of NR4A gene mRNAs and of intracellular cAMP. Analysis of β2ADR protein expression demonstrated that B(a)P rapidly decreased membrane expression of β2ADR with a subsequent degradation of receptor protein. B(a)P exposure concomitantly rapidly increased the β2ADR mRNA levels. The use of the β-blockers, propranolol and ICI 118.551, demonstrated the involvement of β2ADR itself in this increase. However, sustained exposure to B(a)P induced a diminution of β2ADR mRNA steady-state as a result of the acceleration of its degradation. Together, these results show that, beside the well-known activation of the aryl hydrocarbon receptor, PAH deleterious effects may involve the dysfunction of adrenergic responses through, in part, the desensitization of β2ADR. This may be taken in consideration when β2-agonists/antagonists are administered in patients exposed to important concentrations of PAHs, e.g. in cigarette smokers.

Figure 1

B(a)P decreased β2ADR-mediated induction of NR4As expression triggered by epinephrine (EN). (a,b,c) HMEC-1 cells were either untreated or exposed to indicated concentrations of EN for 1 h, in the presence or not of 10 µM propranolol (nonselective β-blocker) or 10 µM ICI 118.551 (selective β2-bloker). mRNA expressions of β2ADR-target genes, NR4A1 (a), NR4A2 (b) and NR4A3 (c) were next determined by RT-qPCR. (d,e,f) HMEC-1 cells were exposed to indicated concentrations of B(a)P or vehicule (DMSO) for 24 h before being co-exposed, or not, to 10 µM EN for 1 h. mRNA expressions of β2ADR-target genes, NR4A1 (d), NR4A2 (e) and NR4A3 (f) were next determined by RT-qPCR. Data are expressed relatively to mRNA levels found in untreated cells, arbitrarily set to 1 unit, and are the means ± S.D of at least three independent assays. *p < 0.05; **p < 0.01 when compared to B(a)P-untreated cells.

Figure 2

B(a)P pre-treatment decreased EN-induced intracellular cAMP production. HMEC-1 cells were exposed to 10 μM B(a)P or vehicule (DMSO) for 24 h, and were then co-exposed, or not, to 10 µM EN for 10 min. cAMP levels (pmol/well) were next determined, as described in Materials and Methods section. Data are the means ± S.D of three independent assays. *p < 0.05 when compared to untreated cells; ^# p < 0.05 when compared to EN treated counterparts.

Figure 3

B(a)P triggered an early reduction of β2ADR expression at cell membrane and a late degradation of β2ADR protein. (a) HMEC-1 cells were exposed to 10 µM EN, 1 µM B(a)P or vehicle (DMSO) for 45 min. β2ADR was then analyzed by immunolocalization for the expression at cell membrane. Data shown are representative of three independent assays. (b) HMEC-1 cells were exposed to 1 µM B(a)P or vehicle (DMSO) for 45 min. β2ADR protein content was then determined by Western-blotting. (c) HMEC-1 cells were exposed to indicated concentrations of B(a)P or vehicle (DMSO) for 24 h. β2ADR protein content was then determined by Western-blotting. (b and c) Bottom panel, a representative blot is shown for β2ADR and P38 protein levels. Upper panel, for each concentration of B(a)P, data were quantified by densitometric analysis, normalized to P38 protein level and expressed relative to β2ADR found in untreated cells, arbitrarily set at the value of 100%. Results are the means ± S.D of values from three independent assays. *p < 0.05 when compared to untreated cells.

Figure 4

B(a)P exposure influenced β2ADR mRNA level. (a) HMEC-1 cells were exposed to 10 µM EN, 1 µM B(a)P or vehicle (DMSO) for indicated times before analyzing β2ADR mRNA expressions by RT-qPCR. Data are expressed relatively to mRNA levels found in corresponding control cells, arbitrarily set to 1 unit, and are the means ± S.D of at least three independent assays. *p < 0.05 when compared to corresponding control cells. (b) HMEC-1 cells were exposed to indicated concentrations of B(a)P or vehicle (DMSO) for 1 h. mRNA expressions of β2ADR were then analyzed by RT-qPCR. Data are expressed relatively to mRNA levels found in control cells, arbitrarily set to 1 unit, and are the means ± S.D of at least three independent assays. (c,d) HMEC-1 cells were exposed to 10 µM EN, 1 µM B(a)P or vehicle (DMSO) for 1 h, in the presence or absence of 10 µM actinomycin D (c), or in the presence or absence of 10 µM propranolol or 10 µM ICI 118.551 (d). mRNA expressions of β2ADR were then analyzed by RT-qPCR. Data are expressed relatively to mRNA levels found in corresponding control cells, arbitrarily set to 1 unit, and are the means ± S.D of at least three independent assays. *p < 0.05 when compared with conotrol cells. ^# p < 0.05 when compared to EN- or B(a)P-treated counterparts. (e,f) HMEC-1 cells were either untreated or exposed (1 h) to 10 µM EN (e), or were exposed to indicated concentrations of B(a)P or vehicle (DMSO) (f), Half-lives of β2ADR mRNA were then calculated as described in Materials and Methods section. Data correspond to mRNA half-lives, expressed in minutes, and are the means ± S.D of three independent assays. *p < 0.05 when compared with untreated cells. ^# p < 0.05 when compared to EN- or B(a)P-treated counterparts. Representative curves of mRNA decay kinetics in the absence (Ct) or presence of EN are given in the insert (e).