Cyclic-AMP mediated regulation of ABCB mRNA expression in mussel haemocytes

Abstract

Background: The multixenobiotic resistance system (MXR) allows aquatic organisms to cope with their habitat despite high pollution levels by over-expressing membrane and intracellular transporters, including the P-glycoprotein (Pgp). In mammals transcription of the ABCB1 gene encoding Pgp is under cAMP/PKA-mediated regulation; whether this is true in mollusks is not fully clarified.

Methodology/principal findings: cAMP/PKA regulation and ABCB mRNA expression were assessed in haemocytes from Mediterranean mussels (Mytilus galloprovincialis) exposed in vivo for 1 week to 0.3 ng/L fluoxetine (FX) alone or in combination with 0.3 ng/L propranolol (PROP). FX significantly decreased cAMP levels and PKA activity, and induced ABCB mRNA down-regulation. FX effects were abolished in the presence of PROP. In vitro experiments using haemocytes treated with physiological agonists (noradrenaline and serotonin) and pharmacological modulators (PROP, forskolin, dbcAMP, and H89) of the cAMP/PKA system were performed to obtain clear evidence about the involvement of the signaling pathway in the transcriptional regulation of ABCB. Serotonin (5-HT) decreased cAMP levels, PKA activity and ABCB mRNA expression but increased the mRNA levels for a putative 5-HT1 receptor. Interestingly, 5-HT1 was also over-expressed after in vivo exposures to FX. 5-HT effects were counteracted by PROP. Forskolin and dbcAMP increased PKA activity as well as ABCB mRNA expression; the latter effect was abolished in the presence of the PKA inhibitor H89.

Conclusions: This study provides the first direct evidence for the cAMP/PKA-mediated regulation of ABCB transcription in mussels.

Figure 1. The pharmaceutical FX alters cell signaling endpoints and ABCB mRNA expression in mussel haemocytes under in vivo exposures.

(A) cAMP content (B) relative PKA activities (C) relative ABCB mRNA expression. All biological endpoints were assessed in haemocytes collected from mussels exposed for 7 days to 0.3 ng/L FX 0.3 ng/L PROP, or to the mixture FX+PROP (0.3 ng/L+0.3 ng/L). A group of control (water) exposed mussels were included in the analysis. Data represent mean ± SEM (N = 5). Different superscripts letters indicate statistically significant differences (p<0.05, one-way ANOVA followed by Bonferroni's test). Basal PKA activity = 0.88±0.07 nmol/min/mg protein.

Figure 2. Effects of NOR and 5-HT on cAMP-related parameters and ABCB mRNA expression in mussel haemocytes incubated in vitro.

(A) cAMP content; (B) relative PKA activities; (C) relative ABCB mRNA expression. All biological endpoints were assessed after 1 h of exposure to 1 µM NOR or 5-HT. Data are derived from three independent experiments (mean ± SEM); *p<0.05 vs control. Basal PKA activity = 1.12±0.17 nmol/min/mg protein.

Figure 3. Effects of PROP pre-incubation on 5-HT modulated parameters in mussel haemocytes incubated in vitro.

(A) cAMP content; (B) relative PKA activities; (C) relative ABCB mRNA expression; (D) relative 5-HT₁ mRNA expression. All biological endpoints were assessed after 1 h of exposure to 1 µM 5-HT in the presence/absence of a 15-min pre-incubation with 100 µM PROP. Data are derived from three independent experiments (mean ± SEM); *p<0.05 between pairs of samples. Basal PKA activity  =  1.12±0.17 nmol/min/mg protein.

Figure 4. Relative mRNA levels of a 5-HT₁ receptor are affected by 5-HT or FX exposure.

(A) Relative 5-HT₁ mRNA expression assessed in haemocytes incubated in vitro for 1 h with 1 µM 5-HT in the presence/absence of a 15-min pre-incubation with 100 µM PROP. Data are derived from three independent experiments (mean ± SEM); *p<0.05 between pairs of samples. (B) Relative 5-HT₁ mRNA expression evaluated in haemocytes of mussels exposed in vivo for 7 days to FX (0.3 ng/L), PROP (0.3 ng/L) or to the mixture FX+PROP (0.3 ng/L+0.3 ng/L). A group of control (water) exposed mussels were included in the analysis. Data are reported as mean ± SEM. Different superscripts letters indicate statistically significant differences (p<0.05, N = 5).

Figure 5. Effects of FSK on cAMP-related parameters and ABCB mRNA expression in mussel haemocytes incubated in vitro.

(A) cAMP content; (B) relative PKA activities; (C) relative ABCB mRNA expression. All biological endpoints were assessed after 4 h of exposure to 20 µM FSK. Data are derived from three independent experiments (mean ± SEM); *p<0.05 vs control. Basal PKA activity = 1.12±0.17 nmol/min/mg protein.

Figure 6. Effects of H89 pre-incubation on dbcAMP induced stimulation of PKA activity and ABCB mRNA expression in mussel haemocytes incubated in vitro.

(A) relative PKA activities; (B) relative ABCB mRNA expression. Both biological endpoints were assessed after 4 h of exposure to 100 µM dbcAMP in the presence/absence of a 30-min pre-incubation with 10 µM H89. Data are derived from three independent experiments (mean±SEM); *p<0.05 between pairs of samples. Basal PKA activity = 1.12±0.17 nmol/min/mg protein.

Figure 7. Untranslated 5′ regulatory region of ABCB genes from the mussels M. galloprovincialis and M. californianus showing promoter elements.

Numbering is relative to the transcription initiation site. Gene Bank Accession Numbers are FM999809 (M. galloprovincialis ABCB1) and EF52141 (M. californianus ABCB). Sequence of the human ABCB1 promoter in the same region is reported for comparison (GenBank Ac. Numb. NM000927). Furthermore, a more complete sequence analysis of the human ABCB1 promoter is reported by Scotto and Johnson and Rohlff and Glazer .

Figure 8. Schematic representation of the cAMP-dependent pathway leading to ABCB transcriptional regulation in mussel haemocytes.

5-HT₁: type 1 serotonin receptor; 5-HT: serotonin; PROP: propranolol; Gi: inhibitory G-protein; AC: adenylyl cyclase; FSK: forskolin; cAMP: cyclic-AMP; PKA(i): inactivated cAMP-dependent protein kinase (PKA); PKA(a): activated PKA (catalytic subunit); dbcAMP: dybutyril cAMP; H89: N-[2-((p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; CRF: cAMP-responsive factors.