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 [6] and Rohlff and Glazer [28].
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.
Table 1.
List of primers used in real time PCR analyses.