PMC:3625153 / 6588-15530 JSONTXT

{"project":"2_test","denotations":[{"id":"23593491-16257063-88440234","span":{"begin":442,"end":444},"obj":"16257063"},{"id":"23593491-18280486-88440235","span":{"begin":448,"end":450},"obj":"18280486"},{"id":"23593491-2888864-88440236","span":{"begin":2628,"end":2630},"obj":"2888864"},{"id":"23593491-8391422-88440237","span":{"begin":2634,"end":2636},"obj":"8391422"},{"id":"23593491-8562304-88440238","span":{"begin":7276,"end":7278},"obj":"8562304"},{"id":"23593491-8562304-88440239","span":{"begin":7886,"end":7888},"obj":"8562304"},{"id":"23593491-8562304-88440240","span":{"begin":8057,"end":8059},"obj":"8562304"},{"id":"23593491-9458366-88440241","span":{"begin":8573,"end":8575},"obj":"9458366"},{"id":"T79814","span":{"begin":442,"end":444},"obj":"16257063"},{"id":"T82223","span":{"begin":448,"end":450},"obj":"18280486"},{"id":"T27426","span":{"begin":2628,"end":2630},"obj":"2888864"},{"id":"T85711","span":{"begin":2634,"end":2636},"obj":"8391422"},{"id":"T53271","span":{"begin":7276,"end":7278},"obj":"8562304"},{"id":"T79686","span":{"begin":7886,"end":7888},"obj":"8562304"},{"id":"T37090","span":{"begin":8057,"end":8059},"obj":"8562304"},{"id":"T72931","span":{"begin":8573,"end":8575},"obj":"9458366"}],"text":"Results\n\nEffects of FX, PROP or the mixture FX+PROP on cAMP-related parameters and ABCB mRNA expression under in vivo exposures\nMussels were exposed in vivo to fluoxetine (FX), a selective serotonin reuptake inhibitor, to propranolol (PROP), a β-adrenergic receptor blocker, or to their mixture (FX+PROP). The tested concentration for both compounds (0.3 ng/L) falls within the low environmental range reported in surface and coastal waters [13], [25].\nHaemocytes collected from mussels exposed to FX showed significantly decreased cAMP levels and PKA activity above control values, and also ABCB mRNA down-regulation ( Fig. 1 ). Mussel exposure to the mixture FX+PROP did not induce significant variations of the biological endpoints with respect to control levels ( Fig. 1 ).\n10.1371/journal.pone.0061634.g001 Figure 1 The pharmaceutical FX alters cell signaling endpoints and ABCB mRNA expression in mussel haemocytes under in vivo exposures.\n(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\u003c0.05, one-way ANOVA followed by Bonferroni's test). Basal PKA activity = 0.88±0.07 nmol/min/mg protein.\n\nExpression of the mussel ABCB gene product is affected by serotoninergic but not by adrenergic agonists\nHaemocytes from control M. galloprovincialis were treated with either noradrenaline (NOR) or serotonin (5-HT) as selective agonists of adrenergic (AR) and 5-HT receptors, respectively. NOR did not increase either cAMP levels or PKA activities, whereas 5-HT addition significantly reduced both endpoints ( Fig. 2A,B ). Accordingly, ABCB mRNA expression was not affected by NOR while it was significantly down-regulated by 5-HT ( Fig. 2C ).\n10.1371/journal.pone.0061634.g002 Figure 2 Effects of NOR and 5-HT on cAMP-related parameters and ABCB mRNA expression in mussel haemocytes incubated in vitro.\n(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\u003c0.05 vs control. Basal PKA activity = 1.12±0.17 nmol/min/mg protein. The effects of 5-HT on mussel haemocytes were further evaluated in the presence of PROP, used as a 5-HT1 receptor antagonist [26], [27]. PROP significantly blocked the reduction in cAMP levels and PKA activities and the down-regulation of the ABCB gene product triggered by 5-HT ( Fig. 3A,B,C ).\n10.1371/journal.pone.0061634.g003 Figure 3 Effects of PROP pre-incubation on 5-HT modulated parameters in mussel haemocytes incubated in vitro.\n(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\u003c0.05 between pairs of samples. Basal PKA activity  =  1.12±0.17 nmol/min/mg protein.\n\nExpression of a mussel 5-HT1 gene product is up-regulated by 5-HT\nAs shown in Fig. 4A , a 1-hour incubation with 5-HT in vitro also induced a significant up-regulation of a transcript encoding a putative mussel 5-HT1 receptor (5-HT1) in haemocytes, while cell pre-treated with PROP significantly blocked the 5-HT effect on its receptor. In agreement, also haemocytes from mussels exposed in vivo to FX showed a significant up-regulation the 5-HT1 transcript ( Fig. 4B ). This effect was not observed in mussels exposed to the mixture FX+PROP ( Fig. 4B ).\n10.1371/journal.pone.0061634.g004 Figure 4 Relative mRNA levels of a 5-HT1 receptor are affected by 5-HT or FX exposure.\n(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\u003c0.05 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\u003c0.05, N = 5).\n\nThe ABCB gene product is over-expressed following FSK-activated cAMP/PKA signaling\nThe adenylyl cyclase (AC) activator forskolin (FSK) was used to investigate the effect of cAMP/PKA stimulation on ABCB mRNA expression. Indeed, FSK induced significant increases in cAMP levels and PKA activities in mussel haemocytes ( Fig. 5A,B ), which was correlated with a significant ABCB transcript up-regulation ( Fig. 5C ).\n10.1371/journal.pone.0061634.g005 Figure 5 Effects of FSK on cAMP-related parameters and ABCB mRNA expression in mussel haemocytes incubated in vitro.\n(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\u003c0.05 vs control. Basal PKA activity = 1.12±0.17 nmol/min/mg protein.\n\nExpression of the ABCB gene product follows induction and inhibition of PKA activity\nHaemocytes were exposed for 4 h to the PKA activator dbcAMP in the presence/absence of H89, a selective PKA inhibitor ( Fig. 6 ). As predicted, dbcAMP significantly increased PKA activities, while no significant effects were detected in the presence of H89 ( Fig. 6A ). ABCB mRNA levels were significantly increased by haemocyte dbcAMP treatment, while cells treated with dbcAMP in the presence of H89 had activities not significantly different from the control ( Fig. 6B ).\n10.1371/journal.pone.0061634.g006 Figure 6 Effects of H89 pre-incubation on dbcAMP induced stimulation of PKA activity and ABCB mRNA expression in mussel haemocytes incubated in vitro.\n(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\u003c0.05 between pairs of samples. Basal PKA activity = 1.12±0.17 nmol/min/mg protein.\n\nSequence analysis of the 5′-untranslated region of mussel ABCB genes\nThe computational analysis of the 5′-untranlasted regions of ABCB genes from two mytilid species M. galloproviancialis and M. californianus revealed the presence of several putative PKA-related regulatory elements within the ABCB promoter region ( Fig. 7 ). Amongst these, putative binding sites for the PKA-dependent CRE-BP transcription factor were found in both M. galloprovincialis (−76) and M. californianus (−96 and −170) ABCB promoter regions; binding sites for this transcription factor are also present within the human ABCB1 gene promoter ([28]; Fig. 7 ).\n10.1371/journal.pone.0061634.g007 Figure 7 Untranslated 5′ regulatory region of ABCB genes from the mussels M. galloprovincialis and M. californianus showing promoter elements.\nNumbering 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]. Regulatory elements binding the AP-1 and AP-2 transcription factors, which occur in the human ABCB1 promoter and are regulated by PKA in a manner similar to CREB ([28] and reference therein), were also found in the 5′untranslated regions of both mussel ABCB gene sequences analysed ( Fig. 7 ). Moreover, binding sites for the Sp1 transcription factor were also found at position −35 (M. galloprovincialis), and at positions −55 and −177 (M. californianus) ( Fig. 7 ). Sp1 is a PKA-regulated nuclear transcription factor that binds to TATA-less promoters, including ABCB gene promoters, and it is primarily involved in constitutive rather than stress-induced gene regulation [6], [29]. Binding sites for the HSF transcription factor were found both in M. galloprovincialis (−95, −174) and M. californianus (−125) ABCB promoter regions ( Fig. 7 ). Finally, binding sites for SRF and NF-kB transcription factors were also found in ABCB promoter regions from both mussel species ( Fig. 7 ), consistent with reports for the human ABCB gene promoter [6].\n"}