PMC:3625153 / 1799-6586 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3625153","sourcedb":"PMC","sourceid":"3625153","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3625153","text":"Introduction\nEnvironmental physiologists often link the endogenous processes of organisms with exogenous stimuli affecting them in order to understand the impact of pollution on the broader distributions of populations and species. However, only a few molecular mechanisms driving stress tolerance and adaptation in aquatic organisms have been elucidated to date; these include pathways of response to thermal [1] or metal [2] stress.\nMarine mussels are intertidal organisms living in rapidly fluctuating habitats where protection from natural and anthropogenic stressors (i.e. temperature or salinity daily and/or seasonal variations, metals, PAHs and other contaminants) is essential for survival. Although the responses are often behavioral or metabolic, a powerful mechanism employed by these organisms to cope with environmental challenges is the regulation of genes and proteins related to cytoprotection. These include the multixenobiotic resistance system (MXR), which prevents the cellular accumulation of potentially harmful xenobiotics by active export from the cell of parental or metabolized forms of the compounds. In mussels the MXR response was investigated to infer stress tolerance in animals inhabiting contaminated environments [3], [4], and experimental evidence indicated that this system provides a powerful adaptive advantage for marine bivalves to cope with environmental challenges [5].\nSeveral proteins are involved in the MXR system, and the most studied in an environmental context is the P-glycoprotein (Pgp) [5]. Pgp induction in mussels has been reported in response to a wide range of chemical and physical stressors, including metals, pesticides, as well as temperature or salinity variations [3], [5], suggesting that this transporter may be part of a general and broad-spectrum cellular stress response.\nEvidence indicates that in mammals transcriptional regulation of the ABCB1 gene encoding Pgp is mediated through the phosphorylation activity of the cAMP-dependent protein kinase (PKA). This regulatory pathway was well characterized in tumor cells displaying a constitutive ABCB1 over-expression that acquired chemoresistance [6]. Conversely, the involvement of cAMP has only been hypothesized in mollusks [7]–[9].\nIn the present work, in vivo and in vitro experiments were performed to investigate the modulation of the cAMP/PKA pathway and the putative downstream effects on mRNA expression of an ABCB gene in Mediterranean mussels (Mytilus galloprovincialis).\nIn vivo experiments were carried out exposing mussels to fluoxetine (FX) alone or in combination with propranolol (PROP). FX has recently received considerable attention in the framework of risk assessment investigations with emerging contaminants due to its frequent detection in aquatic environments [10]; moreover, it is recognized as one of the human pharmaceuticals with the highest acute toxicity toward non target organisms [10]. FX is the active ingredient of the antidepressant Prozac®, the most widely prescribed psychoactive drug in the market, acting as selective serotonin reuptake inhibitor (SSRI) in the treatment of depression and other mood disorders by increasing the serotonin levels in neuron synaptic space [11]–[13]. Serotonin (5-hydroxytryptamine, 5-HT) is involved in hormonal and neuronal mechanisms and plays a key role in regulating food intake, metabolism and reproductive success in invertebrates [14], [15]. By interfering with serotoninergic regulation, FX has, therefore, the potential to impair relevant physiological functions in invertebrates.\nPROP is a β adrenergic receptor antagonist used in human therapies to counteract cardiovascular pathologies [16], but it can also act as a 5-HT receptor antagonist [14]. PROP is widely detected in aquatic environments [17]–[19]. The drug was recently reported to bioconcentrate up to about 360 µg/g w.w. in mussel tissues [20], and also to affect cAMP signaling and ABCB mRNA expression [8].\nTo specifically address different steps of the pathway potentially leading to regulation of ABCB mRNA expression, in vitro experiments using several physiological agonists and pharmacological modulators of cAMP/PKA signaling were carried out on isolated haemocytes. Besides the advantages provided by their employment as a cell model for both in vivo and in vitro investigations of ABCB gene regulation in a nonconventional model species as the marine mussel [21], haemocytes represent an attractive model for the purposes of this study. These cells are known to have a complex cell signaling network that allows them to modulate their own functions [22]. These signaling pathways show high homology with those of vertebrates [23], [24]; however, their physiological roles in mussel haemocytes need further 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