ABSTRACT
In fish of freshwaters environments, the accumulation and toxic effects of arsenite (AsIII) can be attenuated by detoxification proteins such as GST and ABCC transporters. We studied the effects of AsIII on the middle intestine of O. mykiss in ex-vivo and in vivo/ex vivo assays. For the ex vivo assays, we measured the transport rate of the ABCC substrate DNP-SG and GST activity in intestinal strips and everted sacs. AsIII inhibited DNP-SG transport in a concentration-dependent manner, specifically when we applied it on the basolateral side. GST activity increased when we applied a maximum concentration of AsIII. For the in vivo/ex vivo assays, we kept fish in water with or without 7.7⯵molâ¯L-1 of AsIII for 48â¯h. Then, we measured DNP-SG transport rate, GST activity, and PP1 activity in intestine strips during one hour. For PP1 activity, we incubated the strips with or without microcystin-LR (MCLR), a toxin excreted through ABCC2 proteins. We also analyzed Abcc2 and Gst-π mRNA expression in intestine and liver tissue. In the group exposed in vivo to AsIII, DNP-SG transport rate and GST activity were higher and the effect of MCLR over PP1 activity was attenuated. AsIII significantly induced only Abcc2 mRNA expression in both middle intestine and liver. Our results suggest that, in the middle intestine of O. mykiss, AsIII is absorbed mainly at the basolateral side of the enterocytes, excreted to the lumen by ABCC2 transporters, and is capable of modulating Abcc2 mRNA expression by a transcriptional mechanism.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Arsenites , Glutathione S-Transferase pi/metabolism , Intestines/enzymology , Liver/metabolism , Oncorhynchus mykiss/metabolism , Animals , Arsenites/metabolism , Arsenites/pharmacokinetics , Arsenites/toxicity , Fish Proteins/metabolism , Gene Expression Regulation , RNA, Messenger , Xenobiotics/metabolism , Xenobiotics/pharmacokinetics , Xenobiotics/toxicityABSTRACT
RATIONALE: Experimental evidence indicates that nicotine causes long-lasting changes in the brain associated with behavior. Although much has been learned about factors participating in this process, less is known concerning the mechanisms and brain areas involved in nicotine preference. OBJECTIVES: The objective of this study is to examine the participation of brain structures during the development of nicotine-conditioned place preference (CPP). METHODS: To identify brain regions activated in CPP, we have measured the levels of phosphorylated cyclic AMP response element binding protein (pCREB) and Fos protein using a behavioral CPP and conditioned place aversion (CPA) paradigms. RESULTS: Rats developed reliable and robust CPP and also CPA. During nicotine preference and reinstatement behaviors, a significant increase of both pCREB and Fos protein expression occurs in the nucleus accumbens (NAc) and ventral tegmental area (VTA) and also in the prefrontal cortex (PFC), dorsal striatum (DStr), amygdala, and hippocampus. These increases were abolished by the administration of mecamylamine or by a CPA protocol, showing a specific activation of pCREB in drug preference animals, mediated by nicotinic receptors. Specifically in the VTA, nicotine-induced preference and reinstatement of the preference caused the activation of dopaminergic and GABAergic cells in different proportions. CONCLUSION: The results indicate that the phosphorylation of CREB and expression of Fos protein, as indicators of neural activity, accompany the acquisition and maintenance of nicotine-induced CPP but not CPA in mesolimbic areas (NAc, VTA, PFC, and DStr) as well as in memory consolidation structures (hippocampus and amygdala) and nicotinic receptor are involved in this process. Taken together, these studies identify the brain regions where pCREB activity is essential for nicotine preference.
Subject(s)
Brain/drug effects , CREB-Binding Protein/metabolism , Conditioning, Operant/drug effects , Nicotine/administration & dosage , Nicotinic Agonists/administration & dosage , Oncogene Proteins v-fos/metabolism , Animals , Brain/anatomy & histology , Brain/metabolism , Cell Count/methods , Conditioning, Operant/physiology , Extinction, Psychological/drug effects , Gene Expression Regulation/drug effects , Glutamate Decarboxylase/metabolism , Male , Mecamylamine/pharmacology , Nicotinic Antagonists/pharmacology , Phosphorylation/drug effects , Rats , Rats, Sprague-Dawley , Statistics, Nonparametric , Time Factors , Tyrosine 3-Monooxygenase/metabolismABSTRACT
Hypothalamic norepinephrine (NE) release regulates arterial pressure by altering sympathetic nervous system activity. Because angiotensin (Ang) (1-7) decreases hypothalamic NE release and this effect may be correlated with a diminished NE synthesis, we hypothesize that Ang-(1-7) down-regulates tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamines biosynthesis. We investigated the effect of Ang-(1-7) on centrally TH activity and expression. TH activity was evaluated by the release of tritiated water from (3)H-l-tyrosine. TH expression and phosphorylation were determined by western blot. Hypothalami from normotensive or spontaneously hypertensive rats pre-incubated with Ang-(1-7) showed a significant decrease in TH specific activity. Ang-(1-7) caused a decrease in TH phosphorylation at Ser19 and Ser40 residues. The heptapeptide induced a decrease in TH expression that was blocked by an AT(2) receptor antagonist and not by an AT(1) or Mas receptor antagonist, suggesting the involvement of AT(2) receptors. The proteasome inhibitor MG132 blocked the Ang-(1-7)-mediated TH reduction. In addition, Ang-(1-7) increased the amount of TH-ubiquitin complexes, indicating that the Ang-(1-7)-mediated TH degradation involves ubiquitin conjugation prior to proteasome degradation. We conclude that Ang-(1-7) down-regulates TH activity and expression centrally leading to a decrease in the central NE system activity.
