The mechanisms in the central
The mechanisms in the central nervous system (CNS) controlling the sympathetic tone of the gpr120 agonist are associated to behavioral strategies, such as those involved in response to stress. In this regard, morphine withdrawal induces profound and severe stress reactions evidenced by enhancement of NA turnover in PVN and an increase in the number of TH neurons co-expressing c-Fos. In parallel with the changes observed in the CNS after morphine withdrawal present results demonstrated an increase in NMN concentration and NMN/NA ratio after morphine withdrawal in right ventricle from wild-type mice. In addition, our results provided evidence for TH phosphorylation after naloxone-precipitated morphine withdrawal in the noradrenergic nerve terminals innervating the right ventricle. Changes in the state of phosphorylation of TH, the rate-limiting enzyme in the synthesis of catecholamines, are critically involved in the regulation of catecholamine synthesis and function. TH activity is regulated by phosphorylation of specific serine residues (particularly serine 40) (Dunkley et al., 2004), therefore assessment of phosphorylation at serine 40 may represent a direct and sensitive measure of TH activity. Altogether, these data suggest that the phosphorylation of TH at serine 40 residue may be an important modulator of the enzyme catalytic activity during naloxone-precipitated morphine withdrawal and might be directly involved in the regulation of NA turnover in the heart. There is strong evidence suggesting that PKA phosphorylates TH at serine 40 and leading to TH activation and catecholamines synthesis (Dunkley et al., 2004). According to previous data (Milanés et al., 2000, Almela et al., 2008) present results demonstrated that naloxone-precipitated morphine withdrawal is associated with an increase of PKA in parallel with an enhancement of TH phosphorylated at serine 40 and NA turnover. Therefore, our results suggest that PKA pathway might be implicated in the cardiac adaptive changes that occur after naloxone-precipitated morphine withdrawal. It is known that CRF system is important in the response to both stress and drugs of abuse (Sarnyai et al., 2001); specifically, CRF1R mediates neuroendocrine and behavioral response to stress as well as to drug abuse (Kreibich et al., 2009). Beyond CRF function as primary activators of the HPA system, CRF and its analogues also have effects on the heart and vasculature by increasing the secretion of NA and adrenaline from the sympathetic nervous system and adrenal medulla, respectively, and play a role within the heart as local mediators (Yang et al., 2010). Thus, the results of the present study demonstrate that the deletion of CRF1R blocks the increased noradrenergic activity in PVN and VLM during morphine withdrawal. These findings suggest that the activation of CRF1R could mediate the elevated NA neurotransmission from the brainstem to PVN and the activation of TH neurons in the VLM following the proposed loop. Our results are consistent with previous findings in mice (Funada et al., 2001) showing that the CRF1R antagonist CRA1000 attenuated noradrenergic activity in the cerebral cortex. In contrast with our data, CP-154526 (CRF1R antagonist), failed blocking the increase noradrenergic activity in the PVN during morphine withdrawal in rats (Navarro-Zaragoza et al., 2010). This disagreement may be the result of species differences (mice versus rats) and/or the different protocols used (CP-154,526 administration or CRF1R gene deletion). In addition, CRF has interesting effects on cardiovascular system (Morimoto et al., 1993). In this regard, CRF and its analogues have effects on the heart and vasculature by increasing the secretion of NA and adrenaline from the sympathetic nervous system and adrenal medulla, respectively, and play a role within the heart as local mediators (Yang et al., 2010). It has been described that an increase in sympathetic input to the heart ventricles originating in the hypothalamus is enough to precipitate cardiac arrhythmias (Xavier et al., 2013, Fontes et al., 2014). However, it is unclear which type of CRF receptor is responsible for the CRF-induced autonomic response. It is known that CRF1R activation in selected brain regions contributes to the stress induced sympathetic activation (Nijsen et al., 2000, Chu et al., 2004). Present results, demonstrated that naloxone-precipitated morphine withdrawal caused an increase in myocardial NMN content and NMN/NA ratio in parallel with an increased phosphorylation of TH suggesting that TH phosphorylation at serine 40 may be important modulators of TH activity which might be involve in the enhancement of medial arterial blood pressure and heart rate observed during morphine withdrawal in wild-type mice (Martínez-Laoden et al., 2014). According with previous data (Martínez-Laoden et al., 2014) the enhancement of NA turnover was abolished in CRF1R KO mice. In addition, the increased PKA levels and the activation of TH were also decreased in mice lacking functional CRF1R levels. Taken together these results suggest that CRF1R may be responsible for the cardiac sympathetic hyperactivity observed during naloxone-precipitated morphine withdrawal.