Novel interactions between the endocannabinoid system and oxytocin

VALERIA RETTORI; MARÍA ZORRILLA ZUBILETE; JAVIER FERNANDEZ-SOLARI; BERENICE BURDET; ANDREA DE LAURENTIIS

Max Planck Institute, Munich, Germany

Congreso; 14th Annual Meeting of the Neuroendocrinology Section of the German Society of Endocrinology (DGE) and 20th Anniversary of an Argentine-German Collaboration.; 2010

German Society of Endocrinology

The endocannabinoid system is a neuroactive lipid signaling system that functions as modulator of synaptic neurotransmitter release and recent studies reveal an interaction between this system and stress response. It consists mainly of two principal endogenous ligands that are called anandamide (AEA) and 2-arachidonylglycerol (2-AG) that bind to cannabinoid CB1 and CB2 receptors. The CB1 receptors are expressed throughout most regions of the brain and also in some peripheral tissues. CB2 receptors are predominantly expressed in immune cells as well as in microglia. Both receptors are Gi protein coupled receptors which main function is to inhibit adenylyl cyclase activity and therefore inhibit many neurotransmitters release. The well known hypothalamo-pituitary-adrenal axis (HPA) as well as the hypothalamo-neurohypophyseal axis (HNH) are essential to maintain the homeostasis and both are activated by different kind of stress. Magnocellular neurons of the hypothalamic supraoptic nuclei (SON) and paraventricular nuclei (PVN) synthesize vasopressin and oxytocin (OXT) and send axons that terminate in the posterior pituitary from where these hormones are released into blood and regulate different functions such as reproduction and body fluid homeostasis. In addition, the PVN contains another group of OXT producing neurons, the parvocellular neurons whose dendrits and axons terminate in different regions of limbic system and other regions of CNS to intervene in memory and behavior. There is abundant evidence that nitric oxide (NO) functions as a local modulator of neuronal activity. Neuronal nitric oxide synthase is also expressed in the SON and PVN where it was found localized in OXT producing neurons. On the other hand, it has been reported that cannabinoid receptors are localized in the SON and PVN nuclei of the hypothalamus as well as in the pituitary suggesting that endocannabinoids could modulate the hormone release. Furthermore, we measured the activity of anandamide synthase in the hypothalamus and found its activity increased after systemic injection of lipopolysaccharide (LPS) or TNF alpha. Therefore the first aim of our research was to study the interaction between anandamide and NO on the regulation of OXT release from neurohypophysis (NH) and hypothalamus (HYP) of adult male rats in vitro. We found that AEA increased (p<0.01) NOS activity in both HYP and NH. Sodium nitroprusside, a NO donor, decreased (p<0.01) OXT release from HYP and NH. In addition, haemoglobin a scavenger of NO, augmented the increase of OXT release induced by AEA in HYP while blocked the decrease induced in NH. The presence of AM251, a CB1 antagonist, completely prevented the stimulatory effect of AEA on OXT release in HYP but did not modify the inhibitory effect of AEA on OXT release from NH. On the contrary, AM630, a CB2 antagonist, reverted the inhibitory effect in NH and was without effect in HYP. The evaluation of CB1 and CB2 mRNA by quantitative RT-PCR showed that both are present in the HYP but in NH there is a higher quantity of CB2 mRNA. Western blot studies yielded higher amount of CB2 protein in NH that was almost undetectable in HYP. We concluded that the differences of the effect of AEA on the release of OXT between HYP and NH could be due to the different subtype of receptor activated by anandamide. The second aim was to study the hypothalamic endocannabinoid system in the response to LPS challenge on OXT and TNF alpha plasma levels in vivo and their release from HYP fragments in vitro. We found that in vivo administration of LPS i.p. after one hour increased highly significantly OXT (p<0.001) and TNF alpha (p<0.01) plasma levels. The central administration of AEA into third central ventricle (i.c.v.) did not modified significantly basal OXT nor TNF alpha plasma levels but the inhibitor URB597 that inhibits the enzyme that hydrolyses AEA increased further the LPS-induced OXT and TNF alpha plasma levels. The observed increase in plasma OXT levels after LPS was partially blocked when AM251, a CB1 antagonist, was injected i.c.v., and completely blocked when AM630, a CB2 antagonist, was injected together with AM215. Similarly, the increase on TNF alpha was partially prevented when AM251 was injected alone or together with AM630. In vitro studies performed only with hypothalamic fragments showed that LPS or TNF alpha increased OXT release that was completely blocked by CB1 antagonist. Also, nitric oxide synthase activity was increased by LPS or TNF alpha and this activation was only partially prevented by the CB1 antagonist. The expression analysis of CB1 and CB2 receptors mRNA by quantitative RT-PCR in hypothalamic fragments from saline and LPS injected rats showed that LPS increased only CB1 mRNA. Also, Western blot studies showed an increase in CB1 protein expression after LPS administration. In conclusion, our finding revealed an interaction between oxytocin, endocannabinoid and NO-ergic systems at hypothalamic level and this interaction provides a mechanism of hypothalamic-neurohypophyseal activity regulation under basal and stress conditions. The third aim was to study the effect of chronic restraint stress on some stress parameters such as cytokines and corticosterone, and evaluate CB1 receptor in hippocampus and amygdala. Restraint stress was applied to adult male rats two hours daily during 7, 14 and 21 days. Blood samples were collected and the hippocampi and amygdala dissected at the end of each period. The results showed that corticosterone plasma levels were increased in all animals submitted to stress at the end of each period. Plasma OXT levels were only significantly increased at 7 and 21 days periods. IL-6 and CB1 receptors total mRNA analyzed by real time RT-PCR and protein levels by Western blot analysis were increased in hippocampi at 7, 14 and 21 days of stress. IL-1 beta and IL-6 mRNA were significantly increased in amygdala only at 14 days of stress. Also the animals showed a reduced habituation in the open field test. In summary all these results suggest that endocannabinoid system enhances the neuroimmunoendocrine response to stress at the limbic and pituitary levels showing a complex interaction between all the mediators that participate in the control of homeostasis. (Grants: BID-PICT 06-0258, CONICET PIP 02546).