Modulation of neuroendocrine stress response by endocannabinoid system

V. BESUHLI RETTORI,; MARÍA A. ZORRILLA ZUBILETE; ANDREA DE LAURENTIIS

Riberao Preto, San Pablo

Encuentro; Scientific Meeting: ?Recent Advances in the Study of the Integrative Physiology with Emphasis on the Neuroendocrine Control of Energy Metabolism and Body Fluid Homeostasis; 2012

University de Sao Paulo, School of Medicine of Riberao Preto, Department of Physiology

The endocannabinoid system is a neuroactive lipid signaling system that functions as modulator of synaptic neurotransmitter release. It consists mainly of two principal endogenous ligands: 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. Recent studies reveal an interaction between this system and stress response. The 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. There is abundant evidence that nitric oxide (NO) functions as a local modulator of neuronal activity. Neuronal nitric oxide synthase (NOS) is also expressed in the SON and PVN where it was found localized in OXT producing neurons. The cannabinoid receptors are also localized in the SON and PVN nuclei of the hypothalamus as well as in the pituitary suggesting that endocannabinoids could modulate the hormone release. 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 blocked the inhibitory effect of AEA 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 AM251. Similarly, the increase on TNF alpha was partially prevented when AM251 was injected alone or together with AM630. 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. Our in vitro studies using hypothalamic fragments showed that LPS or TNF alpha increased OXT release that was completely blocked by CB1 antagonist. Also, NOS 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 findings 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. (Grants: BID-PICT 06-0258, CONICET PIP 02546).