TheNa-K-2Clcotransporter2(NKCC2)wasthoughttobekidneyspecific.Hereweshowexpressioninthebrainhypothalamo-neurohypophyseal system (HNS), wherein upregulation follows osmotic stress. The HNS controls osmotic stability through the synthesis and release of the neuropeptide hormone, arginine vasopressin (AVP). AVP travels through the bloodstream to the kidney, where it promotes water conservation. Knockdown of HNS NKCC2 elicited profound effects on fluid balance following ingestion of a high-salt solution-rats produced significantly more urine, concomitant with increases in fluid intake and plasma osmolality. Since NKCC2 is the molecular target of the loop diuretics bumetanide and furosemide, we asked about their effects on HNS function following disturbed water balance. Dehydration-evoked GABAmediated excitation of AVP neurons was reversed by bumetanide, and furosemide blocked AVP release, both in vivo and in hypothalamic explants. Thus, NKCC2-dependent brain mechanisms that regulate osmotic stability are disrupted by loop diuretics in rats.

Nitric oxide (NO) negatively modulates the secretion of vasopressin (AVP), oxytocin (OT) and atrial natriuretic peptide (ANP) induced by the increase in extracellular osmolality, whereas carbon monoxide (CO) and hydrogen sulphide (H 2 S) act to potentiate it; however, little information is available for the osmotic challenge model about whether and how such gaseous systems modulate each other. Therefore, using an acute ex vivo model of hypothalamic and neurohypophyseal explants (obtained from male 6/7-week-old Wistar rats) under conditions of extracellular iso-and hypertonicity, we determined the effects of NO (600 μmol L -1 sodium nitroprusside), CO (100 μmol L -1 tricarbonylchloro[glycinato]ruthenium [II]) and H 2 S (10 mmol L -1 sodium sulphide) donors and nitric oxide synthase (NOS) (300 μmol L -1 N ω -methyl-l-arginine [LNMMA]), haeme oxygenase (HO) (200 μmol L -1 Zn(II) deuteroporphyrin IX 2,4-bisethylene glycol [ZnDPBG]) and cystathionine β-synthase (CBS) (100 μmol L -1 aminooxyacetate [AOA]) inhibitors on the release of hypothalamic ANP and hypothalamic and neurohypophyseal AVP and OT, as well as on the activities of NOS, HO and CBS. LNMMA reversed hyperosmolality-induced NOS activity, and enhanced hormonal release by the hypothalamus and neurohypophysis, in addition to increasing CBS and hypothalamic HO activity. AOA decreased hypothalamic and neurohypophyseal CBS activity and hormonal release, whereas ZnDPBG inhibited HO activity and hypothalamic hormone release; however, in both cases, AOA did not modulate NOS and HO activity and ZnDPBG did not affect NOS and CBS activity. Thus, our data indicate that, although endogenous CO and H 2 S positively modulate AVP, OT and ANP release, only NO plays a concomitant role of modulator of hormonal release and CBS activity in the hypothalamus and neurohypophysis and that of HO activity in the hypothalamus during an acute osmotic stimulus, which suggests that NO is a key gaseous controller of the neuroendocrine system. K E Y W O R D S atrial natriuretic peptide, carbon monoxide, hydrogen sulphide, nitric oxide, oxytocin, vasopressin 2 of 16 | COLETTI ET aL. | INTRODUC TI ONThe release of vasopressin (AVP) and oxytocin (OT) by the hypothalamus and the neurohypophysis is a very complex and robust process that is controlled by many factors produced both within and outside these brain sites. 1 Among such factors, atrial natriuretic peptide (ANP) stands out as established modulator of thirst and AVP release, 2-4 whereas a novel class of small polar molecules, mainly represented by nitric oxide (NO), carbon monoxide (CO) and hydrogen sulphide (H 2 S), 5 have achieved prominence more recently.NO is produced from the amino acid l-arginine via a reaction catalysed by the enzyme nitric oxide synthase (NOS), which presents three major isoforms (neuronal [nNOS], endothelial [eNOS] and inducible [iNOS]), where, in the central nervous system (CNS), nNOS is considered the main isoform. 6 NOS active form is usually dimeric and exhibits an oxygenase domain, in which, in addition to ...

The present study investigated the type 1 cannabinoid receptor (CB1R) as a potential candidate to mediate the homeostatic responses triggered by 24 h of water deprivation, which constitutes primarily a hydroelectrolytic challenge and also significantly impacts energy homeostasis. The present results demonstrated for the first time that CB1R mRNA expression is increased in the hypothalamus of water-deprived (WD) rats. Furthermore, the administration of ACEA, a CB1R selective agonist, potentiated WD-induced dipsogenic effect, whereas AM251, a CB1R antagonist, attenuated not only water but also salt intake in response to WD. In parallel with the modulation of thirst and salt appetite, we confirmed that CB1Rs are essential for the development of appropriated neuroendocrine responses. Although the administration of ACEA or AM251 did not produce any effects on WD-induced arginine vasopressin (AVP) secretion, oxytocin (OXT) plasma concentrations were significantly decreased in WD rats treated with ACEA. At the genomic level, ACEA significantly decreased AVP and OXT mRNA expression in the hypothalamus of WD rats, whereas AM251 potentiated both basal and WD-induced stimulatory effects on the transcription of AVP and OXT genes. In addition, we showed that water deprivation alone upregulated proopiomelanocortin, Agouti-related peptide, melanin-concentrating hormone, and orexin A mRNA levels in the hypothalamus, and that CB1Rs regulate main central peptidergic pathways controlling food intake, being that most of these effects were also significantly influenced by the hydration status. In conclusion, the present study demonstrated that CB1Rs participate in the homeostatic responses regulating fluid balance and energy homeostasis during water deprivation.

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