Sex Differences in the Regulation of Vasopressin and Oxytocin Secretion in Bile Duct-Ligated Rats.

INTRODUCTION: Hyponatremia due to elevated arginine vasopressin (AVP) secretion increases mortality in liver failure patients. No previous studies have addressed sex differences in hyponatremia in liver failure animal models. OBJECTIVE: This study addressed this gap in our understanding of the potential sex differences in hyponatremia associated with increased AVP secretion. METHODS: This study tested the role of sex in the development of hyponatremia using adult male, female, and ovariectomized (OVX) female bile duct-ligated (BDL) rats. RESULTS: All BDL rats had significantly increased liver to body weight ratios compared to sham controls. Male BDL rats had hyponatremia with significant increases in plasma copeptin and FosB expression in supraoptic AVP neurons compared to male shams (all p < 0.05; 5-7). Female BDL rats did not become hyponatremic or demonstrate increased supraoptic AVP neuron activation and copeptin secretion compared to female shams. Plasma oxytocin was significantly higher in female BDL rats compared to female sham (p < 0.05; 6-10). This increase was not observed in male BDL rats. Ovariectomy significantly decreased plasma estradiol in sham rats compared to intact female sham (p < 0.05; 6-10). However, circulating estradiol was significantly elevated in OVX BDL rats compared to the OVX and female shams (p < 0.05; 6-10). Adrenal estradiol, testosterone, and dehydroepiandrosterone (DHEA) were measured to identify a possible source of circulating estradiol in OVX BDL rats. The OVX BDL rats had significantly increased adrenal estradiol along with significantly decreased adrenal testosterone and DHEA compared to OVX shams (all p < 0.05; 6-7). Plasma osmolality, hematocrit, copeptin, and AVP neuron activation were not significantly different between OVX BDL and OVX shams. Plasma oxytocin was significantly higher in OVX BDL rats compared to OVX sham. CONCLUSIONS: Our results show that unlike male BDL rats, female and OVX BDL rats did not develop hyponatremia, supraoptic AVP neuron activation, or increased copeptin secretion compared to female shams. Adrenal estradiol might have compensated for the lack of ovarian estrogens in OVX BDL rats.

Brain-Derived Neurotrophic Factor and Supraoptic Vasopressin Neurons in Hyponatremia.

Hyponatremia due to elevated arginine vasopressin (AVP) secretion increases mortality in liver failure patients. The mechanisms causing dysregulation of AVP secretion are unknown. Our hypothesis is that inappropriate AVP release associated with liver failure is due to increased brain-derived neurotrophic factor (BDNF) in the supraoptic nucleus (SON). BDNF diminishes GABAA inhibition in SON AVP neurons by increasing intracellular chloride through tyrosine receptor kinase B (TrkB) activation and downregulation of K+/Cl- cotransporter 2 (KCC2). This loss of inhibition could increase AVP secretion. This hypothesis was tested using shRNA against BDNF (shBDNF) in the SON in bile duct ligated (BDL) male rats. All BDL rats had significantly increased liver weight (p < 0.05; 6-9) compared to shams. BDL rats with control -shRNA injections (BDL scrambled [SCR]) developed hyponatremia with increased plasma AVP and copeptin (CPP; all p < 0.05; 6-9) compared to sham groups. This is the first study to show that phosphorylation of TrkB is significantly increased along with significant decrease in phosphorylation of KCC2 in BDL SCR rats compared to the sham rats (p < 0.05;6-8). Knockdown of BDNF in the SON of BDL rats (BDL shBDNF) significantly increased plasma osmolality and hematocrit compared to BDL SCR rats (p < 0.05; 6-9). The BDL shBDNF rats had significant (p < 0.05; 6-9) decreases in plasma AVP and CPP concentration compared to BDL SCR rats. The BDNF knockdown also significantly blocked the increase in TrkB phosphorylation and decrease in KCC2 phosphorylation (p < 0.05; 6-8). The results indicate that BDNF produced in the SON contributes to increased AVP secretion and hyponatremia during liver failure.

Sniffer cells for the detection of neural Angiotensin II in vitro.

Neuropeptide release in the brain has traditionally been difficult to observe. Existing methods lack temporal and spatial resolution that is consistent with the function and size of neurons. We use cultured "sniffer cells" to improve the temporal and spatial resolution of observing neuropeptide release. Sniffer cells were created by stably transfecting Chinese Hamster Ovary (CHO) cells with plasmids encoding the rat angiotensin type 1a receptor and a genetically encoded Ca2+ sensor. Isolated, cultured sniffer cells showed dose-dependent increases in fluorescence in response to exogenously applied angiotensin II and III, but not other common neurotransmitters. Sniffer cells placed on the median preoptic nucleus (a presumptive site of angiotensin release) displayed spontaneous activity and evoked responses to either electrical or optogenetic stimulation of the subfornical organ. Stable sniffer cell lines could be a viable method for detecting neuropeptide release in vitro, while still being able to distinguish differences in neuropeptide concentration.

Effects of salt-loading on supraoptic vasopressin neurones assessed by ClopHensorN chloride imaging.

Salt-loading (SL) impairs GABAA inhibition of arginine vasopressin (AVP) neurones in the supraoptic nucleus (SON) of the hypothalamus. Based on previous studies, we hypothesised that SL activates tyrosine receptor kinase B (TrkB), down-regulating the activity of K+ /Cl- co-transporter2 (KCC2) and up-regulating Na+ /K+ /Cl- co-transporter1 (NKCC1). These changes in chloride transport would result in increased [Cl- ]i in SON AVP neurones. The study combined virally-mediated chloride imaging with ClopHensorN with a single-cell western blot analysis. An adeno-associated virus with ClopHensorN and a vasopressin promoter (AAV2-0VP1-ClopHensorN) was bilaterally injected in the SON of adult male Sprague-Dawley rats that were either euhydrated (Eu) or salt-loaded (SL) for 7 days. Acutely dissociated SON neurones expressing ClopHensorN were tested for decreases or increases in [Cl- ]i in response to focal application of the GABAA agonist muscimol (100 µmol L-1 ). SON AVP neurones from Eu rats showed muscimol-induced chloride influx (P < 0.05;23/35). SON AVP neurones from SL rats either significantly increased chloride efflux (P < 0.05;27/39) or did not change chloride flux (12/39). The SON AVP neurones that responded to muscimol appeared to be viable and expressed KCC2 and ß-actin. Neurones that did not respond during chloride imaging did not show KCC2 and ß-actin protein expression. The KCC2 antagonist (VU0240551,10 µmol L-1 ) significantly blocked the chloride influx in cells from Eu rats but did not affect cells from SL rats. A NKCC1 antagonist (bumetanide,10 µmol L-1 ) significantly blocked the chloride efflux in cells from SL rats but had no effect on cells from Eu rats. Blocking NKCC1 using bumetanide had less of an effect on the muscimol-induced Cl- influx in Eu rat neurones compared to the KCC2 antagonist. The TrkB antagonist (AnA-12) (50 µmol L-1 ) and protein kinase inhibitor (K252a) (100 nmol L-1 ) each significantly blocked chloride efflux in SON AVP neurones from SL rats. Salt-loading increases [Cl- ]i in SON AVP neurones via a TrKB-KCC2-NKCC1-dependent mechanism in rats.

AT1a influences GABAA-mediated inhibition through regulation of KCC2 expression.

The median preoptic nucleus (MnPO) is an integrative site involved in body fluid homeostasis, cardiovascular control, thermoregulation, and sleep homeostasis. Angiotensin II (ANG II), a neuropeptide shown to have excitatory effects on MnPO neurons, is of particular interest with regard to its role in body fluid homeostasis and cardiovascular control. The present study investigated the role of angiotensin type 1a (AT1a) receptor activation on neuronal excitability in the MnPO. Male Sprague-Dawley rats were infused with an adeno-associated virus with an shRNA against the AT1a receptor or a scrambled control. In vitro loose-patch voltage-clamp recordings of spontaneous action potential activity were made from labeled MnPO neurons in response to brief focal application of ANG II or the GABAA receptor agonist muscimol. Additionally, tissue punches from MnPO were taken to asses mRNA and protein expression. AT1a receptor knockdown neurons were insensitive to ANG II and showed a marked reduction in GABAA-mediated inhibition. The reduction in GABAA-mediated inhibition was not associated with reductions in mRNA or protein expression of GABAA ß-subunits. Knockdown of the AT1a receptor was associated with a reduction in the potassium-chloride cotransporter KCC2 mRNA as well as a reduction in pS940 KCC2 protein. The impaired GABAA-mediated inhibition in AT1a knockdown neurons was recovered by bath application of phospholipase C and protein kinase C activators. The following study indicates that AT1a receptor activation mediates the excitability of MnPO neurons, in part, through the regulation of KCC2. The regulation of KCC2 influences the intracellular [Cl-] and the subsequent efficacy of GABAA-mediated currents.

Angiotensin II induces membrane trafficking of natively expressed transient receptor potential vanilloid type 4 channels in hypothalamic 4B cells.

Transient receptor potential vanilloid family type 4 (TRPV4) channels are expressed in central neuroendocrine neurons and have been shown to be polymodal in other systems. We previously reported that in the rodent, a model of dilutional hyponatremia associated with hepatic cirrhosis, TRPV4 expression is increased in lipid rafts from the hypothalamus and that this effect may be angiotensin dependent. In this study, we utilized the immortalized neuroendocrine rat hypothalamic 4B cell line to more directly test the effects of angiotensin II (ANG II) on TRPV4 expression and function. Our results demonstrate the expression of corticotropin-releasing factor (CRF) transcripts, for sex-determining region Y (SRY) (male genotype), arginine vasopressin (AVP), TRPV4, and ANG II type 1a and 1b receptor in 4B cells. After a 1-h incubation in ANG II (100 nM), 4B cells showed increased TRPV4 abundance in the plasma membrane fraction, and this effect was prevented by the ANG II type 1 receptor antagonist losartan (1 µM) and by a Src kinase inhibitor PP2 (10 µM). Ratiometric calcium imaging experiments demonstrated that ANG II incubation potentiated TRPV4 agonist (GSK 1016790A, 20 nM)-induced calcium influx (control 18.4 ± 2.8% n = 5 and ANG II 80.5 ± 2.4% n = 5). This ANG II-induced increase in calcium influx was also blocked by 1 µM losartan and 10 µM PP2 (losartan 26.4 ± 3.8% n = 5 and PP2 19.7 ± 3.9% n = 5). Our data suggests that ANG II can increase TRPV4 channel membrane expression in 4B cells through its action on AT1R involving a Src kinase pathway.