The "metabolic sensor" function of rat supraoptic oxytocin and vasopressin neurons is attenuated during lactation but not in diet-induced obesity.

The oxytocin (OT) and vasopressin (VP) neurons of the supraoptic nucleus (SON) demonstrate characteristics of "metabolic sensors". They express insulin receptors and glucokinase (GK). They respond to an increase in glucose and insulin with an increase in intracellular [Ca(2+)] and increased OT and VP release that is GK dependent. Although this is consistent with the established role of OT as an anorectic agent, how these molecules function relative to the important role of OT during lactation and whether deficits in this metabolic sensor function contribute to obesity remain to be examined. Thus, we evaluated whether insulin and glucose-induced OT and VP secretion from perifused explants of the hypothalamo-neurohypophyseal system are altered during lactation and by diet-induced obesity (DIO). In explants from female day 8 lactating rats, increasing glucose (Glu, 5 mM) did not alter OT or VP release. However, insulin (Ins; 3 ng/ml) increased OT release, and increasing the glucose concentration in the presence of insulin (Ins+Glu) resulted in a sustained elevation in both OT and VP release that was not prevented by alloxan, a GK inhibitor. Explants from male DIO rats also responded to Ins+Glu with an increase in OT and VP regardless of whether obesity had been induced by feeding a high-fat diet (HFD). The HFD-DIO rats had elevated body weight, plasma Ins, Glu, leptin, and triglycerides. These findings suggest that the role of SON neurons as metabolic sensors is diminished during lactation, but not in this animal model of obesity.

Supraoptic oxytocin and vasopressin neurons function as glucose and metabolic sensors.

Neurons in the supraoptic nuclei (SON) produce oxytocin and vasopressin and express insulin receptors (InsR) and glucokinase. Since oxytocin is an anorexigenic agent and glucokinase and InsR are hallmarks of cells that function as glucose and/or metabolic sensors, we evaluated the effect of glucose, insulin, and their downstream effector ATP-sensitive potassium (KATP) channels on calcium signaling in SON neurons and on oxytocin and vasopressin release from explants of the rat hypothalamo-neurohypophyseal system. We also evaluated the effect of blocking glucokinase and phosphatidylinositol 3 kinase (PI3K; mediates insulin-induced mobilization of glucose transporter, GLUT4) on responses to glucose and insulin. Glucose and insulin increased intracellular calcium ([Ca(2+)]i). The responses were glucokinase and PI3K dependent, respectively. Insulin and glucose alone increased vasopressin release (P < 0.002). Oxytocin release was increased by glucose in the presence of insulin. The oxytocin (OT) and vasopressin (VP) responses to insulin+glucose were blocked by the glucokinase inhibitor alloxan (4 mM; P ≤ 0.002) and the PI3K inhibitor wortmannin (50 nM; OT: P = 0.03; VP: P ≤ 0.002). Inactivating K ATP channels with 200 nM glibenclamide increased oxytocin and vasopressin release (OT: P < 0.003; VP: P < 0.05). These results suggest that insulin activation of PI3K increases glucokinase-mediated ATP production inducing closure of K ATP channels, opening of voltage-sensitive calcium channels, and stimulation of oxytocin and vasopressin release. The findings are consistent with SON oxytocin and vasopressin neurons functioning as glucose and "metabolic" sensors to participate in appetite regulation.

Multiple alpha1-adrenergic receptor subtypes support synergistic stimulation of vasopressin and oxytocin release by ATP and phenylephrine.

Simultaneous exposure of explants of the hypothalamo-neurohypophyseal system (HNS) to ATP and the α(1)-adrenergic receptor (α(1)-R) agonist, phenylephrine (ATP+PE) induces a synergistic stimulation of vasopressin and oxytocin (VP/OT) release that is sustained for hours. The current studies confirm that the synergism is dependent upon activation of α(1)-R by demonstrating that an α(1)-R antagonist prevents the response. The role of the α(1)A, B, and D-adrenergic receptor subtypes in the synergistic effect of ATP+PE on intracellular calcium ([Ca(2+)](i)) in supraoptic nucleus (SON) neurons and VP/OT release from neural lobe was evaluated. The increase in [Ca(2+)](i) induced by PE in SON predominantly reflects release from intracellular stores and is mediated by activation of the α(1)A adrenergic receptor subtype. The α(1)A subtype is also required for the sustained elevation in [Ca(2+)](i) induced by ATP+PE. In contrast, although synergistic stimulation of VP/OT release was eliminated by removal of PE and was blunted by benoxathian, an α(1)-R antagonist that is not subtype selective, no single α(1)-R subtype selective antagonist prevented sustained stimulation of VP/OT release by ATP+PE. Thus, sustained activation of α(1)-R is essential for the synergistic VP and OT response to ATP+PE, but multiple α(1)-R subtypes can support the response. Redundancy amongst the α(1)-R subunits in supporting this response is consistent with the predicted importance of the response for sustaining the elevated VP release required to prevent cardiovascular collapse during hemorrhage and sepsis.

Role of purinergic P2Y1 receptors in regulation of vasopressin and oxytocin secretion.

Pharmacological studies demonstrated that ATP elevates intracellular calcium ([Ca(2+)](i)) in supraoptic nucleus (SON) neurons primarily by activation of P2X2 and P2Y1 purinergic receptors [P2Y1R]. The current studies provide evidence for the presence of P2Y1R protein in SON neurons, evidence that activation of these P2Y1Rs induces an increase in [Ca(2+)](i) from both intracellular stores and Ca(2+) influx, and functional evidence that activation of P2Y1Rs induces vasopressin (VP) and oxytocin (OT) hormone release. Pretreatment of Fura-2 AM-loaded explants of the hypothalamo-neurohypophyseal system (HNS) with thapsigargin (TG) significantly (approximately 80%) reduced the increase in [Ca(2+)](i) induced by the P2Y1R-specific agonist, 2-methylthio-ADP (2-MeSADP). In contrast, the increase in [Ca(2+)](i) was slightly (approximately 20%) decreased in calcium-free medium. The calcium response to 2-MeSADP was completely blocked by the P2Y1R-specific antagonist, MRS2179 or by a combination of TG pretreatment and calcium-free medium. It was absent in P2Y1R knockout mice (P2Y1R(-/-)). 2-MeSADP significantly increased VP and OT release from perifused rat and wild-type mouse HNS explants compared with control. MRS2179 prevented this response in wild-type mouse, but it did not prevent ATP-induced hormone release from rat explants. 2-MeSADP did not induce hormone release from P2Y1R(-/-) explants. These findings support a potential role for P2Y1Rs in regulation of VP and OT release. The finding that P2Y1R activation induces a small Ca(2+) influx suggests that P2Y1Rs may regulate VP release by modifying ion channels such as stretch-inactivated cation channels.

Sustained stimulation of vasopressin and oxytocin release by ATP and phenylephrine requires recruitment of desensitization-resistant P2X purinergic receptors.

Coexposure of hypothalamo-neurohypophyseal system explants to ATP and phenylephrine [PE; an alpha1-adrenergic receptor (alpha1-AR) agonist] induces an extended elevation in vasopressin and oxytocin (VP/OT) release. New evidence is presented that this extended response is mediated by recruitment of desensitization-resistant ionotropic purinergic receptor subtypes (P2X-Rs): 1) Antagonists of the P2X2/3 and P2X7-Rs truncated the sustained VP/OT release induced by ATP+PE but did not alter the transient response to ATP alone. 2) The P2X2/3 and P2X7-R antagonists did not alter either ATP or ATP+PE-induced increases in [Ca(2+)](i). 3) P2X2/3 and P2X7-R agonists failed to elevate [Ca(2+)](i), while ATP-gamma-S, an agonist for P2X2-Rs increased [Ca(2+)](i) and induced a transient increase in VP/OT release. 4) A P2Y1-R antagonist did not prevent initiation of the synergistic, sustained stimulation of VP/OT release by ATP+PE but did reduce its duration. Thus, the desensitization-resistant P2X2/3 and P2X7-R subtypes are required for the sustained, synergistic hormone response to ATP+PE, while P2X2-Rs are responsible for the initial activation of Ca(2+)-influx by ATP and ATP stimulation of VP/OT release. Immunohistochemistry, coimmunoprecipitation, and Western blot analysis confirmed the presence of P2X2 and P2X3, P2X2/3, and P2X7-R protein, respectively in SON. These findings support the hypothesis that concurrent activation of P2X2-R and alpha1-AR induces calcium-driven recruitment of P2X2/3 and 7-Rs, allowing sustained activation of a homeostatic circuit. Recruitment of these receptors may provide sustained release of VP during dehydration and may be important for preventing hemorrhagic and septic shock.