Celia Sladek - US grants

The objectives of this research program are 1) to define the neurological mechanisms operating at the hypothalamic level to control vasopressin (VP) release and 2) to ascertain the relationship of these control mechanisms to the osmotic regulation of VP release. Two culture systems will be used in these studies: organ cultured explants of the adult rat hypothalamo-neurohypophyseal system (HNS) and primary dispersed monolayer co-cultures of anterior hypothalamus (including the supraoptic nucleus) with either pituicytes or ventral medullary neurons (including the Al catecholamine neurons.) The effect of various agents on VP release from these cultures will be monitored with a VP radioimmunoassay. Norepinephrine release from the dispersed cultures containing Al neurons will be monitored with HPLC techniques. Specific agents to be evaluated are neurotransmitters, centrally active brain peptides, prostaglandins, and agents used clinically in the treatment of abnormalities of VP release. These studies will provide information on the mechanisms which allow a single target neuron to integrate multiple afferent signals. They will also provide basic information about the physiological mechanisms involved in the osmotic control of VP release, and thereby, they may provide the basis for understanding clinically observed abnormalities in VP release.

Existing evidence suggests that vasopressin (VP, antidiuretic hormone) release is elevated in several types of experimental hypertension as well as in humans with hypertension. Furthermore, the VP system is hyperresponsive to certain stimuli in rats genetically disposed to spontaneous development of hypertension (e.g. the spontaneously hypertensive rats (SHRs) developed by Okamoto) but relationships between this hyperresponsiveness and the hypertension is unclear. Therefore, experiments are proposed to evaluate this relationship. Two approaches will be used: 1). The importance of the hyperresponsiveness to the development of hypertension will be evaluated by chronically blocking the vasopressin effects of VP with an analog of VP which has specific pressor antagonist actions. Similarly the contribution of elevated VP release to the maintenance of blood pressure at the different stages of the hypertensive process will be analyzed by acutely infusing the VP pressor antagonist during the development and chronic phases of hypertension. 2). The opposite relationship also will be examined, e.g. the potential role of the hypertension in initiating the hyperresponsiveness of the VP system or in the eventual disappearance of the hyperresponsiveness. This will be evaluated by using three interventions to prevent development of the hypertension and examining the responsiveness of the VP system in rats with these interventions. The interventions employed will be chronic converting enzyme inhibition, intraventricular 6-hydroxydopamine injections, and renal denervation. In rats maintained normotensive with some of these interventions, the responsiveness of the renin-angiotensin system and the NE innervation of VP neurons also will be evaluated, because abnormalities in these factors have also been observed in SHRs.

vasopressins; neural information processing; hormone regulation /control mechanism; secretion;

DESCRIPTION (provided by applicant): Most neuronal systems are innervated by multiple afferent pathways that utilize a wide variety of neurotransmitters and neuropeptides. In addition, other agents such as hormones and cytokines influence neurons via non-synaptic mechanisms, and many neurons release multiple neuroactive agents. A major challenge for neuroscientists is to understand the mechanisms that allow target neurons to integrate these regulatory signals into appropriate responses. This proposal focuses on two aspects of this using the vasopressin (VP) neurons of the hypothalamo-neurohypophseal system (HNS) as a model system: 1) The interactions between ionotropic and G-protein mediated afferents; and 2) the modulatory effects of steroid hormones on these afferent signals. The proposed experiments build on two important findings from the past funding cycle: 1) Impressive synergism between ATP and phenylephrine (to mimic the action of norepinephrine on alpha1-adrenergic receptors) and preliminary evidence for synergism between glutamate and AII; and 2) Evidence that estrogen receptor beta (ER-beta) is inhibitory to VP release and that its expression is inversely correlated with osmotic stimulation of the HNS. The specific aims of the proposal are: 1. To address the hypothesis that the synergistic responses between ATP plus PE and glutamate plus AII reflect activation of similar intracellular signal cascades that converge to increase intracellular Ca++. 2. To evaluate the hypothesis that the synergistic responses to ATP plus PE and glutamate plus AII reflect alterations in intracellular Ca++. 3. To test the hypothesis that gonadal steroids inhibit the response to ionotropic agents that allow Ca++ influx via an ER-beta mediated mechanism. 4. To test the hypothesis that ER-beta expression is inhibited by activation of afferent pathways that stimulate VP and/or OT release. Perifused explants of the HNS will be used to study VP release. Live cell imaging of SON neurons in HNS explants and acutely dissociated preparations will address the role of Ca++ as an integrator of signal cascades for ionotropic and G-protein coupled ligands. Immunocytochemistry and in situ hybridization techniques will be used to study regulation of ER-beta expression.

DESCRIPTION (provided by applicant): Vasopressin (VP) and oxytocin (OT) secretion from the posterior pituitary occurs as a result of a variety of neurotransmitters and neuropeptide afferents to the supraoptic and paraventricular nuclei in the hypothalamus. Many of these afferents co-localize and co-release multiple neuroactive substance. In evaluating the responses to agents colocalized in the A1 catecholamine neurons (e.g. norepinephrine, ATP, neuropeptide Y (NPY), and substance P (SP), we found evidence for differential potentiation of hormone release by these substances as well as a potent and sustained response to SP. Specifically, combined exposure to SP and ATP or to NPY and phenylephrine (PE, an alpha adrenergic receptor agonist) resulted in synergistic stimulation of VP and OT release. The goals of the current proposal are to determine the relative importance of NPY and SP in eliciting VP and OT responses to physiological challenges and to elucidate the cellular mechanisms responsible for these synergistic responses. Since the A1 pathway transmits information about moderate decreases in blood pressure/volume to the VP and OT neurons, SP and NPY may be important to this physiological response. Aim 1 will test the hypothesis that release of SP and NPY from A1 terminals is required for activation of VP neurons in response to hypotension. The effect of unilateral supraoptic nucleus (SON) injections of SP and NPY receptor antagonists on Fos expression in SON will be assessed following hemorrhage or an acute osmotic stimulus. Aim 2 will evaluate the cellular mechanisms responsible for the synergistic responses to co-exposure to SP/ATP and NPY/PE. Explants of the hypothalamo-neurohypophyseal system will be used to: identify the receptor types required for synergism; determine the roles of protein kinase C and IP3-mediated Ca++ release; and evaluate the role of gene transcription. Aim 3 will test the hypothesis that SP and NPY receptor expression in SON is altered by stimulation of the A1 pathway. Western blots, immunocytochemistry, and in situ hybridization will be used to assess the expression of the neurokinin 3 tachykinin receptor and Y1/Y5 NPY receptors in rats following water deprivation, chronic saline ingestion, and hemorrhage. Aim 4 will test the hypothesis that dehydration-induced increases in Y1/Y5 receptor expression in SON alter the VP and OT response to NPY. The VP, and OT response to NPY will be evaluated using HNS explants obtained from rats exposed to chronic water deprivation. Inadequate or inappropriate VP secretion contributes to pathologies such as orthostatic hypotension and congestive heart failure. Understanding the role of specific neurotransmitters in relaying hemodynamic information to the VP and OT neurons is central to manipulating the system for treatment of pathological conditions.

DESCRIPTION (provided by applicant): The goal of this application is to develop background information to determine the physiological significance of the unexpected observation that a physiological stimulus (hypotension) and a selective neurokinin 3 receptor (NK3-R) agonist (senktide) induce the appearance of NK3-R immunoreactivity (ir) in the nucleus of neurons in the supraoptic nucleus (SON) of the hypothalamus. Since NK3-Rs are G-protein coupled receptors (GPCR), this suggests the intriguing possibility that extracellular NK3-R ligand induces translocation of NK3-R from the cell membrane to the nucleus for direct regulation of gene expression by a GPCR. This is exciting, because it has important implications for other GPCRs, and therefore could impact on numerous GPCR-regulated cellular functions. Also, the observation could be important to a wide range of CNS functions, because NK3-Rs are widely distributed in the CNS and have been implicated in diverse and important CNS functions and pathophysiology including motivated behaviors such as salt, cocaine and alcohol intake;emotional states such as anxiety and depression;locomotion and dystonia;fluid, electrolyte, and cardiovascular homeostasis;and analgesia. Therefore, this observation has the potential to impact the treatment of diverse and debilitating neurologic, psychiatric, and homeostatic disorders. In order to assess the potential importance of this observation, we must 1) fully characterize the NK3-R-ir observed in the nucleus of SON neurons, and 2) determine if the same phenomenon occurs in other CNS regions. Therefore, the Specific Aims for this proposal will test the following hypotheses: Hypothesis 1: The ligand-induced appearance of NK3-R-ir in the nuclei of SON neurons represents all or a portion of NK3-R that translocates to the nucleus following receptor internalization. To test this hypothesis, we have generated antibodies to different epitopes in the N-terminal and C-terminal regions of NK3-R. These antibodies will be used for immunohistochemical (IHC) and western blot analysis of SON. SON from normotensive, hypotensive, and senktide (a selective NK3-R agonist)-treated rats will be compared. Western blots will be used to analyze cytosolic and nuclear extracts of SON to determine the size and cellular location of NK3-R-ir. Hypothesis 2: NK3-R activation in other brain regions induces nuclear localization of NK3-R immunoreactivity. Senktide injection into the lateral ventricle has been shown to activate cfos in numerous regions of the CNS that also express H3-senktide binding and NK3-R mRNA and immunoreactivity. This approach will be used to screen for nuclear translocation of NK3-R-ir in areas such as lateral septum, amygdala, hypothalamus (median preoptic nucleus, paraventricular, supraoptic, and arcuate nuclei), substantia nigra, ventral tegmental area, periaqueductal gray, and nucleus tractus solitarius. PUBLIC HEALTH RELEVANCE The proposed studies have potential significance to a wide range of human diseases as a result of 1) the possibility that a new method of regulation of gene expression by neurokinin 3 receptors (NK3-R) may be identified that is applicable to other G-protein coupled receptors and 2) the evidence that NK3-Rs are involved in a large number of neurological and psychiatric disorders including addiction, depression, anxiety, movement disorders, and hypertension.

DESCRIPTION (provided by applicant): Glucokinase and insulin receptors (InsR) are abundant in the hypothalamic supraoptic nucleus (SON). The goal of this application is to develop background information to determine if the oxytocin (OT) neurons in SON utilize these molecules to monitor body nutrient status. The presence of glucokinase in other neurons and cells that function as glucose sensors (e.g. pancreatic beta cells and neurons in recognized appetite regulating centers) suggests that glucose-sensitivity may allow the OT neurons to monitor extracellular glucose and thereby respond appropriately to induce anorexia after a meal. Insulin is also recognized as a satiety-inducing signal. Thus, the presence of InsR in OT neurons may provide a second mechanism for the OT neurons to monitor changes in body nutrient status. Since OT is a recognized anorexic agent (e.g. suppresses food intake), alterations in the control of OT secretion may contribute to obesity and/or provide alternate treatment strategies to prevent or reverse obesity. The specific aims of the proposal are: 1. To test the hypothesis that magnocellular OT neurons function as glucose sensors and monitor hormonal indices of body nutrient stores. 2. To test the hypothesis that lactation alters the effect of glucose and insulin on OT release. 3. To test the hypothesis that the role of glucokinase and InsR in SON is altered by diet- induced obesity. Explants of the hypothalamo-neurohypophyseal system (HNS) will be used to determine the effect of glucose and insulin on OT and VP release and to determine if intracellular calcium ([Ca2+]i) signaling is altered in OT and VP SON neurons by glucose and/or insulin. Both hypothalamic and neural lobe hormone release will be monitored, because OT acts centrally to induce anorexia, and dendritic and/or en passant axonal OT release is thought to be the source of ligand for OT receptors (OTR) in 'satiety neurons' of the ventromedial nucleus. Since lactation is associated with both stimulation of OT release and increased food intake, it is possible that the impact of glucose and insulin on OT release is altered during lactation and that similar changes contribute to the difficulty that obese individuals experience in reducing food intake.