Marc E. Freeman - US grants

In most spontaneously ovulating mammals the corpora lutea formed at ovulation secrets progesterone for approximately 2 weeks. The corporalutea formed at ovulation in the rat secrete progesterone for only a few hours during the estrous cycle. However, a prolonged luteal phase or pseudopregnancy of approximately 2 weeks can be induced in the rat by mating or alternatively, by mechanical or electrical stimulation of the cervix on proestrus. Such stimulation leads to 10 days of daily nocturnal and diurnal surges of prolactin secretion from the pituitary gland. This hormone is responsible for maintenance of the corpus luteum. The cervical stimulus changes the activity of 2 areas of the hypothalamus to initiate these surges. Within the medial preoptic area, it turns off inhibitory neurons for nocturnal and diurnal surges and excites stimulatory neurons for diurnal surges of prolactin. Moreover, the stimulus excites stimulatory neurons for both surges within the dorsomedial-ventromedial areas of the hypothalamus. The prolactin-secretory response to cervical stimulation is further modified by ovarian and uterine input. The ovarian steroids either sensitize or inhibit prolactin secretion while the uterus secretes an unidentified activity which inhibits prolactin secretion directly at the pituitary level. Utilizing radioimmunologic estimates of hormone levels in blood, the chemical identity of the inhibitory and stimulatory input from the hypothalamus to the pituitary gland will be clarified. Moreover, the physiological role, the mechanism of action, and the chemistry of the uterine factor responsible for inhibiting prolactin secretion will be identified. The importance of these studies is that they will describe the nature of the hypothalamic response to a brief exteroceptive stimulus and furthermore will view the non-pregnant uterus as an endocrine organ whoe secretory product modifies pituitary prolactin secreted in response to that stimulus. Such studies also may suggest new methods of regulating fertility.

[unreadable] DESCRIPTION (provided by applicant): The central hypothesis of this application is that under physiological conditions, prolactin secretion from the anterior lobe of the pituitary gland is inhibited by dopamine and stimulated by oxytocin and that the activities of these neurons are regulated by clock genes, higher hypothalamic input and prolactin itself. This will be accomplished by pursuing 4 specific aims: (1) To characterize the role of OT neurons in control of PRL secretion under physiological conditions. To accomplish this, the incidence of FOS or FRAs staining of magnocellular and parvocellular OT neurons will be characterized during periods of physiologically induced PRL secretion. Antagonists of OT will be used to determine if endogenous OT controls PRL secretion under physiological circumstances. (2) To determine the nature of the afferent control of OT and DA neuronal activities under physiological conditions. This will be accomplished by first determining if there are VIP receptors on OT neurons and then administer antisense oligonucleotides to VIP (see Progress Report and Preliminary Data) and determine their effects on OT and DA neuronal activity as indicated by FOS or FRAs staining. (3) To determine if PRL feeds back on either OT neurons and/or their afferent controls to regulate their activities. First, it will be determined if OT neurons have PRL receptors and if so, immunoneutralize circulating PRL or administer ovine PRL and determine the effect on activity of these neurons. (4) To determine the molecular mechanisms controlling the circadian rhythm of neuroendocrine DA neurons involved in PRL secretion. The expression of clock genes and the phenotype of the neurons expressing them in the SCN, ARM or PeVN in a constant environment will first be characterized. Then, using antisense technology, their disruption will show function of the expressing neurons. The health relatedness of this project is to suggest new ways of treating diseases related to hyperprolactinemia and breast cancer. [unreadable] [unreadable]

The long-term objective of this proposal is to characterize the role of endothelins (ETs) in regulating reproductive hormone secretion. The aims of this proposal revolve around elucidation of the mechanisms whereby ETs' affect pituitary prolactin (PRL) secretion. It is submitted under the area of Reproductive Endocrinology in response to the Special Emphasis Area solicitation of the NICHD. The first specific aim of this proposal is to characterize the physiological role of endothelins in the control of lactotroph function during the estrous cycle. This will be accomplished by examining the effects of specific ET antagonists on PRL secretion in vivo. The second specific aim is to characterize the autocrine and paracrine actions of ETs in controlling PRL secretion. This will be assessed in vitro (a) at the single cell level by using reverse hemolytic plaque assay methods on pituitary cells collected during different stages of the estrous cycle and (b) in a multicellular environment by using pituitary cell micro-aggregate cultures in a cell-perfusion system. The effects of ET receptor antagonists on the secretory activity of lactotrophs will be tested in basal and secretagogue-induced conditions. The third specific aim is to identify signal transduction mechanisms by which ETs affect PRL secretion. We will use dispersed pituitary cells enriched in lactotrophs and/or an established pituitary cell line of lactotroph origin expressing ETA receptors. We will employ specific inhibitors of protein kinase cascades thought to be involved in the regulation of PRL secretion. Specific emphasis will be made on investigating the role of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) dependent signaling pathways. The fourth specific aim is to establish the ionic basis for the effects of endothelins on pituitary lactotrophs. In these studies we will seek to confirm our hypothesis that ET-induced multiple signaling pathways converge upon a unique population of BKCa channels and that modulation of these channels is responsible for most of the effects of ETs on PRL secretion. Using cultured lactotrophs, we will employ patch clamp methods in cell-attached and inside-out configuration to gain information on gating kinetics at the single channel level. The fifth specific aim is to establish the mechanism of cross-talk between dopaminergic D2 and endothelin ETA receptors. The investigation will focus on the mechanisms by which dopamine transforms the response of lactotrophs to ET from inhibitory to stimulatory. These studies will provide, for the first time, a thorough characterization of the physiological role of ETs in regulating pituitary hormone secretion. Accomplishment of these specific aims will clarify a heretofore undescribed cellular mechanism regulating PRL secretion and suggest novel therapeutic approaches to control secretory functions of the hypothalamo-pituitary axis.