Phyllis Callahan - US grants

The specific aim of this proposal is to characterize age-related alterations in anterior pituitary lactotroph responses. A major focus of this work will be on the cell signal transduction mechanism of the lactotroph, specifically the hydrolysis of phosphatidyl-inositol-4,5- bisphosphate (PIP2). This produces an accumulation in inositol phosphates (IPs), notably inositol 1,4,5-triphosphate (IP3) which serves as an intracellular second messenger. Prolactin secretion and cell content, inositol phosphate accumulation and phospholipid metabolism will be determined in primary cultures derived from anterior pituitary glands from three different aged donor groups, i.e. young (3-4 months), mature (7-8 months) and old (22-24 months) male and female Sprague-Dawley rats. Cultures will be treated with secretagogues (TRH, Angiotensin II or neurotensin) which have been shown to increase prolactin release via PIP2 hydrolysis. The stimulatory activity of these secretagogues, as well as the ability of dopamine to inhibit these responses will be determined. In order to distinguish between possible differences in receptor binding characteristics of these secretagogues, the receptors will be by passed by administration of phorbol esters. The addition, the involvement of estrogen on pituitary responsiveness will be determined. This well be accomplished in two different ways. First, a number of female rats will be ovariectomized at 3-4 months and included as a separate group of pituitary donors for each of the older groups (i.e. mature and old). Second, the direct effects of estrogen on the pituitary will be assessed by adding it to the cultures. Lactotroph number will be quantified in all donor groups and all cells will be incubated in steroid free media. Aging produces a number of alterations in many physiologic functions. Significant changes occur in the endocrine system, including alterations in anterior pituitary secretory function, most notable, an increase in prolactin secretion. Aging is also clearly associated with an increase in pituitary adenomas (which involves the lactotroph population). Thus, alterations in the responsiveness of the anterior pituitary lactotroph may be an early marker of pituitary adenomas. It is important to understand the characteristic(s) and mechanism(s) associated with the aging process so that the relationship between normal aging and the pathogenesis of age- related diseases can be determined, especially taking into consideration the growing shift in demographics toward the elderly population in this country.

DESCRIPTION (Adopted from applicant's abstract): Angiotensin II (AII), an octapeptide that is a component of the renin-angiotensin system, is produced within the anterior pituitary gland of rats and humans, and appears to play a paracrine (rat) or autocrine (human) role in regulation of prolactin secretion. This research proposes to examine the effects of estrogen on rat lactotroph sensitivity to the paracrine mediator AII. Specifically, the hypothesis to be tested is that the pattern and levels of estrogen secretion (or replacement) influences anterior pituitary lactotroph sensitivity to AII-and LHRH-induced stimulation of prolactin secretion. Ovariectomized, prepubertal female rats will be injected with estrogen and progesterone to induce LH surges in a pattern that mimics estrous cyclicity. A second group of ovariectomized animals will receive no steroids, and a third group will not be ovariectomized. Anterior pituitary cells from these animals at six months of age will be exposed to physiological doses of AII and LHRH to determine the prolactin secretory response. Accumulation of inositol phosphates (IP), the second messenger which mediates the AII stimulation of lactotrophs, and AII receptor binding characteristics will also be investigated.

The goal of this ILI proposal is to offer new laboratory exercises that utilize state of the art technology in four upper level undergraduate courses: Endocrinology, Neuroanatomy, Neurophysiology and Parasitology. Students play an active role in incorporating this technology into practical and experiential learning and thus are better prepared for careers in science. The proposed laboratories are based on three pedagogical principles: student-designed inquiry, collaboration among learners and use of new technology. Students test hypotheses, conduct experiments, collect and analyze their data and present their results in written and/or oral reports to their class and to lower-level students in workshops. Thus other undergraduates also benefit from this project. Literature review and assimilation is a critical aspect of these laboratory exercises and reports. With these innovations in our courses, students are experiencing real science as it is performed by research scientists. All of these courses, except Parasitology, are included in the interdisciplinary Neuroscience Minor, jointly offered with the Department of Psychology. Approximately 100 students enroll in these courses each year. Most of these students continue their education in post-baccalaureate programs across the nation, such as medical veterinary, dental, physical therapy and Ph.D. programs. The results and impact of this project will be assessed by formative and summate evaluations conducted in collaboration with an evaluation coordinator. Results will be shared with colleagues through publications in education journals, presentations at professional meetings and the Miami University website.

DESCRIPTION (provided by applicant): The hypothesis to be tested is that OFQ/N plays a physiologically significant role in the regulation of prolactin secretion and that it exerts this control at the hypothalamic level, specifically by inhibiting the tuberoinfundibular dopaminergic neurons. To accomplish this goal, male and female OFQIN knockout mice, and their wild type and heterozygous littermates, will be used in these proposed studies. The suckling-induced prolactin increase wifl be determined since suckling is a potent and important physiological stimulus for prolactin release. If OFQ/N is important for this response, then the suckling-induced increase will be attenuated in OFQ/N knockout mice. Furthermore, OFQIN administration will stimulate prolactin release and enhance the suckling-induced increase, even in knockouts because they still express the receptors. However, the possibility that compensatory mechanisms occur, e.g. due to the actions of other opiate peptides will be addressed by pretreating mice with opiate receptor antagonists or antiserum to eliminate each one of the other endogenous opiates. Sex differences in the sensitivity of the prolactin secretory response to OFQIN will be determined by conducting dose response studies in male and female mice. The ability of the OFQIN receptor to function in knockout mice, that develop and mature in the absence of the peptide, will be determined. Finally, the neural mechanism of action will be determined by quantifying specific amines, particularly dopamine, in the median eminence and other select regions of the brain.The iong term objective of this research is to understand the neural regulation of prolactin secretion, particularly the interaction among the different endogenous opiate peptides and their receptors. Prolactin has a number of important physiological roles. It is involved in reproduction, as well as being important in mediating the stress response, immunocompetence and maintaining homeostasis. To understand reproductive and stress-related disorders, it is important to understand the neurochemical regulation of prolactin release.

[unreadable] DESCRIPTION (provided by applicant): Stress affects virtually everyone in society at some point in life and it has major deleterious effects on health. It contributes to numerous pathologies, including disorders in the cardiovascular, immune and reproductive systems, as well as sleep disorders, depression and substance abuse. In spite of this, the neuroendocrine mechanisms regulating the physiological responses to stress remain poorly understood, particularly the regulation and role of prolactin secretion. The overall goal of these studies is to understand the regulation of prolactin secretion during stress and its impact on the response of hypothalamic-pituitary adrenal (HPA) axis to stress. The major hypothesis is that the endogenous opiate, Orphanin FQ/Nociceptin (OFQ/N), is an important neuropeptide regulating prolactin secretion during stress. The OFQ/N knock out model will be used because it is ideally suited to these studies. First, knock out mice do not respond to stress in the same way as wild type mice; this is true for both males and females. Acute stress increased plasma prolactin levels in wild type, but not knock out males. Also, OFQ/N knock out, but not wild type females continued to respond to repeated stress, suggesting PRL feedback is not active when OFQ/N is absent. The role and mechanisms of action of OFQ/N in mediating these gender differences will be examined in Specific Aims 1 and 2. Second, knock out post-partum females are not hyperprolactinemic and do not seem to have increased PRL-R mRNA in the choroid plexus of the brain, making them much different from normal post-partum females. In Specific Aim 3, we will take advantage of this difference to examine the role and mechanisms involved in OFQ/N regulation of hyperprolactinemia and prolactin's role and mechanisms of action in mediating attenuation of the HPA axis response to stress. Plasma corticosterone levels, a reliable indicator of stress activation, and plasma PRL levels will be measured. To determine the mechanisms regulating the stress response, tuberoinfundibular dopaminergic neuronal activity, anterior pituitary prolactin content, prolactin receptor expression in the choroid plexus and paraventricular nucleus (PVN) of the hypothalamus and CRH mRNA levels in the PVN will be determined. Specificity of action will be demonstrated by administering prolactin antiserum to block effects mediated by increased prolactin levels. Although little is known about the neuroendocrine effects of OFQ/N, our studies indicate that OFQ/N plays an important role in prolactin regulation. These studies will determine the mechanisms and physiological significance of OFQ/N in mediating the prolactin response to stress and the effects of prolactin on HPA axis activation. Understanding the neuroendocrine mechanisms regulating the stress response will lead to improved treatments for alleviating stress and anxiety, and hence, have a positive impact on health. Stress affects virtually everyone in society at some point in life, and has major deleterious effects. It contributes to numerous pathologies, including, but not limited to, disorders in the cardiovascular, immune and reproductive systems, as well as sleep disorders, depression and substance abuse. The cost to society in terms of job productivity and health care is staggering. The focus of these studies is on the neuroendocrine response to stress, particularly on the role of the endogenous opiate, Orphanin FQ/Nociceptin, a purported anxiolytic, in prolactin regulation. Prolactin is critically important in the stress response, but its mechanisms of action are poorly understood. Understanding the neuroendocrine regulation of stress will lead to improved treatments to alleviate stress and anxiety, and hence, have a positive impact on health. [unreadable] [unreadable] [unreadable]

ABSTRACT

Miami University's URM program will recruit 12 undergraduates each year for the next five years to provide the students with research and mentoring experience in order to prepare them for graduate school in the biological sciences. As a first step toward institutionalizing the program, NSF funds will be used to support each student for the first two years in the program, after which Miami University will provide support for an additional two years. Activities planned for academic, research and career enrichment will be offered to a larger group of students, especially those from underrepresented minority groups. The major goals of the URM program are to: (i) Increase the number of underrepresented minority students recruited into the biological sciences; (ii) Enhance the preparation of students to enable them to successfully enter and remain in graduate programs; and (iii) Build a better foundation at the institution for promoting interactions among academic, student support and administrative structures. The research programs of about 30 faculty across four departments (Biochemistry, Botany, Microbiology and Zoology) will provide students with a wide and diverse choice of projects, all under a unifying theme of "Signaling mechanisms and cellular responses". URM scholars will be exposed to a variety of model organisms, tools and techniques, and scholars' interest in a research career will be sustained and strengthened through enrichment activities and a strong mentoring component. More information about the program can be found at http://www.units.muohio.edu/urm_assure/.