William C. Engeland - US grants

This project is designed to define the role of mechanisms other than ACTH in the control of adrenal corticosteroid secretion. Plasma ACTH-like bioactivity that increases after hemorrhage in awake dogs will be characterized using gel filtration and high performance liquid chromatography. Bioactive ACTH, measured using a rat adrenocortical cell assay, will be compared to immunoreactive ACTH. Affinity chromatography prior to bioassay will separate ACTH from other ACTH-like factors. To assess the requirement for an increase of ACTH or other pituitary factors, the cortisol secretory response to hemorrhage will be measured in hypophysectomized awake dogs given infusions of ACTH. Control mechanisms for the release of non-ACTH factors will be defined by determining responses to graded hemorrhage, to corticotropin-releasing factor and to steroids. The role of sympathetic neural input on the adrenal response to ACTH will be assessed in anesthetized, hypophysectomized dogs receiving infusions of ACTH by measuring cortisol secretory responses to electrical stimulation of the splanchnic nerve. Direct neural effects on the adrenal cell will be distinguished from effects on adrenal blood flow by monitoring the distribution of flow using non-radioactive microspheres. Episodic cortisol secretion observed under basal conditions will be examined in awake dogs after hypophysectomy. Also, in dogs with bilateral adrenal venous cannula, synchronicity and periodicity of adrenal secretion will be determined using cross-correlation and spectral analysis; effects of unilateral and bilateral adrenal denervation will be examined. To determine how changes in plasma ACTH, in sympathetic neural input and in adrenal blood flow that occur after acoustic startle interact to mediate the cortisol secretory responses, dogs will be exposed to acoustic startle after hypophysectomy or after adrenal denervation. Alteration of the distribution of adrenal blood flow following acoustic startle will be measured. The results of these studies will elucidate the effectiveness of changes in non-ACTH humoral factors, in sympathetic neural input or in adrenal blood flow to influence the cortisol secretory response to ACTH under different physiological conditions. This information, by establishing a more complete view of stimulus-specific modulation of adrenal corticosteroid secretion, will offer a new perspective to the study and treatment of ACTH-independent adrenocortical disease.

9319097 Engeland Stress Activates the neuroendocrine system through a feedback mechanism involving the hypothalamic-pituitary-adrenal system. Corticotropin releasing factor, a peptide hormone, is released from the hypothalamus and travels to the pituitary where it triggers the release of adrenocorticotropic hormone (ACTH). ACTH travels in the blood to the adrenal glands where it stimulates the release of glucocorticoids, such as cortisol and corticosterone. This system is also involved in the maintenance of the body's normal internal functioning. Glucocorticoids not only help the body respond to stress but play an integral part of daily life and adaptation to environmental change. Recently, Dr. Engeland and others have found changes in plasma glucocorticoids in the absence of changes in plasma ACTH under both stressful and non-stress conditions. The finding challenges our simplistic view of adrenocortical secretion and indicates that other mechanisms are involved in the release of adrenal hormones. Recent evidence suggests a role for the adrenal nerves but the specific contribution of adrenal neural input to physiological responses are not known. Using a variety of innovative techniques, Dr. Engeland will characterize the contribution of adrenal neural activity to non-stress and stress- induced secretion of adrenal glucocorticoids. He will then determine which neurotransmitters are mediating its effect. Finally Dr.Engeland will identify the adrenal cellular mechanisms that mediate neural effects on steroidogenesis. These results will provide a new understanding about why exposure to the same environmental conditions that appear to have similar effects on ACTH release may produce different adrenocortical responses. The function of most tissues is dependent on the maintenance of normal plasma glucocorticoids levels. Aberrant adrenal hormone secretion can result in weaken muscles and suppressed organ systems. Understanding the nature of the interaction between adrenal nerve input and ACTH will provide a new perspective on physiological and pathological alternations in adrenocortical secretion. Thus, findings resulting from this basic research may eventually lead to the development of new pharmacological or surgical approaches to control adrenal secretion of glucocorticoids. ***

ABSTRACT IBN-9728132 PI = ENGELAND, William C. Secretion of steroid hormones by the adrenal cortex is required to maintain whole body homeostasis; that is, the ability to maintain blood pressure and volume, carbohydrate, protein and fat metabolism and immune and nervous system function within normal limits is dependent on adrenocortical hormones. The primary goal of the project is to understand the role of adrenal innervation in the control of steroid secretion. The hypothesis to be tested is that under non- stress conditions when reduced steroid secretion is required, neural activity is inhibitory, whereas during stress conditions when elevated steroid secretion is necessary, neural activity is excitatory. The capacity for innervation to produce both inhibitory and excitatory effects suggests that neural input must be encoded differentially; encoding could be dependent on the neurotransmitter released or on the target affected. Experiments are designed: (1) to define the neurotransmitters that modulate non-stress and stress- induced secretion of adrenal corticosteroids and (2) to elucidate the adrenal site of neurotransmitter action. Rats will undergo immunological, surgical, or pharmacological sympathectomies to remove selectively adrenal neurotransmitter systems. The effect of adrenal denervation on non-stress steroid secretion will be assessed using in vivo adrenal microdialysis, a technique developed to measure adrenal secretion in awake animals. It is proposed that circadian variation in non-stress steroidogenesis which is reflected by differences in episodic secretion is regulated in part by the inhibitory effects of innervation. Episodic corticosterone secretion will be assessed by time series analysis and diurnal variations in adrenal sensitivity to ACTH will be determined. It is proposed that adrenal neural elements interact positively with ACTH to mediate acute stress-induced increases in steroid secretio n. Neural input could act directly at the adrenal cell to affect steroidogenesis or act indirectly by changing adrenal blood flow. Experiments will assess the interaction between stress and ACTH and characterize the neurotransmitter system(s) mediating facilitatory control. Experiments will also measure adrenal blood flow during stress using fluorescent microspheres and define the neurotransmitters that mediate stress-induced changes. Neurotransmitter-specific innervation will be monitored using immunofluorescence histochemistry and quantitated by radioimmunoassay. Effects on specific steroidogenic enzymes will be assessed by measuring gene expression using in situ hybridization and RNAse protection assay, by measuring enzyme content using western analysis and by quantifying enzyme activity in adrenal cells. Results of these experiments should delineate the role of autonomic nerves as an extra-ACTH mechanism in the control of adrenal corticosteroid secretion and define the neurotransmitter systems involved. Understanding the nature of the this unique neural-endocrine interaction will provide a new perspective on physiological regulation of corticosteroid secretion.

The overall hypothesis is that adrenal regulation is dependent on a sequence of early cellular events that are characterized by phenotypic plasticity and mediated by interaction between local inflammatory and circulating endocrine factors. To test this hypothesis, three major questions will be addressed. First, what is the mechanism for ACTH-induced promotion of adrenal regeneration? Experiments will delineate the effect of ACTH suppression on phenotypic and proliferative changes in regenerating adrenals. Changes in expression of mRNA and protein for cytochrome P450 and aldosterone synthase, a P450 11beta-hydroxylase, will be used as phenotype markers of glomerulosa and fasiculata cells, respectively. Proliferation of specific cell phenotypes will be monitored using double-label immunohistochemistry that induces proliferation markers. Changes in adrenal melanocortin-2 receptor mRNA and protein will be examined using RNase protection and western analysis, respectively; immunohistochemistry will assess the cellular distribution of the receptor. Second, does the renin-angiotensin (AT) system act to suppress regeneration? Since AT upregulates adrenal P450 aldosterone synthase and promotes expansion of zona glomerulosa, studies will determine the role of specific AT receptor subtypes in regeneration using antagonists of the AT-1 and AT-2 receptors. Experiments will characterize the distribution and binding of AT-1 and AT-2 receptors in regenerating adrenals. Since AT has been implicated in models of fibrosis, studies will assess whether AT produces adrenal fibrosis. Third, what components of inflammatory responses affect adrenal regeneration? Since both adrenal and inflammatory cells can synthesize cytokines, the phenotype of cells expressing cytokine activity during regeneration will be delineated. Cytokines identified in regenerating adrenals including interleukin-1, interleukin-1 receptor antagonist, interleukin-18, macrophage migration inhibitory factory and transforming growth factor-beta will be examined in vitro. The effect of specific cytokines on adrenal cell differentiation and proliferation will be defined using short-term cultures of adrenal capsules with adherent cortical cells; potential cytokine modulation of ACTH and AT receptors will be evaluated. It is proposed that corticosteroids affect macrophage secretory activity at the site of adrenal inflammation. Studies using cultured regenerating adrenals or co-cultures of macrophages and adrenal capsules will assess macrophage-adrenal interactions. Since ACTH and AT may affect regeneration indirectly by altering the local inflammatory response, experiments will assess adrenal cytokine mRNA expression after in vivo blockade of ACTH or AT receptors. The proposed experiments should delineate the interaction between local inflammatory and endocrine factors responsible for adrenal regeneration and offer new perspective on injury-induced processes that result in tissue regeneration or fibrosis.

The hypothalamic-pituitary-adrenal system is characterized by a prominent circadian rhythm with peak plasma corticosterone occurring prior to the onset of the activity cycle. Since corticosterone is the primary glucocorticoid in rodents, daily variation in corticosterone is critical for homeostatic regulation of metabolic, cardiovascular and neural processes. The primary goal of this project is to define the central neural circuitry that underlies circadian changes in corticosterone secretion in rats. Adrenal denervation by splanchnicectomy reduces plasma corticosterone at the peak of the circadian rhythm without affecting ACTH, the pituitary hormone that stimulates the adrenal cortex; these results suggest that splanchnic innervation facilitates corticosterone responses independently of ACTH. The goal of these experiments is to characterize the central neural substrate for diurnal changes in plasma corticosterone mediated by peripheral innervation of the adrenal cortex. The project will determine whether neurons in the paraventricular hypothalamic nucleus receive input from the suprachiasmatic nucleus and project to the spinal cord to provide inhibitory and excitatory input to the adrenal cortex that drives the circadian rhythm. A combination of physiological and anatomical approaches will be used to dissect the pathway; plasma corticosterone and ACTH will be measured in experiments that include chemical lesioning, retrograde tracing and monitoring the expression of Fos, a marker of neuronal activation. This research is required for understanding how the central nervous system regulates adrenal secretion of glucocorticoids, hormones that have a significant impact on a broad spectrum of physiological processes that are essential for homeostasis. Graduate and undergraduate students will participate in all aspects of these studies, providing training that will enhance their opportunities for productive careers in research and teaching.

Daily variation in glucocorticoids is critical for homeostatic regulation of metabolic, cardiovascular and neural processes. An adrenal clock generates glucocorticoid rhythmicity, but elements that entrain the clock are unknown. The overall hypothesis is that sympathetic neural activity (SNA) is a critical adrenal input that transmits rhythmic light-dark and feeding time signals to modulate glucocorticoid rhythms by interacting with ACTH to entrain the adrenal clock. Experiments will use mPER2:Luciferase knockin mice, in which luciferase is rhythmically expressed under the control of the Per2 clock gene. To assess SNA-mediated entrainment, the PER2Luc rhythm will be measured in adrenal explants from intact or adrenal-denervated mice on a 12:12 light:dark cycle and after light-dark phase shifts. Use of hypophysectomized mice replaced with constant ACTH will address whether ACTH rhythms entrain the adrenal clock or augment SNA-mediated entrainment. Using restricted feeding, studies will determine whether the rate of food entrainment is reduced by denervation. Using pharmacological approaches, experiments will determine whether ACTH acts through a cyclic AMP-dependent pathway. Examining responses to adrenergic receptor agonists and antagonists will determine if the adrenal medulla links SNA and the adrenal clock. Broader Impacts: The use of the PER2Luc mice provides an innovative experimental model that will stimulate students to challenge current understanding of neuroendocrine function. With the College of Biological Sciences, the PI will develop an evaluation tool to measure the effectiveness of the research experience to enhance academic achievement in the College; results will be reported in annual NSF reports. The PI and his students also will participate in outreach activities, including Brain Awareness Week for elementary students, Brain Bee for secondary students, and Brain U for middle school science teachers. Finally, to promote information dissemination, the PI will share data with collaborators, like Professor Horacio de la Iglesia, University of Washington to enhance mutual lab productivity and augment student development.