Abstract
     Over the past few years, considerable evidence has accumulated to challenge the accepted view of the regulation of adrenocortical function. Conventionally, the cortex and medulla have been viewed as distinct functional units, with cortical function regulated primarily by the circulating hormones, ACTH, and angiotensin II, acting mainly on the inner adrenocortical zones and the glomerulosa, respectively. However, it has become clear that certain aspects of adrenocortical function cannot be explained in this simplistic manner. Certainly, there are discrepancies between the concentrations of these regulatory hormones and the secretion of the corticosteroids, suggesting that other factors may also be involved.
     As a result of intensive study in recent years, we now know that the regulatory mechanisms that account for such discrepancies are mainly located within the adrenal itself, and that several different components of the gland contribute to these functions. The adrenal produces a wide variety of hormones, neuropeptides, neurotransmitters, and cytokines, and it is evident that the colocalization of these different systems has a profound functional significance. The cells within the adrenal thus communicate with each other and adapt the function of the gland to different situations. The integrated control of adrenocortical function involves cortico-medullary interactions, the gland's vascular supply, its neural input, the immune system, growth factors, and the interglandular renin-angiotensin and CRH-ACTH systems. As well as directly regulating adrenocortical function, these systems influence each other and form complex intraadrenal regulatory circuits.

Techniques

Transfection of Endocrine Cells
     NCI-Transfection with IL-1 receptor compounds Interleukin-1 is a cytokine which was shown to          promote cell proliferation. In addition, we were able to demonstrate that aberrant expression of its signal transducing receptor lead to adenomatous formation of adrenocortical cells. Therefore, it is important to further investigate the effects of this cytokine on proliferation of adrenocortical cells. In a first step we want to transfect cells of the adrenocortical carcinoma cell line NCI-H295 with the type I interleukin-1 receptor. In a second step the activity of this receptor and the rate of proliferation of the transfected cells will be determined. Briefly, this study is designed to disclose the direct effects of interleukin-1 on adrenocortical cells. It can be extended to a third step in which compounds of the receptor, necessary for signal transducing, will be transfected only.

Quantitative PCR

     Quantitative PCR Qualitative investigation of ribonucleic acid expression gives insights in the regulation of biochemical processes. However, qualitative measurements alone often remain unsatisfactory when an alteration of expression from a basal rate, say, that of an enzyme, becomes cruical in reaching a new quality. Therefore, a quantitative determination of ribonucleic acid copies per cell is more and more desired. Real time PCR of reverse transcripts offers the opportunity to easily overcome the classical problems of quantification in comparence to external standarts. In this modern PCR technique a third primer is introduced, which through deposition of a linked flouroescent molecule during the process of PCR, allows quantification via colorimetric measurements. In addition, this method becomes valuable especially when determination must be performed from low quantities of RNA and "classical" methods such as Northern blotting fail.

Laser Capture Microdissection

     Microdissection of human adrenal glands Ribonucleic acid (RNA) obtained from laser capture microdissection of malignant and nonmalignant human adrenals may serve as an indicator for the regulation of gene transcription of certain proteins. Differences in first quality and, secondly, quantity of messanger RNA composition will be investigated using a semiquantitative way. After being established, the above described method of a real time PCR can also be applied to this particular project. This combined approach will be powerful in screening a high number of tumors for any important marker discovered. This project should provide new insights in adrenocortical tumor development.

Northern Blot Analysis

     In this project we study bovine adrenal cytochrom P450 enzyme regulation and kinetics by analyzing substrate-induced optical difference spectrums as a measure of spin state transition due to direct ligand-protein interaction. In contrast to most other investigations in this field, we determine enzyme activity in intact cells and not in mitochondrial or microsomal membrane fractions. Current topics are the effects of leptin on CYP11A, CYP11B, CYP17 and CYP21A activity and the regulation of proteolytic degradation of this enzymes in vitro.

Transgenic Animals

    Metabolic function and the sympathoadrenal system are closely linked. Glucose and fat metabolism as well as thermogenisis are regulated by the sympethatic nervous system. Leptin serves as an important mediator in many of those regulatory mechanisms. However, the interaction between leptin and the sympathoadrenal system is not clearly understood. Therefore, in this project we systematically investigate the effect of catecholamine metabolism on leptin production and the metabolism in vivo. For this, we developed a transgenic mouse model overexpressing the enzyme PNMT resulting in elevated levels of epinephrine. In these mice leptin secretion and metabolic parameters of glucose and fat metabolism will be analyzed. Fat tissue will also be investigated morphologically. In addition, these data will be compared to leptin levels in pheochromocytoma patients and patients with cardiac failure. These studies are aimed to contribute to the understanding of the interaction between the sympathetic system, leptin and metabolism under physiological conditions and in states of catecholamine hypersecretion.

Immunology

     Normal adrenal glands express MHC class II molecules within the androgen producing Zona reticularis. Beside the balanced expression in normal adrenal glands, two altered expression patterns exist in medical conditions. A distinctive feature of adrenocortical neoplasms consists in the complete loss of MHC class II expression, as we could show in recent work. In addition, our studies on apoptosis in normal and neoplastic adrenals reveal a diminished apoptotic rate in such adrenal tumors. Contrary, in Addison's disease a high MHC class II expression has been reported, and the involution of the gland with decreasing hormonal capacity is well known. The adrenal MHC class II molecules provide one prerequisite to intercellular communication with immunocompetent cells, namely the T-Helper cells. Hence, a regulatory circuit between adrenocortical cells and T-Helper cells may guarantee cell and tissue homeostasis in both adrenocortical zones and lymphocytes. The physiologic action of this circuit could protect from both neoplastic degeneration and the danger of excessive involution in adrenals. In lymphocytes a blunting of autoreactive cells might be possible.

Cell Culture

     In adipose people who are suffering from Non Insulin Depending Diabetes Mellitus (NIDDM) caused by insulinresistance high levels of the adipostatic hormone leptin and cortisol are found. Leptin is snythesized and produced by the adipose tissue and belived to regulate food intake. We are currently investigating the possible relationship between leptin and the cortisol producing adrenal gland. We established primary cultures of bovine adrenocortical cells to test the influence of leptin on cortisol secretion and the cortisol producing enzymes. A diabetic mouse model, in which the phenotypic symtomes are similar to human NIDDM, allows us to test the effect of leptin on cortisol production in vivo. Currently ongoing techniques for our experiments are tissue cultures, ELISA, PCR and Northern Blotting.