Recipient Organization
UNIVERSITY OF TENNESSEE
2621 MORGAN CIR
KNOXVILLE,TN 37996-4540
Performing Department
COMPARATIVE MEDICINE
Non Technical Summary
Aberrant adrenocortical disease (ACD) is a disease in which cortisol usually remains at normal concentrations (which is misleading), but excessive adrenal intermediate steroids are secreted causing clinical signs of canine Cushing's syndrome. Melatonin is currently used as a treatment for ACD in dogs however its mechanism of action is unknown and its effectiveness as a treatment for ACD varies from dog to dog. The purpose of this project is to learn if melatonin may exert its effect through inhibition of certain enzymes in the steroidogenic pathway that are needed for conversion of one hormone to another which includes examining the effectiveness of melatonin to decrease adrenal steroid hormone concentrations.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
1. Determine the effect of melatonin on steroid production by human adrenal cells and canine adrenal tissue explants stimulated by ACTH. 2. Investigate melatonin's effect on specific enzymes within the adrenal steroidogenesis pathway and on the extracellular signal-regulated kinase pathway.
Project Methods
Specific objective 1: Steroid-producing human adrenal cells (ATCC, Manassas, VA) will be cultured in Dulbecco's modified Eagle's medium and Ham's F12 supplemented with insulin, transferrin, selenium, bovine serum albumin, linoleic acid, and Nu-serum, and cultivated at 37C. Canine adrenal tissue will be obtained from patients requiring surgery to resolve ACD at the College of Veterinary Medicine, The University of Tennessee. Healthy adrenal tissue will be obtained from euthanized client dogs prior to necropsy or from a source yet to be determined. Primary explants of adrenal tissue will be grown in appropriate growth medium and cultivated at 37C. Cells will be treated with ACTH and melatonin. Initially, ACTH will be used at a concentration of 100 nM, based on work by Torres-Farfan et al.16, and melatonin at 1, 10 and 100 nM. Based on preliminary results, melatonin concentrations may be altered. Different treatment groups will be established: Group 1. Control with no
ACTH or melatonin, Group 2. ACTH (100 nM) only, Group 3. Melatonin (10 nM) only, Groups 4, 5, 6. ACTH (100 nM) and melatonin at 1, 10, 100 nM, respectively. Adrenal cells and explants will be treated for 24 and 48 hours then medium will be collected, centrifuged and supernatant stored at -80C until analyzed using radioimmunoassays to determine cortisol, progesterone, 17-estradiol, 17-hydroxyprogesterone, androstenedione, and testosterone concentrations. Specific objective 2: Adrenal cells will be treated as in specific objective 1; then after 24 and 48 hours, cells will be harvested, lysed and subjected to western blot analysis. Briefly, proteins will be separated on a resolving gel by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a nitrocellulose filter and probed with antibodies specific for 21-hydroxylase, aromatase, Raf, Mek, phosphorylated (active) Mek, Erk1/2 and phosphorylated (active) Erk1/2. Antigen-antibody complexes will be detected by
autoradiography.