The anterior pituitary corticotrope population is controlled by multiple hormones and factors from the brain and peripheral organs. This multifactorial control may allow corticotropes to respond to a variety of stresses including those encountered clinically (trauma, surgery or disease states). Some of these factors and hormones may work synergistically with corticotropin releasing hormone (CRH) to maintain corticotrope function. The long range objective of this proposed study is to determine the effects of stimulatory neuropeptides and growth factors on corticotrope growth and function. A unique, newly developed primary culture system consisting of 87-98% corticotropes will be studied. The use of this culture system will thereby provide stronger evidence for direct effects of these hormones or factors. Responses of corticotropes in enriched cultures will be compared with those in mixed cultures. The first aim will be to determine if epidermal growth factor or nerve growth factor promote corticotrope proliferation and if they act synergistically with CRH or AVP. The study will also determine effects of CRH, AVP, vasointestinal polypeptide (VIP), angiotensin II (A-H) and oxytocin on proliferation. The second and third aims will determine effects of these neuropeptides and growth factors on adrenocorticotropin (ACM) secretion, proopiomelanocotin mRNA, and CRH, AVP or EGF binding by corticotropes in enriched or mixed cultures. The fourth aim will determine if enriched corticotropes are affected by paracrine secretions from other anterior pituitary cells. Conditioned media from cultures of different fractions of pituitary cells will be tested. Corticotrope growth will be assayed by immunocytochemical detection of Bromodeoxyuridine uptake (by mitotic cells), MTT assays that detect changes in cell numbers, and the Coulter Counter. Functional parameters to be assayed include changes in CRH- or AVP-receptor binding, cellular proopiomelanocortin mRNA and adrenocorticotropin (ACTH) storage and release. Receptor binding and mRNA will be detected cytochemically with biotinylated probes and labeled avidin. ACTH storage and release will be detected by radioimmunoassay and immunocytochemistry. It is anticipated that these studies will provide strong direct evidence for multifactorial control of corticotrope growth and function. The results will show how this population is able to respond to multiple stresses encountered clinically or in the environment. It will also provide information about paracrine mediators of corticotrope function.

R01DK039553

1992-03-01

1996-02-29