The applicant recently presented evidence that the intrinsic excitability of chromaffin cells is regulated by stress hormones of the hypothalamic-pituitary-adrenal axis. The proposed study will further test and measure the mechanism linking the stress axis to the mechanics of acute epinephrine responses. The evidence suggests that steroid stress hormones control the assembly of a key ion channel, the BK-type potassium channel. By biasing the alternative splicing of pre-messenger RNA, the hormones """"""""tune"""""""" the channel's structure in a way that increases the ease with which the channel is opened. This results in an increase in the cell's excitability and epinephrine secretion. This study will use molecular biological (RT-PCR and immunocytochemistry) and electrophysiological (patch clamp) techniques in parallel to measure the effects of stress hormones on structure and function of the BK channel. Surgical and hormonal perturbations in vivo and in vitro will identify molecular pathways by which the regulatory signal is transduced, and explore the consequences to cell function. The study benefits from the fact that the same assays can be applied to compare between bovine and rat cells, between rat strains with different stress response profiles, and between rat developmental stages, in addition to between experimental treatment groups. Thus, the goal is to elucidate molecular mechanisms by which stress experiences, life stages, and genes adapt and/or limit the mechanisms of catecholamine secretion.
