Neural stimuli frequently evoke long term alterations in gene expression. It has been exceedingly difficult to understand how a neuron integrates a diverse array of stimuli into an appropriate level of expression. The primary objective of this research is to resolve the molecular mechanisms responsible for neural regulation of gene expression. Cholinergic regulation of the epinephrine-synthesizing enzyme phenylethanolamine N- methyltransferase (PNMT) in bovine adrenal chromaffin cells provides an excellent model system in which to analyze how naturally occurring neurotransmitters modulate transcription of a target gene. Nicotine, muscarine and K+-mediated depolarization each activate distinct intracellular mechanisms capable of stimulating PNMT gene transcription. The proposed studies target these neural responsive elements encoded in the PNMT gene. The constraints of the cellular environment necessary to permit neural regulation are also assessed.
The first aim of these studies is to identify those sequences on the rat PNMT promoter that comprise the cholinergic and depolarization responsive elements. Deletion mapping using PNMT promoter reporter fusion constructs will define sequences conveying inducibility to nicotine, muscarine, and K+ depolarization. Transient transfection and expression of parental and mutated constructs in primary chromaffin cells will confirm the functionality of these sites.
The second aim evaluates the role of multiple cell-specific 'silencer' elements in the high degree of cell specificity for PNMT gene expression. The third goal will delineate intracellular second messengers responsible for neural regulation of PNMT expression by mapping their responsive sites on the PNMT promoter and by assessing the relative loss of neurally stimulated expression when responsive elements are mutated or pathways selectively inhibited. The fourth goal is to investigate the potential requirement of the PNMT gene for a glucocorticoid environment in order to permit regulation by neural (cholinergic) stimuli. Because epinephrine functions both as a hormone and a neurotransmitter, the PNMT gene represents a central regulation point for integration of neural and endocrine systems. Peripherally, neural regulation of this gene coordinates stimuli necessary for the Fight-or-Flight reflex. In brainstem nuclei, epinephrine is postulated to play pivotal roles in maintaining arterial pressure and coordinating cardiovascular and respiratory reflexes. The proposed studies effectively address the critical issue of how PNMT expressing cells respond to neural stimuli. In so doing, this research may reveal underlying pathophysiology for human essential hypertension, SIDS, or other centrally mediated neurological disorders.
| Evinger, M J; Ernsberger, P; Regunathan, S et al. (1994) A single transmitter regulates gene expression through two separate mechanisms: cholinergic regulation of phenylethanolamine N-methyltransferase mRNA via nicotinic and muscarinic pathways. J Neurosci 14:2106-16 |
| Evinger, M J; Towle, A C; Park, D H et al. (1992) Glucocorticoids stimulate transcription of the rat phenylethanolamine N-methyltransferase (PNMT) gene in vivo and in vitro. Cell Mol Neurobiol 12:193-215 |
