Abstract
In the rat, decreasing transsynaptic activity through adrenal denervation, nicotinic receptor blockade, or explantation is associated with an increase in preproenkephalin mRNA, enkephalin prohormone and peptide. In contrast, catecholamine pathways remain unchanged under similar conditions. Since it is not known whether changes in messenger RNA result from stabilization or increased synthesis, we exploited transcription ‘run-on’ assays to measure the rate of transmitter gene read out. Tyrosine hydroxylase message (TH-mRNA) was found to be the most abundantly produced transcript in the unmanipulated control rat adrenal medulla. TH-mRNA was produced in excess of twice the rate of transcription of the structural gene β-actin. In contrast, preproenkephalin transcription occurred at a much lower rate (60% of the actin gene and only 25% of tyrosine hydroxylase gene transcription). All transcripts were inhibited by the polymerase II inhibitor, α-amanitin. After two days in explant culture, the rate of enkephalin transcription increased approximately 2-fold (to the same level as actin transcription); while tyrosine hydroxylase transcriptional activity fell to 30% of actin levels. To analyze cellular mechanisms, explants were depolarized with potassium chloride. Enkephalin gene transcription was observed to be 2.5-fold less when grown under depolarizing conditions (50 mM KCl) than in control explants. On the other hand, tyrosine hydroxylase gene read-out was unchanged, similar to results obtained when TH catalytic activity was measured. These data indicate that membrane depolarization can selectively regulate expression of a transmitter gene product and are consistent with a proposed transsynaptic regulatory mechanism controlling biosynthesis of adrenal opiate peptides. Additionally, these observations represent a molecular mechanism through which physiologically relevant transsynaptic stimuli (membrane depolarization) can modify transmitter expression to alter neurohumoral function or transmitter phenotypic plasticity.
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