REGULATION OF PNMT ACTIVITY BY ALTERNATIVE MRNA SPLICING

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DESCRIPTION: The long-term goal of this study is to identify molecular mechanisms controlling the tissue-specific expression of phenylethanolamine methyl transferase (PNMT). PNMT has health-related importance, as it is the enzyme that converts noradrenaline to the neurotransmitter adrenaline. We recently discovered that both in vivo in the brainstem, as well as in vitro in cultured PC12 cells, two species of PNMT mRNA are produced by an alternative splicing mechanism known as intron retention. We hypothesize that this splicing mechanism, rarely encountered in mammals, forms the basis for tissue-specific regulation of PNMT expression in various adrenergic tissues. In brainstem, the expression of a spliced intron-less message is down-regulated post- natally, while an intron-retrained mRNA species is constitutively expressed. Hormonal control of this species reaction is indicated by our preliminary data, as in vitro exposure of PC12 cells to the synthetic glucocorticoid (GC), dexamethasone, induce the spliced variant while in control cells, or upon exposure to nerve growth factor (NGF), only the intron-retained variant is observed. The appearance of the spliced variant is accompanied by induction of PNMT activity. In adrenal, the main organ for PNMT activity and epinephrine synthesis, only the spiced variant is evident. We propose to investigate the physiological importance of PNMT mRNA splicing in brainstem, adrenal and superior cervical ganglia using the RT-PCR. All these tissues express PNMT in a developmentally-regulated manner. We will physiologically modulate embryonic PNMT activity by injecting both pregnant animals, as well as their offspring, with dexamethasone or a specific inhibitor of glucocorticoid receptors (GCRs). The relative levels of mRNA spliced variants will be correlated with measurements of function, viz., enzyme activity by radio-enzyme assay, expression of PNMT protein by Western blotting, and catecholamines by HPLC. In vitro, the role of GCs and GCRs in this splicing reaction will be investigated using PC12 cells, purified chromaffin cells and cervical ganglia, cultured with or without dexamethasone and in the presence or absence of GCRs. Relative levels of PNMT mRNA spliced variants will be correlated with PNMT function. Understanding the molecular basis for the differential regulation of PNMT function. Understanding the molecular basis for the differential regulation of PNMT activity in the brain and adrenal may reveal possible targets for therapeutic intervention in diseased states with perturbed adrenaline production.