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Chronic nicotine treatment (two daily subcutaneous injections administered approximately 12 h apart for 14 days) is associated with long-term inductions of tyrosine hydroxylase (TH) protein and TH mRNA in locus ceruleus (LC) neurons. These increases persist for at least 3 days after the final nicotine injection in LC cell bodies and for at least 7 to 10 days in LC nerve terminal regions. We tested whether this long-term response is due to sustained stimulation of TH gene transcription rate. A semiquantitative reverse transcription-polymerase chain reaction assay was developed to assess changes in the levels of TH RNA primary transcripts; these changes are an indirect measurement of changes in TH gene transcription rate. TH RNA primary transcript levels increase rapidly in the LC after a single nicotine administration and return to basal levels by 24 h. A similar rapid and transient induction of LC TH RNA primary transcripts occurs after chronic nicotine administration. In contrast, TH RNA primary transcript levels remain elevated for a sustained period of time (at least 1 day) in the adrenal medulla after chronic nicotine administration. Similar rapid, but transient changes in LC TH RNA primary transcript levels are observed after repeated immobilization stress. These results suggest that TH gene transcription rate in the LC is stimulated rapidly after each nicotine injection; however, in contrast to the adrenal medulla, there is no sustained transcriptional response elicited by chronic nicotine treatment or repeated immobilization stress in the LC, suggesting that post-transcriptional mechanisms may also play a role in these long-term responses.

Nicotine induces tyrosine hydroxylase (TH) mRNA by interacting with nicotinic acetylcholine receptors (nAChRs) in cultured adrenal medullary cell systems; however, the mechanisms responsible for the induction of adrenal TH in response to systemically administered nicotine under in vivo conditions are more complex. In the present study, we tested whether nAChRs and muscarinic acetylcholine receptors (mAChRs) participate in the induction of adrenal TH observed after long-term treatment with nicotine. Chronic nicotine treatment (1.6 mg/kg, two daily injections spaced 12 h apart for 7 days) induced TH mRNA, TH protein and TH activity in rat adrenal medulla. This induction of TH gene expression was totally blocked when an antagonist of either nAChRs or mAChRs was administered prior to each nicotine injection. Repeated injections of the mAChR agonist bethanechol (5 mg/kg injected twice per day for 7 days) also produced increases in TH mRNA levels; however, TH protein levels and TH activity did not increase in response to bethanechol. In denervated adrenal glands chronic nicotine treatment did not lead to induction of either TH mRNA, TH protein or TH activity, whereas chronic bethanechol treatment led to induction of TH mRNA, but not TH protein or activity. These results suggest that agonist occupation of both nAChRs and mAChRs are essential for the complete response of TH gene expression to chronic nicotine treatment in rat adrenal medulla, but that stimulation of either cholinergic receptor by itself is not sufficient to elicit a full response. The results also suggest that both transcriptional and post-transcriptional mechanisms may potentially need to be regulated to induce TH protein in response to some stimuli.

Nicotine is a powerful stimulant of the sympathoadrenal system, causing the release of peripheral catecholamines and activation of catecholamine biosynthesis. In previous reports, we have studied the mechanisms by which short-term nicotine treatment regulates tyrosine hydroxylase (TH) in adrenal medulla. In this report, we study the effects of chronic nicotine treatment on adrenal TH gene expression. Rats were injected with either saline or nicotine twice per day for up to 14 days. Chronic nicotine treatment elicited long-lasting, dose-dependent increases in the levels of adrenal TH mRNA, TH protein, and TH activity. In contrast, a single injection of nicotine elicited only a small increase in adrenal TH mRNA levels, which was transient and did not result in the induction of TH enzyme. Chronic nicotine administration also elicited a sustained increase in adrenal TH gene transcription rate, which persisted for up to 7 days after the final nicotine injection. This sustained transcriptional response correlated with a modest sustained increase in adrenal TH AP1 binding, but not in the levels of Fra-2 or other fos or jun proteins. These results demonstrate that repeated nicotine injections administered chronically over 1 to 2 weeks lead to sustained stimulation of the TH gene and consequent induction of TH gene expression in rat adrenal medulla. These studies support the hypothesis that chronic nicotine administration produces long-lasting cellular changes in adrenal medulla that lead to sustained transcriptional responses.