Chronic nicotine treatment (two daily subcutaneous injections administered ϳ12 h apart for 14 days) is associated with longterm 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.

During prolonged stress or chronic treatment with neurotoxins, robust compensatory mechanisms occur that maintain sufficient levels of catecholamine neurotransmitters in terminal regions. One of these mechanisms is the up-regulation of tyrosine hydroxylase (TH), the enzyme that controls catecholamine biosynthesis. In neurons of the periphery and locus coeruleus, this up-regulation is associated with an initial induction of TH mRNA. In contrast, this induction either does not occur or it is nominal in mesencephalic dopamine neurons. The reasons for this lack of compensatory TH mRNA induction remain obscure, because so little is known about the regulation of TH expression in these neurons. In this study, we test whether activation of the cAMP signaling pathway regulates TH gene expression in two rodent models of midbrain dopamine neurons, ventral midbrain organotypic slice cultures and MN9D cells. Our results demonstrate that elevation of cAMP leads to induction of TH protein and TH activity in both model systems; however, TH mRNA levels are not up-regulated by cAMP. The induction of TH protein is the result of a novel post-transcriptional mechanism that activates TH mRNA translation. This translational activation is mediated by sequences within the 3Ј untranslated region (UTR) of TH mRNA. Our results support a model in which cAMP induces or activates trans-factors that interact with the TH mRNA 3ЈUTR to increase TH protein synthesis. An understanding of this novel regulatory mechanism may help to explain the control of TH gene expression and consequently dopamine biosynthesis in midbrain neurons under different physiological and pathological conditions.

This review discusses the criteria for determining whether a binding site or functional response is directly mediated by either the mu, delta, or kappa opioid receptors. In 1988, Sibinga and Goldstein published the first review that addressed whether cells from the immune system express opioid receptors. The criteria that they used, namely, structure-activity relationships, stereoselectivity, dose- and concentration-dependence, and saturability are still relevant criteria today for determining if an immunological response is mediated by either the mu, delta or kappa opioid receptors. Radioligand receptor binding studies and functional studies that clearly show the presence of an opioid receptor on immunocytes are presented. Selective agonists and antagonists for the mu, delta, and kappa opioid receptors are discussed, and the need for their use in experiments is emphasized. Conditions used in functional assays are very important. Receptor desensitization and downregulation occur within minutes after the application of an agonist. However, many immunological assays are applying an agonist for days before measuring an immunological effect. The results obtained may reflect changes that are results of receptor desensitization and/or downregulation instead of changes that are observed with acute activation of the receptor. The future of receptor pharmacology lies in the crosstalk and dimerization of G protein-coupled receptors. In transfected systems, opioid receptors have been shown to dimerize with chemokine and cannabinoid receptors, resulting in crosstalk between different types of receptors.

Long-term stress leads to the induction of tyrosine hydroxylase (TH) protein and enzymatic activity in the adrenal medulla. This adaptive response is necessary to maintain the catecholamine biosynthetic capacity of adrenal chromaffin cells during periods of sustained catecholamine secretion. In this report we demonstrate that when rats are subjected to short-term stress, TH mRNA is induced for at least 24 h, but TH protein and TH activity (assayed under Vmax conditions) are not increased. In contrast, adrenal TH mRNA, TH protein and TH activity are induced in rats subjected to long-term stress. Using sucrose gradient fractionation, we show that the lack of induction of TH protein after one type of short-term stressor, a single 2-h immobilization stress is associated with a decrease in the percentage of TH mRNA molecules associated with polysomes. In contrast, after repeated immobilizations the polysome profile of TH mRNA is identical to that observed in control animals, even though TH mRNA is induced 2- to 3-fold. These results are consistent with the hypothesis that even though TH mRNA is induced by short-term stressors, mechanisms that control TH mRNA translation must also be appropriately regulated for TH protein to be induced.

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.

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Tyrosine hydroxylase (TH) gene transcription rate increases in response to numerous pharmacological and physiological stimuli. The AP1 site within the TH gene proximal promoter is thought to play an important role in mediating many of these responses; however, it is unclear which AP1 factors are required. To investigate whether c-Fos is essential for the response of the TH gene to different stimuli, c-Fos-deficient PC12 cell lines were produced utilizing an antisense RNA strategy. In these cell lines, stimulus-induced increases in c-Fos protein levels were dramatically attenuated, while c-Jun and CREB levels remained unchanged. TH gene transcription rate increased from four-to eight-fold in control cells after treatment with either 50 mM KCl or TPA. These responses were dramatically decreased in the c-Fos-deficient cell lines. In contrast, c-Fos down-regulation had little effect on the response of the TH gene to forskolin. Stimulation of TH gene promoter activity, which was observed in control cell lines treated with either 50 mM KCl or TPA was also dramatically inhibited in the c-Fos-deficient cells. These results suggest that c-Fos induction is essential for maximal stimulation of the TH gene in response to either depolarization or PKC activation in PC12 cells.