QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sun, B. Articles by Tank, A. W. Search for Related Content PubMed PubMed Citation Articles by Sun, B. Articles by Tank, A. W.

CELLULAR AND MOLECULAR

Chronic Nicotine Treatment Leads to Sustained Stimulation of Tyrosine Hydroxylase Gene Transcription Rate in Rat Adrenal Medulla

Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York

Received August 24, 2002 ; accepted October 28, 2002.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
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.

A number of studies have shown that chronic nicotine treatment leads to induction of TH in adrenal medulla. An early report showed that nicotine, when it is administered twice daily for 14 days, induces adrenal TH activity (Seidler and Slotkin, 1976). More recently, it has been shown that nicotine induces adrenal TH mRNA, when it is injected subcutaneously once or twice daily for 3 to 5 days, but not when it is administered by continuous infusion for up to 27 days (Stachowiak et al., 1988; Hiremagalur and Sabban, 1995; Serova et al., 1999). This latter result suggests that the induction of adrenal TH is dependent on intermittent, repetitive exposure to nicotine. However, the mechanisms responsible for this long-term modulation of gene expression by chronic, intermittent, repeated administration of nicotine have not been investigated.

Previous research in our laboratory has focused on the mechanisms that regulate TH gene expression in response to acute nicotine treatments (Fossom et al., 1991a; Piech-Dumas et al., 1999; Sterling and Tank, 2001). We have shown that a single injection of nicotine rapidly stimulates TH gene transcription rate in rat adrenal medulla, but that this transcriptional response lasts for less than 3 h and, most importantly, does not lead to measurable changes in TH mRNA or TH protein. This finding is consistent with some of the previous studies, which have shown that a single injection of nicotine does not induce adrenal TH gene expression (Seidler and Slotkin, 1976; Stachowiak et al., 1988). However, when nicotine is injected repeatedly over a 3-h period, adrenal TH gene transcription rate remains stimulated for a more sustained period of time (3–6 h), resulting in 2- to 3-fold inductions of TH mRNA and TH protein.

Because our results suggest that a single injection of nicotine is not sufficient to induce adrenal TH and because the half-life of the drug is relatively short in rats (60 min (Kyerematen et al., 1988)), it is surprising that TH is induced when a single injection of nicotine is administered once or twice daily over several days. However, recent studies have indicated that a single injection of nicotine does induce adrenal TH mRNA (Hiremagalur and Sabban, 1995; Serova et al., 1999), although in these reports it was not determined whether a single nicotine injection induces TH protein or enzymatic activity. The studies in this article were undertaken to investigate these discrepancies and to test the hypothesis that chronic nicotine treatment results in sustained stimulation of the TH gene leading to the observed changes in TH expression.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Administration of Nicotine to Rats. Male Sprague-Dawley rats (175–250 g) purchased from Charles River Laboratories, Inc. (Wilmington, MA) were used in this study. Rats were injected subcutaneously with doses of nicotine ranging from 0.4 to 2.3 mg/kg (dosages were expressed in terms of nicotine free base, even though the bitartrate salt was used for injections). A short, mild seizure was observed using 1.6 mg/kg nicotine, whereas longer and more convulsive seizures were noted when using 2.3 mg/kg nicotine. No seizures were observed using 0.8 mg/kg nicotine or lower doses. These injections were made twice daily with injections spaced 10 to 14 h apart. Nicotine bitartrate was dissolved in phosphate-buffered saline (150 mM NaCl and 10 mM potassium phosphate) and the solutions were buffered to pH 7.5. The injections were made using a volume of 1 ml/kg. Control rats were injected with the same volume of phosphate-buffered saline. Rats were injected for periods of time ranging from one injection to 29 injections administered over 14 to 15 days. The final injections were always administered in the morning. Rats were euthanized using an overdose of sodium pentobarbital (150 mg/kg, injected intraperitoneally) 3 h after the final injection of nicotine, unless otherwise specified. Adrenal glands were removed while the animals were anesthetized before death and immediately frozen on dry ice, except for the nuclear run-on assays (see below).

Immobilization Stress. Immobilizations were performed as described in Nankova et al. (1994). Briefly, the four limbs of each rat were taped to a metal board and the head was restrained in metal rings fixed to the board. The rats were immobilized for 2 h each day in the morning. Immediately after the seventh immobilization, rats were euthanized and adrenal medullae were dissected.

All procedures and drug administrations with rats were performed in accordance with the guidelines and approval of the University of Rochester Committee on Animal Resources.

Assays for TH Enzyme Activity and TH Protein. TH activity was assayed as described previously (Fossom et al., 1991a). Briefly, frozen adrenal glands were homogenized in 250 µl of 30 mM potassium phosphate (pH 6.8), 50 mM NaF, and 10 mM EDTA, and the homogenate was centrifuged at 15,000g for 10 min. TH activity was assayed by a coupled decarboxylation assay. Endogenous catecholamines and other small molecules that might interfere with the assay were removed by subjecting the adrenal supernatants to gel filtration using Sephadex G-50 columns, equilibrated with 30 mM potassium phosphate (pH 6.8), 10 mM NaF, and 0.1 mM EDTA. A 50-µl aliquot of the gel-filtered supernatant was used for the assay. Protein was measured by the method of Bradford (1976), using bovine serum albumin as a standard. Adrenal TH activity was expressed as nanomoles of ¹⁴CO₂ formed per minute per milligram of protein.

TH protein was measured using Western analysis as described in previous publications (Fossom et al., 1991a; Piech-Dumas et al., 1999; Sterling and Tank, 2001). A representative group of the same adrenal supernatants used for assaying TH activity were used for measuring TH protein. Briefly, for each sample three different concentrations of supernatant protein (5–25 µg) were loaded onto separate lanes of a 10% SDS-polyacrylamide gel. In addition, a known amount of purified rat pheochromocytoma TH protein was loaded onto a separate lane for each gel. The samples were subjected to electrophoresis, transferred to nitrocellulose, and immunoblotted using rabbit antiserum specific for TH. The antibody-TH complexes were detected using the ECS system (Amersham Biosciences, Inc., Piscataway, NJ) and autoradiography as described by Piech-Dumas et al. (1999). The autoradiographic bands were quantitated by scanning the autoradiograms with a Hewlett Packard ScanJet 4C scanner with a transparency adaptor along with computer-assisted imaging analysis using NIH Image software to calculate the density units. Care was taken to use only those density values that were within the linear range of the autoradiographic film. The density units for each TH protein band were normalized to the amount of total soluble protein loaded onto the gel for that sample and then divided by the density units for the known amount of purified TH protein loaded onto that gel. TH protein was expressed as the micrograms of TH protein per milligram of protein.

Measurement of Adrenal TH mRNA Using RNase Protection Assays. RNase protection assays were performed essentially as described by Piech-Dumas et al. (1999). Briefly, total cellular RNA was isolated from rat adrenal glands as described previously (Fossom et al., 1991a) and hybridized to radiolabeled antisense TH and actin riboprobes. The signals from the actin antisense riboprobe were used to control for differences in RNA input into the hybridization reactions and for recovery of RNA duplexes during the procedure. Antisense riboprobes were synthesized using [³²P]UTP according to the manufacturer (Promega, Madison, WI). A nonradioactive sense riboprobe encoding TH sequences complementary to the antisense TH riboprobe was also synthesized using these standard procedures; this sense riboprobe was used to generate a standard curve. All riboprobes were purified on a denaturing 5% polyacrylamide/8 M urea gel before use. For TH mRNA antisense and sense riboprobes, pTH.3 was used as a template; this plasmid contains a 280-bp insert encoding the 3' region of rat TH mRNA (nucleotides 1241 to 1540) inserted into the multiple cloning site of pGEM3 (Fossom et al., 1991a). The actin mRNA antisense riboprobe was synthesized using a template containing 125 bp of rat actin cDNA; this cDNA template was purchased from Ambion (Austin, TX). Hybridizations were performed for 16 to 20 h at 42 to 45°C in the presence of 10 to 15 µg of total cellular RNA, 500 pg of rat TH antisense riboprobe, 500 pg of rat actin antisense riboprobe, 80% deionized formamide, 300 mM sodium acetate (pH 6.4), 100 mM sodium citrate, and 1 mM EDTA. Other reaction tubes contained known amounts (0–20 pg) of sense TH riboprobe and 10 µg of total cellular RNA isolated from rat liver, in place of the rat adrenal RNA samples; these reactions were used to construct a standard curve. Unhybridized RNA was digested with a mixture of RNase A (5 units/ml) and RNase T1 (200 units/ml) for 30 min at 37°C. The protected radiolabeled RNA duplexes were precipitated, resuspended, and separated on a 5% nondenaturing polyacrylamide gel as described by Piech-Dumas et al. (1999). The radiolabeled RNA duplexes were detected with the use of a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) or autoradiographically. Radioactive bands representing protected RNA species were densitometrically quantitated by scanning the autoradiogram with a Hewlett Packard ScanJet 4C scanner as described above. Care was taken to use only those density values within the linear range of the autoradiographic film. The density units obtained for the TH mRNA duplex bands were normalized to the density units obtained for the standard curve using known amounts of TH sense riboprobe to calculate the picograms of TH mRNA present in the hybridization reactions. These values were converted to attomoles of TH mRNA and then normalized to femtomoles of actin mRNA, which were calculated from the density units obtained for the actin mRNA duplex bands in the same samples.

Measurement of TH mRNA Using Semiquantitative RT-PCR. Semiquantitative RT-PCR assays were performed as described previously by Bowyer et al. (1998) with only minor modifications, except that TH mRNA signals were normalized to 28S ribosomal RNA signals (instead of GAPDH mRNA signals as presented in the previous study). Briefly, 0.2 µg of adrenal RNA was subjected to RT using random hexamer primers. Aliquots of the resulting single-stranded cDNA products were used along with the appropriate primers (see below) in the PCR to incorporate [³²P]dATP (0.5 µCi/reaction) into double-stranded products encoding 519 bp of TH cDNA or 295 bp of 28S cDNA. The 5' and 3' primers used for the PCR for TH mRNA were selected such that they encoded regions of different exons separated by an intron; therefore, PCR products derived from genomic DNA contaminating the isolated RNA would produce a detectably larger PCR band. These bands were never observed.

For each RT reaction, 6 µl of 4 ng/µl hexamer random primers (Invitrogen, Carlsbad, CA) were added to 2 µl of RNA in a thin-walled PCR tube. The reactions were heated to 70°C for 5 min and then cooled to 5°C to anneal the primers. Subsequently, 12 µl of reaction buffer was added to the reaction mixtures (final volume 20 µl). Final concentrations of reactants were as follows: 50 mM Tris (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 10 mM dithiothreitol, 0.5 mM dNTPs, 2.5 units/µl RNase inhibitor, and 50 units Superscript II (Invitrogen). The reaction mixtures were warmed to 42°C for 15 min and then placed on ice. PCR amplifications of TH and 28S cDNAs were performed in separate reaction tubes. A 2-µl aliquot of the 20-µl RT reaction was used as the first-strand template for the PCR amplification. The PCRs were performed in a 50-µl reaction volume containing (final concentrations) 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 3 mM MgCl₂, 200 µM dNTPs, 1.5 units of TaqDNA polymerase (Invitrogen), 0.2 µM of both 5' and 3' primers, and 0.5 µCi (3000 Ci/mmol) of [-³²P]dATP (PerkinElmer Life Sciences, Boston, MA). The primers for the PCR amplification of TH and 28S cDNAs were selected using the cDNA sequences for these mRNAs in GenBank (National Cancer Institute/Frederick Biomedical Supercomputing Center, Frederick, MD) and the program OligoR. The 5' TH mRNA sense primer encoded cDNA sequences 390 to 410; the 3' antisense primers were complementary to TH cDNA sequences 888 to 908. The sequences for these primers were as follows: 5' TH primer, 5'-ccc cac ctg gag tat ttt gtg-3'; 3' TH primer, 5'-atc acg ggc gga cag tag acc-3'. The 5' 28S primer encoded sequences 1 to 22 of 28S cDNA (5'-gtg aac agc agt tga aca tggg-3'); the 3'' 28S primer were complementary to 28S cDNA sequences 295 to 276 (5'-aac cgc gac gct ttc caag-3'). All PCRs were performed using an Eppendorf Mastercycler and PerkinElmer enzymes and protocols with slight modifications. For TH cDNA amplification, each PCR cycle, with the exception of the first cycle which had a longer denaturing period at 94°C for 2 min and the final cycle which had an extension period of 7 min, consisted of three steps: 1 min at 94°C (denaturing), 1 min at 55°C (annealing), and 1 min at 72°C (primer extension). The number of cycles used for PCR were between 23 to 26, depending upon the assay. After the last PCR cycle the reactions were allowed to cool to room temperature and then subjected to electrophoresis using a 6% nondenaturing polyacrylamide gel, as described in Bowyer et al. (1998). The same protocol was used for PCR amplification of 28S cDNA, except that the number of cycles used was 14 to 16. For each experiment, the linearity of the RT-PCR for both TH mRNA and 28S rRNA was assessed with respect to both micrograms of RNA added to the RT reaction and the number of PCR cycles. After electrophoresis, the gels were dried down onto Whatman 1MM paper (Schleicher & Schuell, Keene, NH). The levels of ³²P-labeled RT-PCR products separated on the gels were quantified using the PhosphorImager system from Molecular Dynamics (Sunnyvale) after exposure to the phosphor screens for 2 to 24 h. The ImageQuant software (Molecular Dynamics) methods of volume integration were used to quantify the intensity of the RT-PCR bands. Alternatively, the dried-down gels were exposed to X-ray film for 1 to 3 days and the autoradiographic signals were quantified using a scanner and NIH Image software as described above.

Measurement of TH Gene Transcription Rate in Adrenal Medulla Using Nuclear Run-On Assays. Nuclear run-on assays were performed essentially as described in previous publications (Fossom et al., 1991a; Piech-Dumas et al., 1999; Sterling and Tank, 2001). Briefly, adrenal glands were removed and medullae were dissected away from the cortex under a dissecting microscope. Nuclei were isolated from the adrenal medullae from a single animal and incubated for 90 min with [³²P]UTP and appropriate buffers to promote the elongation of nascent RNA strands. Radiolabeled RNA was isolated and hybridized to a nitrocellulose filter on which the following plasmids were applied using a slot blotter: pTHg6.3, p28S1.5, and pGem7Zf. pTHg6.3 is a genomic clone encoding 6.3 kilobases of the rat TH gene (Fossom et al., 1991a). p28S1.5 encodes 1.5 kilobases of the human 28S rRNA gene and was purchased from American Type Culture Collection (catalog no. 77235; Manassas, VA). The 28S rRNA cDNA was used to provide signals for normalization of the TH signals, so as to control for loss of radiolabeled RNA during the assay and for hybridization efficiency. pGem7Zf was purchased from Promega and was used to provide background hybridization signals. After hybridization, the filters were washed, and the hybridized radioactivity was visualized and quantitated using either a PhosphorImager or autoradiography as described above. The signals for pGem7Zf hybrids were subtracted from the pTHg6.3 or p28S1.5 hybrid signals to calculate signals that represented radiolabeled RNA specifically hybridized to either TH or 28S gene sequences, respectively. The specifically hybridized TH signal was then divided by the specifically hybridized 28S signal for each sample to obtain the relative TH gene transcription rate.

Electrophoretic Mobility Shift Assays (EMSAs) and Western Analysis of Transcription Factor Levels. Adrenal medullae were dissected and immediately frozen on dry ice. Whole cell proteins were extracted by homogenizing the medullae in a buffer containing 20 mM HEPES (pH 7.4), 25% glycerol, 0.5 M KCl, 1.5 mM MgCl₂, 50 mM NaF, 1 mM EDTA, 0.5 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml pepstatin. Electrophoretic mobility shift assays were performed as described previously (Nagamoto-Combs et al., 1997). Briefly, 0.2 pmol (50,000–100,000 cpm) of ³²P-radiolabeled TH AP1 oligonucleotide probe was incubated together with 10 µg of whole cell extracts in a reaction buffer containing 25 mM HEPES (pH 7.5), 6% glycerol, 0.13 M KCl, 0.1% Triton X-100, 2 mM EDTA, 0.4 mM MgCl₂, 12.5 mM NaF, 0.6 mM spermidine, 0.2 mM spermine, 0.1 mM phenylmethylsulfonyl fluoride, 0.25 µg/ml leupeptin, 0.25 µg/ml pepstatin, 0.4 mM dithiothreitol, and 1 µg of poly(dIdC). The binding reaction was carried out first on ice for 30 min and then at room temperature for 5 min. The reaction mixture was subjected to 5% polyacrylamide gel electrophoresis. After drying the gel onto Whatman paper, the radiolabeled protein-DNA complexes were visualized by autoradiography, and the densities of the bands were measured using either NIH Image software or the PhosphorImager system as described above.

Western analysis was used to measure levels of AP1 transcription factors essentially as described in previous publications (Nagamoto-Combs et al., 1997; Sun and Tank, 2002). Briefly, whole cell extracts (10 µg of protein) were subjected to SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were soaked in 5% dry milk dissolved with TBS-T (10 mM Tris, pH 7.4, 150 mM NaCl, 0.05% Triton X-100) for 1 h, and then rinsed with TBS-T. The primary antibodies for Fos or Jun proteins [purchased from Santa Cruz Biotechnology (Santa Cruz, CA)] were diluted (1:1000) with 3% dry milk dissolved in TBS-T and incubated with the membranes on a shaker for 1 h. After washing for three times with TBS-T, the membranes were incubated for 45 min with horseradish peroxidase-conjugated secondary antibody (diluted 1:3000 with 3% dry milk dissolved in TBS-T), and then thoroughly washed with TBS-T and rinsed with TBS (10 mM Tris, pH 7.4, 150 mM NaCl). Immunolabeled proteins were detected using a chemiluminescent detection protocol provided with the enhanced chemiluminescence detection kit from Amersham Biosciences, Inc., and visualized by exposing the membranes to X-ray film.

Statistical Analyses. The results were analyzed by one-way analysis of variance, using the computer program INSTAT. Comparisons between groups were made using the Student-Newman-Kuels or Dunnett's multiple comparisons test. A level of p < 0.05 was considered statistically significant.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Repeated Injections of Nicotine Administered Chronically over 14 Days Lead to Long-Term Induction of TH Protein and Enzymatic Activity in Rat Adrenal Medulla. Rats were injected subcutaneously twice per day for 14 days (injections were spaced 10–14 h apart) with different doses of nicotine. Adrenal glands were removed under sodium pentobarbital anesthesia 3 h after the final injection, which was given on the morning of the 15th day. Control rats were injected with saline according to the same schedule. Adrenal TH activity increased in a dose-dependent manner in animals treated chronically with nicotine (Table 1). Treatment with 0.4 mg/kg nicotine did not produce a significant response; however, treatment with 0.8 mg/kg nicotine elicited an 1.5-fold increase in TH activity. Treatment with greater doses (1.6 or 2.3 mg/kg) produced 2- to 4-fold increases in adrenal TH activity. This increase in TH activity was sustained for a long period of time, even after the nicotine injections were terminated (Fig. 1). Adrenal TH activity remained elevated by 2-fold for up to 3 days after the last nicotine injection and was still significantly increased 7 days after cessation of treatment. Adrenal TH activity returned to basal levels 10 days after the final nicotine injection.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Adrenal TH activity increases in response to chronic nicotine treatment. Rats were injected saline or 1.6 mg/kg nicotine twice per day (injections spaced 10–14 h apart) for 14 days. A final injection was administered on the morning of the 15th day and then adrenal glands were removed from the animals under sodium pentobarbital anesthesia at different times after this final injection. TH activity was assayed using 4 mM 6MPH4. The results represent the means ± S.E. from 5 to 12 rats. **, p < 0.01 compared with saline-treated controls.

In these experiments adrenal TH activity was assayed using a saturating concentration of pterin cofactor; hence, the observed changes in activity represent changes in the V_max of the enzyme. Because activation of adrenal TH by nicotine or other stimuli is primarily due to an apparent decrease in K_m for pterin cofactor (Masserano and Weiner, 1979; Fossom et al., 1991b), these changes in V_max are thought to be principally representative of changes in the amount of enzyme. To test directly whether the increases in TH activity were due to changes in the amount of TH protein, we performed Western analysis on some of the same adrenal supernatants used for the measurement of TH activity. A representative Western blot for adrenal TH is presented in Fig. 2A. A single band representing TH immunoreactive protein was observed using adrenal gland supernatants. It is clear from this figure that TH protein increased after chronic nicotine treatment. The densities of the TH bands on this Western blot along with those derived from other animals were quantified. TH protein and enzymatic activity values obtained from the same adrenal supernatants are presented in Fig. 2B. The increases in TH protein levels observed after chronic nicotine treatment were approximately equivalent to the increases in TH enzymatic activity.

View larger version (36K): [in this window] [in a new window]   Fig. 2. Adrenal TH protein levels increase in response to chronic nicotine treatment. Rats were injected with saline or the designated dose of nicotine twice per day for 14 days. A final injection was administered on the morning of the 15th day and the adrenal glands were removed under sodium pentobarbital anesthesia 3 h after this final injection. A, representative autoradiogram of a Western blot depicting adrenal TH protein isolated from either saline-treated or nicotine-treated rats is shown. The signal obtained from a known concentration of a purified TH standard is shown in the rightmost lane. The micrograms of soluble adrenal protein loaded onto each lane of the gel are presented under each TH band. B, bar graph depicts TH enzymatic activity and TH protein levels measured in the same adrenal supernatants obtained from either saline-treated or nicotine-treated rats. Each data point represents the means ± S.E. from three animals. *, p < 0.05 compared with saline-treated controls. **, p < 0.01 compared with saline-treated controls.

Chronic, Repeated Injections of Nicotine Lead to Induction of TH mRNA in Rat Adrenal Medulla. Adrenal TH mRNA levels were first measured using an RNase protection assay, an example of which is presented in Fig. 3A. In this set of experiments, actin mRNA levels were used to normalize TH mRNA signals between samples, so as to control for losses of RNA during the assay. It is evident from the example shown in Fig. 3A that chronic nicotine treatment increased adrenal TH mRNA levels in a dose-dependent manner. Data from a number of experiments are presented in Table 1. The lowest dose of nicotine (0.4 mg/kg) did not produce any change in TH mRNA levels. However, the higher doses (0.8–2.3 mg/kg) produced 2- to 4-fold increases in adrenal TH mRNA levels after 14 days of treatment. One problem with this assay was that even though actin mRNA levels were not significantly changed by chronic treatment with lower doses of nicotine, 2.3 mg/kg nicotine produced a 1.7-fold decrease in actin mRNA levels (actin mRNA values in atmol/µg total cellular RNA: saline-treated rats, 83 ± 9; nicotine-treated rats, 50 ± 7, p < 0.05). In a previous article, we developed a semiquantitative RT-PCR assay for measuring TH mRNA in midbrain samples. Hence, we modified this assay to measure TH mRNA in adrenal samples and used 28S ribosomal RNA as a normalization standard. This assay provided a more sensitive and rapid procedure for the reliable measurement of TH mRNA in adrenal samples and was used to verify the TH mRNA results obtained using the RNase protection assays.

View larger version (56K): [in this window] [in a new window]   Fig. 3. Adrenal TH mRNA levels increase in response to chronic nicotine treatment. Rats were injected with saline or the designated dose of nicotine twice per day for 14 days. A final injection was administered on the morning of the 15th day and the adrenal glands were removed under sodium pentobarbital anesthesia 3 h after this final injection. A, representative autoradiogram depicting adrenal TH mRNA and actin mRNA levels measured using RNase protection assays is shown. B, representative autoradiogram depicting adrenal TH mRNA and 28S rRNA measured using semiquantitative RT-PCR is shown.

Great care was taken to verify the quantitative nature of this RT-PCR assay. For every experiment preliminary assays were performed on selected samples to verify that the assay conditions were linear with respect to both total cellular RNA input into the assay and with respect to PCR cycle number. These parameters varied slightly between experiments but the linear ranges were always relatively wide. For instance, the linear range for measuring TH mRNA with respect to PCR cycle number using 0.2 µg of adrenal RNA in the initial RT step ranged from 22 to 28 or 24 to 30 in different experiments. Likewise, the linear range for RNA input ranged from 0.1 to 0.8 µg of RNA using 24 cycles in the PCR. All assays were performed within these linear ranges for RNA input and cycle number. Furthermore, for each assay known amounts of sense strand RNA encoding either TH mRNA or 28S rRNA sequences were used in separate RT-PCR reactions (Fig. 3B). The signals from these reactions were used to construct standard curves.

We measured TH mRNA and 28S rRNA in the same adrenal samples that were assayed using the RNase protection assay (Table 1). Almost identical results were obtained. TH mRNA levels were not induced significantly by 0.4 mg/kg nicotine, whereas 0.8 and 1.6 mg/kg nicotine elicited 2-fold increases in TH mRNA levels. One difference between the two assays is the result using 2.3 mg/kg nicotine. As noted above, using the RNase protection assay, an 4-fold induction of TH mRNA was observed; only a 2-fold effect was observed using the RT-PCR assay. Presumably, this discrepancy is due to the use of 28S rRNA as a normalization standard in the RT-PCR reaction as opposed to actin mRNA. As noted above, the 1.7-fold decrease in actin mRNA levels observed after treatment with 2.3 mg/kg nicotine accounted for this larger induction; we did not note any changes in 28S rRNA levels after any of the nicotine treatments.

Dependence of the Induction of TH Gene Expression on the Number of Nicotine Injections. Rats were administered either a single injection of 1.6 mg/kg nicotine or repeatedly injected with 1.6 mg/kg nicotine twice per day (injections spaced 10–14 h apart) for 2 to15 days (equivalent to 3, 5, 15, or 29 injections). The final injection was always administered in the morning of the last day and adrenal glands were dissected from the animals either 3 or 24 h after this final injection. As controls, rats were administered saline according to the same schedule. There were no significant differences in TH activity or TH mRNA levels in rats treated with saline for 1 to 14 days; hence, the data obtained from the saline-treated animals at all the different time points were pooled. The results of these experiments are presented in Fig. 4.

View larger version (34K): [in this window] [in a new window]   Fig. 4. Dependence of the nicotine-mediated induction of TH and TH mRNA on the number of nicotine injections. Rats were injected with either saline or 1.6 mg/kg nicotine twice per day for the number of injections noted in the figure. Adrenal glands were removed under sodium pentobarbital anesthesia either 3 or 24 h after the final injection. TH activity was measured using 4 mM 6MPH4 and TH mRNA levels were measured using RNase protection assays. The results represent the means ± S.E. from 4 to 13 rats. *, p < 0.05 compared with saline-treated controls. **, p < 0.01 compared with saline-treated controls.

A single injection of nicotine did not induce TH activity in adrenal medulla at either the 3- or 24-h time point after drug administration. Three nicotine injections elicited a significant 1.5-fold induction of adrenal TH activity. Five or more nicotine injections elicited apparent maximal induction (2-fold) of adrenal TH. Adrenal TH mRNA was induced slightly after a single injection of nicotine; however, this effect was transient, in that TH mRNA levels returned to baseline 24 h after the single injection. Three or more injections of nicotine elicited a 2- to 3-fold induction of adrenal TH mRNA, and this induction was sustained for at least 24 h after 29 injections.

Chronic Nicotine Treatment Elicits a Long-Term, Sustained Stimulation of TH Gene Transcription Rate. The results presented in the previous sections indicated that when nicotine was chronically administered twice daily, TH mRNA, and TH protein were induced in rat adrenal medulla for a sustained period of time. These effects were not observed after a single injection of nicotine, suggesting that chronic administration of the drug produced alterations in the mechanisms responsible for regulating TH expression. We tested the hypothesis that transcriptional mechanisms regulating the TH gene participate in this long-term induction of TH.

In our first set of studies, rats were injected twice daily with either saline or 1.6 mg/kg nicotine for 7 days. On the morning of day 8, the rats were administered saline or different doses of nicotine and killed 20 min after this final injection. TH gene transcription rate was measured in the adrenal medullae of these rats using nuclear run-on assays (Fig. 5). In animals treated chronically with saline for 7 days, there was a dose-dependent increase in TH gene transcription rate observed after the challenge nicotine injections. Even the lowest dose of nicotine (0.4 mg/kg) produced a 2- to 3-fold stimulation of the TH gene in these animals. The greatest dose of nicotine (1.6 mg/kg) produced a 5- to 6-fold response. A similar dose-response curve for the challenge nicotine injections was obtained in animals treated chronically with nicotine for 7 days, except that TH gene transcription rate was significantly elevated even in animals administered saline as the challenge injection. This result is consistent with the hypothesis that TH gene transcription rate remained elevated for a prolonged period of time after chronic nicotine administration.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Dose-response curves for the final challenge nicotine injection in animals treated chronically with either saline or nicotine. Rats were injected with saline or 1.6 mg/kg nicotine twice per day for 7 days. In the morning of the 8th day, the animals were injected with either saline or different doses of nicotine as noted in the figure. Adrenal glands were removed under anesthesia 20 min after this final challenge injection and adrenal medullae were dissected. TH gene transcription rate was assayed using nuclear run-on assays. The results represent the means ± S.E. from six to eight rats. The TH gene transcription rate values derived from all the nicotine-treated rats were significantly greater (p < 0.01) than that measured in saline-treated rats. *, p < 0.05 compared with animals treated with a final injection of saline. **, p < 0.01 compared with rats chronically administered saline and injected with a final challenge injection of saline.

A second set of experiments was performed to test further this hypothesis and to verify that the effect of saline in Fig. 5 was not due to a conditioned response to the subcutaneous nicotine injections. These experiments were also performed to determine how long after the final nicotine injection TH gene transcription rate remained elevated (Table 2). Rats were injected with saline or 1.6 mg/kg nicotine for 7 days and administered a final injection on the morning of the 8th day. Then, animals were euthanized at different times after this final injection. As expected, TH gene transcription rate increased dramatically (4.6-fold) 20 min after a challenge nicotine injection in animals chronically treated with saline. However, this response was transient, in that TH gene transcription rate returned to basal activity 24 h after the nicotine injection. Contrasting results were obtained in animals treated chronically with nicotine. In agreement with the data in Fig. 5, TH gene transcription rate was elevated 3-fold in these animals 20 min after a challenge injection of saline; this response was 5-fold 20 min after a final challenge injection of 1.6 mg/kg nicotine. The increase in TH gene transcription rate was sustained for many days in the animals chronically treated with nicotine. Relative to saline-treated controls, TH gene transcription rate remained stimulated 4-fold 12 or 24 h after the final injection of the drug. This response was sustained for at least 7 days, during which time TH gene transcription rate was elevated 2- to 3-fold over that observed in control animals. It is likely that this sustained stimulation of the TH gene at least partially explains the long-term induction of TH expression observed in the animals chronically treated with nicotine.

Protein Binding to the TH AP1 Site Increases after Chronic Nicotine Administration. As a first attempt to investigate the mechanisms responsible for this sustained stimulation of the TH gene after chronic nicotine treatment, we tested whether adrenal medullary protein binding to specific regions of the TH gene promoter were altered in nicotine-treated animals. We chose to study protein binding to the TH cAMP response element site at position –45 to –37 and the TH AP1 site at position –201 to –195 within the TH gene proximal promoter. These sites were first investigated, because they are generally recognized as the major sites within the TH gene proximal promoter that respond to extracellular stimuli (Kumer and Vrana, 1996). No differences in TH CRE binding were observed at any time point using adrenal medullary proteins from animals treated chronically with nicotine (data not shown). Nor did we observe changes in cAMP response element-binding protein phosphorylation using western analysis at either 2 or 24 h after the final nicotine injection (data not shown). However, we did observe a modest increase in the binding of adrenal medullary proteins to the TH AP1 site. An autoradiogram from an EMSA depicting TH AP1 binding is observed in Fig. 6A; data obtained from three separate experiments are shown in the bar graph in Fig. 6B. A single major band corresponding to TH AP1 binding to whole cell proteins isolated from the adrenal medulla was observed in animals treated for 7 days with saline. This TH AP1 binding was significantly increased 2 h after a single injection of nicotine in the animals chronically administered saline. However, this increase in TH AP1 binding was transient, in that TH AP1 binding returned to control levels 24 h after the single nicotine injection. In animals treated chronically for 7 days with nicotine, TH AP1 binding also increased modestly 2 h after the final nicotine injection; however, this increase in TH AP1 binding was sustained for at least 2 days.

View larger version (32K): [in this window] [in a new window]   Fig. 6. Adrenal medullary TH AP1 binding increases in response to chronic nicotine treatment. Rats were injected twice per day with 1.6 mg/kg nicotine for 7 days. In the morning of the 8th day, the animals were injected with either saline or 1.6 mg/kg nicotine and adrenal glands were removed 2 or 24 h after this final injection. Adrenal medullae were dissected, whole cell proteins were isolated and EMSA were performed. A, representative autoradiogram depicting an EMSA of adrenal medullary protein binding to a double-stranded oligonucleotide probe encoding the TH AP1 site is presented. The adrenal proteins were derived from animals chronically treated with saline and then administered a final injection of either saline (Sal/Sal) or 1.6 mg/kg nicotine (Sal/Nic), or from animals chronically treated with nicotine and administered a final injection of nicotine (Nic/Nic). The time points at which the animals were killed after the final challenge injection are denoted in the figure. Proteins extracted from untreated PC12 cells or PC12 cells treated for 1 h with 50 mM KCl were used as positive controls. B, bar graph represents EMSA data obtained over three experiments. The data are the means ± S.E. from five to six rats. *, p < 0.05 compared with controls.

We next measured the levels of different AP1 factors in the adrenal medulla to determine whether any of them changed in a manner that correlated with the sustained increase in TH gene transcription rate observed after chronic nicotine treatment. Our first set of experiments focused on Fra-2 (Fig. 7), because Nankova et al. (2000) recently showed a sustained induction of adrenal Fra-2 after chronic immobilization stress. We used two different antibodies to measure Fra-2 levels. The results using an antibody [SC-604, Fra-2 (Q20); Santa Cruz Biotechnology, Inc., Santa Cruz, CA] that was designed to recognize epitopes mapping to the amino terminus of Fra-2 identified a number of adrenal medullary bands on a Western blot (Fig. 7A). The slowest migrating bands corresponded with Fra-2 immunoreactive proteins that were inducible by nerve growth factor in PC12 cells; NGF was shown to induce Fra-2 in PC12 cells (Boss et al., 2001). When rats were treated chronically with either saline or nicotine for 7 days and then administered a challenge injection of nicotine in the morning of the 8th day, the densities of the slowest migrating, Fra-2 immunoreactive bands increased dramatically 2 h after the challenge nicotine injection (S/N2 or N/N2 in Fig. 7A). However, the levels of these Fra-2 immunoreactive proteins were not increased at either 24 or 48 h after the challenge nicotine injection in rats treated chronically with either saline or nicotine (Fig. 7A). When we used an antibody [SC-171, Fra-2 (L-15); Santa Cruz Biotechnology, Inc.] designed to recognize the carboxy terminus of Fra-2, a single major adrenal medullary, Fra-2-immunoreactive protein was identified on the Western blot (Fig. 7B). The level of this protein did not increase significantly at either 2 or 24 h after the challenge nicotine injection in the animals chronically administered saline. In contrast, the level of this Fra-2 immunoreactive protein increased 2 h after the challenge nicotine injection in animals chronically administered nicotine for 7 days, but its level was not elevated above controls at 24 or 48 h after the challenge nicotine injection (Fig. 7B). As a positive control, we observed significant increases in adrenal medullary Fra-2 immunoreactive proteins in animals that were subjected to immobilization stress for 7 days (Fig. 7C), as reported by Nankova et al. (2000). Taken together, these results suggest that the sustained increase in TH gene transcription rate elicited by chronic nicotine treatment was not due to increased levels of Fra-2.

View larger version (36K): [in this window] [in a new window]   Fig. 7. Effect of chronic nicotine administration on Fra-2 levels in adrenal medulla. Rats were injected twice per day with saline or 1.6 mg/kg nicotine for 7 days. In the morning of the 8th day, the animals were injected with either saline or 1.6 mg/kg nicotine and adrenal glands were removed under anesthesia at different time points after this final challenge injection. PC12 cells were treated for 24 h with 100 ng/ml NGF or control vehicle before harvest and whole cell proteins were isolated as described for adrenal medulla. In C, rats were either handled or immobilized for 2 h daily for 7 days as described by Nankova et al. (2000). Adrenals were isolated immediately after the final immobilization. Western analysis was used to measure changes in Fra-2 levels. A, autoradiogram depicts the changes in Fra-2 immunoreactive proteins using an antibody that recognizes the amino terminus of Fra-2. B and C, autoradiograms depict changes in Fra-2 immunoreactive proteins using an antibody that recognizes the carboxy terminus of Fra-2. The rats chronically treated with saline and administered a final injection of either saline or nicotine are designated S/S or S/N, respectively. The rats chronically treated with nicotine and administered a final injection of nicotine are designated N/N. The numbers after these designations represent the time point after the final injection at which the animals were killed. Each lane represents an adrenal medullary sample from a different rat. These results are representative of three separate experiments.

We next measured the effects of chronic nicotine injections on other fos and jun family members. Adrenal c-Fos levels increased dramatically 2 h after a single challenge injection of nicotine in animals that were treated chronically with saline for 7 days (Fig. 8, A and B). This induction of c-Fos was transient, in that c-Fos levels returned to baseline after 24 h. When animals were treated chronically with nicotine for 7 days, adrenal c-Fos levels increased only slightly at 2 h after the challenge nicotine injection and no effect was observed at 24 h. This diminished response of c-Fos after repeated nicotine injections compared with that observed after a single injection suggests that the signaling pathways leading to c-Fos induction were desensitized after this chronic drug treatment. Using an antibody that recognized all members of the fos gene family [SC-253, c-Fos (K-25); Santa Cruz Biotechnology, Inc.], we were able to observe increases in the levels of FosB and other Fras 2 h after either a single injection or repeated injections of nicotine (Fig. 8B). However, the levels of these fos proteins were not reproducibly increased 24 h after the challenge nicotine injection. Finally, we measured the levels of Jun family members in the adrenal medulla after either single or repeated nicotine injections. We were unable to detect reproducible changes in any of the jun family member proteins after either acute or chronic nicotine treatment (data not shown).

View larger version (55K): [in this window] [in a new window]   Fig. 8. Effect of chronic nicotine administration on fos protein levels in adrenal medulla. Rats were injected twice per day with saline or 1.6 mg/kg nicotine for 7 days. In the morning of the 8th day, the animals were injected with either saline or 1.6 mg/kg nicotine and adrenal glands were removed under anesthesia at different time points after this final challenge injection. The designations for the different treatment paradigms are the same as those used in Fig. 7. A, autoradiogram depicts changes in c-Fos immunoreactive proteins using an antibody [c-Fos (4), sc-52; Santa Cruz Biotechnology, Inc.] that specifically recognizes c-Fos protein and not other fos family members. B, autoradiogram depicts changes in c-Fos, fosB and Fra proteins using an antibody [c-Fos (K-25), sc-253; Santa Cruz Biotechnology, Inc.] that recognizes epitopes in common with most c-fos family members. Each lane represents an adrenal medullary sample from a different rat. These results are representative of three separate experiments.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Previous work in our laboratory has focused on the acute effects of nicotine and other cholinergic agonists on TH gene expression in rat adrenal medulla (Fossom et al., 1991a; Piech-Dumas et al., 1999; Sterling and Tank, 2001). The results of these studies have led us to conclude that TH gene transcription rate must be elevated for at least3hto produce a change in TH gene expression and that a single nicotine injection is not sufficient to produce this sustained response. However, other workers have observed induction of adrenal TH or TH mRNA when animals are chronically injected with nicotine once or twice daily over a number of days or weeks (Seidler and Slotkin, 1976; Stachowiak et al., 1988; Hiremagalur and Sabban, 1995; Serova et al., 1999). As discussed in Introduction, based on our studies on the acute effects of nicotine, this chronic response would not be expected, because the daily injections are spaced far enough apart to anticipate only transient (<3 h) stimulation of TH gene transcription rate after each injection, which should not induce TH. In this report, we have tried to clarify this discrepancy by testing the hypothesis that repeated nicotine injections administered chronically produce long-lasting changes in the adrenal medulla, leading to sustained stimulation of the TH gene and consequent induction of TH mRNA and TH protein.

TH Is Induced in the Adrenal Medulla in Response to Chronic Nicotine Administration, but not after a Single Nicotine Injection. Our results clearly show that repeated injections of nicotine administered chronically over 3 to 14 days elicit a long-lasting induction of TH protein and TH mRNA in the adrenal medulla. In contrast, a single injection of nicotine elicits only a small and transient induction of adrenal TH mRNA, which does not lead to induction of TH. To our knowledge this study is the first to closely correlate changes in adrenal TH mRNA with those in TH protein and enzyme activity in response to chronic nicotine.

With respect to the response to chronic nicotine administration, our findings confirm and extend those in previous studies (Seidler and Slotkin, 1976; Stachowiak et al., 1988; Hiremagalur and Sabban, 1995). The major discrepancy between the different studies is the response to a single injection of the drug. In the present study, a single nicotine injection elicits a small, but significant induction of adrenal TH mRNA. We did not observe this induction in our previous report (Fossom et al., 1991a), probably because we used Northern analysis and dot blots to measure TH mRNA rather than the more sensitive and highly resolving RNase protection and RT-PCR assays used in the present study. However, the induction of TH mRNA observed after a single nicotine injection is small (1.5-fold), is observed at 3 h after the injection, but not at 24 h, and does not lead to induction of TH protein. In contrast, Hiremagalur et al. (1995) show that one nicotine injection results in large and long-lasting increases in TH mRNA levels; they did not measure whether TH protein is induced. The reason for this discrepancy is not clear; however, it may be due to the use of a larger dose of nicotine (5 mg/kg subcutaneously) in their studies. Nevertheless, the results of the present study and previous work from our laboratory support the hypothesis that adrenal TH is induced only after repeated injections of nicotine either acutely over a 3-h period or chronically over at least a 3-d period.

Chronic Administration of Nicotine Leads to a Sustained Stimulation of TH Gene Transcription Rate. The most significant finding in this study is that repeated nicotine injections administered chronically over 7 days leads to a sustained stimulation of the TH gene in rat adrenal medulla. This response is very long-lasting; TH gene transcription rate is elevated for at least 7 days after the final nicotine injection. It is reasonable to conclude that the inductions of TH mRNA and TH protein that occur after chronic nicotine administration are due at least partially to this sustained stimulation of the TH gene.

Similarly, a long-lasting transcriptional response is observed in the adrenal medulla after repeated immobilization stress in both rats and mice (Osterhout et al., 1997; Nankova et al., 1999). Sabban and coworkers (Nankova et al., 1994; Sabban and Kvetnansky, 2001) were the first to note that even though a single immobilization induces adrenal TH mRNA, the response is greatly enhanced and persists for a prolonged period of time when the animals are immobilized repeatedly over 2 to 7 days. Using transgenic mice, our laboratory confirmed these results and showed that repeated immobilization stress results in a sustained stimulation of TH gene promoter activity. Subsequently, Nankova et al. (1999) demonstrated a sustained transcriptional response to repeated immobilization in rat adrenal medulla.

Taken together, these results support the hypothesis that repeated stimulation of the adrenal medulla either by stress or nicotine leads to cellular changes in adrenal chromaffin cells, resulting in enhanced transcription of the TH gene and consequent induction of TH mRNA and TH protein. It is likely that the transcription rates of other genes are also modified by this repeated daily stimulation of the adrenal medulla. It is also reasonable to speculate that similar sustained transcriptional responses to these stimuli may occur in central catecholaminergic neurons, providing the basis for some aspects of drug addiction, depression and other long-term behavioral responses.

Molecular Alterations in Adrenal Chromaffin Cells That Mediate the Sustained Transcriptional Response to Chronic Nicotine Treatment. The pharmacological and cellular changes that mediate this sustained transcriptional response remain mysterious. Acute treatment of adrenal medullary derived cells with nicotine is associated with membrane depolarization and subsequent activation of a number of different protein kinases (Eiden et al., 1984; Stachowiak et al., 1990; Hiremagalur et al., 1993). These protein kinases activate signaling pathways that stimulate the TH gene via the TH CRE and TH AP1 sites within the TH gene proximal promoter (Kumer and Vrana, 1996). Both cAMP response element-binding protein and c-Fos, transcription factors that bind to and activate CRE and AP1 sites, respectively, participate in the regulation of the TH gene in response to acute activation of these signaling pathways (Icard-Liepkalns et al., 1992; Nagamoto-Combs et al., 1997; Piech-Dumas and Tank, 1999; Piech-Dumas et al., 2001).

In animals treated chronically with saline and administered a single challenge injection of nicotine, we have observed a modest increase in TH AP1 binding at 2 h, but not at 24 h after the challenge injection. We have also observed an increase in the levels of c-Fos, as well as the levels of almost all the other fos family member proteins 2 h, but not 24 h after the challenge nicotine injection in animals chronically treated with saline. Because we have not observed changes in jun proteins, our results are consistent with the hypothesis that increased binding of fos proteins accounts for the increased TH AP1 complex formation after a single nicotine injection; however, more work is needed to test this hypothesis. These results essentially agree with those of Hiremagalur et al. (1995).

In animals treated chronically with nicotine, we have observed a sustained increase in TH AP1 binding after the final nicotine injection. This increase is modest (1.3-fold), but is reproducibly observed over many experiments. Based on the studies of Nankova et al. (2000), we hypothesized that this increase may be due to sustained induction of Fra-2, which occurs after repeated immobilization stress. However, we have not been able to detect any long-term changes in Fra-2 levels after chronic nicotine treatment. Furthermore, there are no long-term changes in any of the fos or jun proteins detected by the antibodies used in this study. These results suggest that the modest sustained increase in TH AP1 binding after chronic nicotine administration may be due to other factors that interact with fos and jun proteins, of which there are over 50 that have been identified (Chinenov and Kerppola, 2001). It is also likely that other regions of the TH gene promoter participate in this sustained response, particularly because the sustained increase in TH AP1 binding is so modest.

The lack of a sustained induction of Fra-2 is particularly intriguing, because both immobilization stress and nicotine treatment increase adrenal medullary gene transcription at least partially by stimulation of the splanchnic nerve and consequent transsynaptic activation of adrenal chromaffin cell membrane receptors. Hence, even though both long-term stress and chronic nicotine treatment elicit sustained transcriptional responses and persistent induction of TH gene expression in adrenal medulla, the mechanisms responsible for these sustained transcriptional responses may differ.

	Acknowledgements

 
We acknowledge the excellent technical assistance of Deanne Mickelsen in performing some of these experiments. We also acknowledge the assistance of Drs. T. C. Tai and Dona Lee Wong (Department of Psychiatry, Harvard Medical School) in the performance of the immobilization stress study.

	Footnotes

 
This work was supported by National Institutes of Health Grants DA05014 and NS39415 and the Smokeless Tobacco Research Council Grant 0481.

DOI: 10.1124/jpet.102.043596.

ABBREVIATIONS: TH, tyrosine hydroxylase; bp, base pair(s); RT-PCR, reverse transcriptase-polymerase chain reaction; RT, reverse transcription; PCR, polymerase chain reaction; EMSA, electrophoretic mobility shift assay; TBS-T, Tris-buffered saline/Triton X-100.

Address correspondence to: Dr. Baoyong Sun, Department of Pharmacology and Physiology, Box 711, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. E-mail: baoyong_sun{at}urmc.rochester.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Balfour DJ and Ridley DL (2000) The effects of nicotine on neural pathways implicated in depression: a factor in nicotine addiction? Pharmacol Biochem Behav 66: 79–85.[CrossRef][Medline]
Boss V, Roback JD, Young AN, Roback LJ, Weisenhorn DM, Medina-Flores R, and Wainer BH (2001) Nerve growth factor, but not epidermal growth factor, increases Fra-2 expression and alters Fra-2/JunD binding to AP-1 and CREB binding elements in pheochromocytoma (PC12) cells. J Neurosci 21: 18–26.[Abstract/Free Full Text]
Bowyer JF, Frame LT, Clausing P, Nagamoto-Combs K, Osterhout CA, Sterling CR, and Tank AW (1998) The long-term effects of amphetamine neurotoxicity on tyrosine hydroxylase mRNA and protein in aged rats. J Pharmacol Exp Ther 286: 1074–1085.[Abstract/Free Full Text]
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.[CrossRef][Medline]
Chinenov Y and Kerppola TK (2001) Close encounters of many kinds: Fos-Jun interactions that mediate transcription regulatory specificity. Oncogene 20: 2438–2452.[CrossRef][Medline]
Dani JA and De Biasi M (2001) Cellular mechanisms of nicotine addiction. Pharmacol Biochem Behav 70: 439–446.[CrossRef][Medline]
Eiden LE, Giraud P, Dave JR, Hotchkiss AJ, and Affolter HU (1984) Nicotinic receptor stimulation activates enkephalin release and biosynthesis in adrenal chromaffin cells. Nature (Lond) 312: 661–663.[CrossRef][Medline]
Fossom LH, Carlson CD, and Tank AW (1991a) Stimulation of tyrosine hydroxylase gene transcription rate by nicotine in rat adrenal medulla. Mol Pharmacol 40: 193–202.[Abstract]
Fossom LH, Sterling C, and Tank AW (1991b) Activation of tyrosine hydroxylase by nicotine in rat adrenal gland. J Neurochem 57: 2070–2077.[CrossRef][Medline]
Haass M and Kubler W (1997) Nicotine and sympathetic neurotransmission. Cardiovasc Drugs Therapy 10: 657–665.
Harlan RE and Garcia MM (1998) Drugs of abuse and immediate-early genes in the forebrain. Mol Neurobiol 16: 221–267.[Medline]
Hiremagalur B, Nankova B, Nitahara J, Zeman R, and Sabban EL (1993) Nicotine increases expression of tyrosine hydroxylase gene. Involvement of protein kinase A-mediated pathway. J Biol Chem 268: 23704–23711.[Abstract/Free Full Text]
Hiremagalur B and Sabban EL (1995) Nicotine elicits changes in expression of adrenal catecholamine biosynthetic enzymes, neuropeptide Y and immediate early genes by injection but not continuous administration. Mol Brain Res 32: 109–115.[Medline]
Icard-Liepkalns C, Faucon Biguet N, Vyas S, Robert JJ, Sassone-Corsi P, and Mallet J (1992) AP-1 complex and c-fos transcription are involved in TPA provoked and trans-synaptic inductions of the tyrosine hydroxylase gene: insights into long-term regulatory mechanisms. J Neurosci Res 32: 290–298.[CrossRef][Medline]
Kumer SC and Vrana KE (1996) Intricate regulation of tyrosine hydroxylase activity and gene expression. J Neurochem 67: 443–462.[Medline]
Kyerematen GA, Taylor LH, deBethizy JD, and Vesell ES (1988) Pharmacokinetics of nicotine and 12 metabolites in the rat. Application of a new radiometric high performance liquid chromatography assay. Drug Metab Dispos 16: 125–129.[Abstract]
Masserano JM and Weiner N (1979) The rapid activation of adrenal tyrosine hydroxylase by decapitation and its relationship to a cyclic AMP-dependent phosphorylating mechanism. Mol Pharmacol 16: 513–528.[Abstract/Free Full Text]
Mitchell SN and Gray JA (1992) Systemic administration of nicotine increases hippocampal noradrenaline release in vivo largely by an action at the locus coeruleus. Br J Pharmacol 105: 9P-0.
Nagamoto-Combs K, Piech KM, Best JA, Sun B, and Tank AW (1997) Tyrosine hydroxylase gene promoter activity is regulated by both cyclic AMP-responsive element and AP1 sites following calcium influx. Evidence for cyclic amp-responsive element binding protein-independent regulation. J Biol Chem 272: 6051–6058.[Abstract/Free Full Text]
Nankova B, Kvetnansky R, McMahon A, Viskupic E, Hiremagalur B, Frankle G, Fukuhara K, Kopin IJ, and Sabban EL (1994) Induction of tyrosine hydroxylase gene expression by a nonneuronal nonpituitary-mediated mechanism in immobilization stress. Proc Natl Acad Sci USA 91: 5937–5941.[Abstract/Free Full Text]
Nankova BB, Rivkin M, Kelz M, Nestler EJ, and Sabban EL (2000) Fos-related antigen 2: potential mediator of the transcriptional activation in rat adrenal medulla evoked by repeated immobilization stress. J Neurosci 20: 5647–5653.[Abstract/Free Full Text]
Nankova BB, Tank AW, and Sabban EL (1999) Transient or sustained transcriptional activation of the genes encoding rat adrenomedullary catecholamine biosynthetic enzymes by different durations of immobilization stress. Neuroscience 94: 803–808.[CrossRef][Medline]
Osterhout CA, Chikaraishi DM, and Tank AW (1997) Induction of tyrosine hydroxylase protein and a transgene containing tyrosine hydroxylase 5' flanking sequences by stress in mouse adrenal gland. J Neurochem 68: 1071–1077.[Medline]
Piech-Dumas KM, Best JA, Chen Y, Nagamoto-Combs K, Osterhout CA, and Tank AW (2001) The cAMP responsive element and CREB partially mediate the response of the tyrosine hydroxylase gene to phorbol ester. J Neurochem 76: 1376–1385.[CrossRef][Medline]
Piech-Dumas KM, Sterling CR, and Tank AW (1999) Regulation of tyrosine hydroxylase gene expression by muscarinic agonists in rat adrenal medulla. J Neurochem 73: 153–161.[CrossRef][Medline]
Piech-Dumas KM and Tank AW (1999) CREB mediates the cAMP-responsiveness of the tyrosine hydroxylase gene: use of an antisense RNA strategy to produce CREB-deficient PC12 cell lines. Mol Brain Res 70: 219–230.[Medline]
Sabban EL and Kvetnansky R (2001) Stress-triggered activation of gene expression in catecholaminergic systems: dynamics of transcriptional events. Trends in Neuroscience 24: 91–98.[CrossRef][Medline]
Seidler FJ and Slotkin TA (1976) Effects of chronic nicotine administration on the denervated rat adrenal medulla. Br J Pharmacol 56: 201–207.[Medline]
Serova L, Danailov E, Chamas F, and Sabban EL (1999) Nicotine infusion modulates immobilization stress-triggered induction of gene expression of rat catecholamine biosynthetic enzymes. J Pharmacol Exp Ther 291: 884–892.[Abstract/Free Full Text]
Stachowiak M, Stricker EM, Zigmond MJ, and Kaplan BB (1988) A cholinergic antagonist blocks cold stress-induced alterations in rat adrenal tyrosine hydroxylase mRNA. Brain Res 427: 193–195.[Medline]
Stachowiak MK, Goc A, Hong JS, Kaplan BB, and Stachowiak EK (1990) Neural and hormonal regulation of the tyrosine hydroxylase gene in adrenal medullary cells: participation of c-fos and AP1 factors. Mol Cell Neurosci 1: 202–213.[CrossRef]
Sterling CR and Tank AW (2001) Adrenal tyrosine hydroxylase activity and gene expression are increased by intraventricular administration of nicotine. J Pharmacol Exp Ther 296: 15–21.[Abstract/Free Full Text]
Sun B and Tank AW (2002) Overexpression of c-Fos is sufficient to stimulate tyrosine hydroxylase (TH) gene transcription in rat pheochromocytoma PC18 cells. J Neurochem 80: 295–306.[CrossRef][Medline]

This article has been cited by other articles:

				R. Kvetnansky, E. L. Sabban, and M. Palkovits Catecholaminergic Systems in Stress: Structural and Molecular Genetic Approaches Physiol Rev, April 1, 2009; 89(2): 535 - 606. [Abstract] [Full Text] [PDF]

				X. Chen, L. Xu, P. Radcliffe, B. Sun, and A. W. Tank Activation of Tyrosine Hydroxylase mRNA Translation by cAMP in Midbrain Dopaminergic Neurons Mol. Pharmacol., June 1, 2008; 73(6): 1816 - 1828. [Abstract] [Full Text] [PDF]

				J. Chevalier, P. Derkinderen, P. Gomes, R. Thinard, P. Naveilhan, P. Vanden Berghe, and M. Neunlist Activity-dependent regulation of tyrosine hydroxylase expression in the enteric nervous system J. Physiol., April 1, 2008; 586(7): 1963 - 1975. [Abstract] [Full Text] [PDF]

				K. E. Inouye, O. Chan, J. T. Y. Yue, S. G. Matthews, and M. Vranic Effects of diabetes and recurrent hypoglycemia on the regulation of the sympathoadrenal system and hypothalamo-pituitary-adrenal axis Am J Physiol Endocrinol Metab, February 1, 2005; 288(2): E422 - E429. [Abstract] [Full Text] [PDF]

				B. Sun, X. Chen, L. Xu, C. Sterling, and A. W. Tank Chronic Nicotine Treatment Leads to Induction of Tyrosine Hydroxylase in Locus Ceruleus Neurons: The Role of Transcriptional Activation Mol. Pharmacol., October 1, 2004; 66(4): 1011 - 1021. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sun, B. Articles by Tank, A. W. Search for Related Content PubMed PubMed Citation Articles by Sun, B. Articles by Tank, A. W.