Induction of tyrosine hydroxylase mRNA by nicotine in rat midbrain is inhibited by mifepristone

Abstract

Repeated nicotine administration induces tyrosine hydroxylase (TH) mRNA in rat midbrain. In this study we investigate the mechanisms responsible for this response using two models of midbrain dopamine neurons, rat ventral midbrain slice explant cultures and mouse MN9D cells. In both models nicotine stimulates TH gene transcription rate in a dose-dependent manner. However, this stimulation is short-lived, lasting for 1 h, but less than 3 h, and is not sufficient to induce TH mRNA or TH protein. Nicotine elevates circulating glucocorticoids, which induce TH expression in some model systems. We tested the hypothesis that the effect of nicotine on midbrain TH mRNA is mediated by the glucocorticoid receptor. When rats are administered the glucocorticoid receptor antagonist mifepristone, the induction of TH mRNA by nicotine in both substantia nigra and ventral tegmentum is inhibited. Furthermore, the glucocorticoid receptor agonist dexamethasone stimulates TH gene transcription for sustained periods of time in both midbrain slices and MN9D cells, leading to induction of TH mRNA and TH protein. Our results are consistent with the hypothesis that nicotine induces TH mRNA in midbrain by elevating glucocorticoids, which then act on glucocorticoid receptors in dopamine neurons leading to transcriptional activation of the TH gene.

Figure 1. Nicotine stimulated TH gene transcription in midbrain slice explant cultures and MN9D cells

(A) Ventral midbrain slices from 7–10 day old mouse pups were cultured for 1 day in vitro and then treated with different concentrations of nicotine for 1 hr. MN9D cells were treated with different concentrations of nicotine for 15 min. Total cellular RNA was extracted and semiquantitative RT-PCR was used to measure changes in TH RNA primary transcripts that expressed intron 2 sequences. The levels of 28S rRNA were also measured using this assay and these values were used for normalization purposes. The autoradiogram depicts representative assays of RNA isolated from either midbrain explant cultures or MN9D cells. RNA from each sample was assayed in the presence (+RT) or absence (−RT) of RT, to verify that the amplified cDNA products were derived from RNA, not genomic DNA. (B) The bar graph represents the means ± SE from 4 slice cultures or 6 dishes of MN9D cells. (C) Ventral midbrain slice cultures or MN9D cells were treated with 100 uM nicotine for different periods of time and TH RNA primary transcripts were measured as described above. The bar graph represents the means ± SE from 3–4 slice cultures and 3–6 dishes of MN9D cells. (D) Ventral midbrain slice cultures or MN9D cells were treated for 1 hr with 100 uM nicotine ± 1 uM methyllycaconitine (MLA). The bar graph represents means ± SE from 3–5 slice cultures or dishes of MN9D cells. a: p < .05 compared to controls.

Figure 2. Nicotine did not induce TH mRNA, TH protein or TH activity in midbrain slice explant cultures or MN9D cells

(A) Ventral midbrain slices from 7–10 day old mouse pups were cultured for 1 day in vitro and then treated with 100 uM nicotine for different periods of time up to 48 hr. MN9D cells were treated with 100 uM nicotine for similar durations of time. Total cellular RNA was extracted and semiquantitative RT-PCR was used to measure changes in TH RNA levels; 28S rRNA levels were also measured using this assay and these values were used for normalization purposes. The autoradiogram depicts representative assays of RNA isolated from either midbrain slice cultures or MN9D cells. (B) The bar graph depicts the means ± SE from 3 explant cultures or 3 dishes of MN9D cells. These results are from a single experiment, and similar results were obtained from at least two other experiments. (C) Midbrain slice explant cultures were treated with 100 uM nicotine for 24, 36 or 48 hr and then assayed for TH protein using western analysis and TH activity using saturating cofactor concentration (4 mM 6MPH₄). The data represent means ± SE from 9–12 cultures. (D) MN9D cells were treated with 100 uM nicotine for the designated periods of time and TH protein and TH activity were assayed as in panel C. The data represent the means ± SE from 6–12 dishes.

Figure 3. Mifepristone inhibited the induction of TH mRNA elicited by nicotine administration to rats in adrenal medulla and midbrain

Rats were injected subcutaneously with 0.8 mg/kg nicotine twice per day for 3 days with injections spaced ~12 hr apart. On the morning of the fourth day, the rats were injected one more time with 0.8 mg/kg nicotine and euthanized using an overdose of sodium pentobarbital (150 mg/kg) 3 hr after this final injection. Control animals were injected with the same volume of saline (1 ml/kg) at the same time points. When appropriate, 10 mg/kg mifepristone (MIF) (suspended in sesame oil) was injected subcutaneously 15 min prior to each injection of either saline or nicotine. The same volume of sesame oil (1 ml/kg) was used to inject animals that were not administered the GR antagonist. Adrenal glands were removed while the animals were anesthetized and then the animals were decapitated and SN and VTA were dissected. TH mRNA, DAT mRNA and GAPDH mRNA were measured using quantitative RT-PCR. GAPDH mRNA values were used to normalize TH mRNA values in adrenal samples, whereas DAT mRNA values were used for normalization in midbrain samples. The data represent the means ± SE from 10 adrenal samples, 7–10 SN samples and 4–5 VTA samples. The experiments were performed at least two times with similar results. a: p < .05 compared to controls

Figure 4. Dexamethasone stimulated TH gene transcription and induced TH mRNA and TH protein in midbrain slice explant cultures

Ventral midbrain slices from 7–10 day old rat pups were cultured for 1 day in vitro and then treated with 0.1 uM dexamethasone for 24 hr. The relative levels of TH mRNA, TH RNA primary transcripts and 28S rRNA were measured using semiquantitative RT-PCR. TH protein was measured using western analysis. Autoradiograms depicting representative assays are presented. TH activity was assayed using 4 mM 6MPH₄. The bar graph represents the means ± SE from 3–5 slice cultures. a: p < .05 compared to control values

Figure 5. Dexamethasone stimulated TH gene transcription and induced TH mRNA and TH protein in MN9D cells

(A) MN9D cells were treated with different concentrations of dexamethasone for 24 hr and TH mRNA and TH primary transcripts were measured using semiquantitative RT-PCR (for the autoradiogram) and quantitative RT-PCR (for the complete concentration-response curve). The data represent the means ± SE from 3 dishes. Dexamethasone concentrations of 30 uM or greater yielded significant increases in both TH mRNA and TH RNA primary transcripts (p < .05). (B) MN9D cells were treated with 0.1 uM dexamethasone for different periods of time and TH mRNA and TH RNA primary transcripts were measured using quantitative RT-PCR. The data represent the means ± SE from 6 dishes. (D) MN9D cells were treated with 0.1 uM dexamethasone for different periods of time and TH protein was measured using western analysis and TH activity was assayed using 4 mM 6MPH₄. The data represents the means ± SE from 3 dishes. (D) MN9D cells were treated with 0.1 uM dexamethasone in the presence or absence of different concentrations of mifepristone for 24 hr. TH RNA primary transcripts expressing genomic intron-2 sequences (filled squares) and mature TH mRNA transcripts (filled circles) were measured using quantitative RT-PCR. GAPDH mRNA transcripts were also measured using this assay and these values were used for normalization purposes. Values for TH RNA primary gene transcripts in control and dexamethasone-treated cells (without mifepristone) and expressed as fold-increases) were 1.0 ± 0.1 and 1.9 ± 0.2, respectively (p < .05). Values for TH mRNA transcripts in control and dexamethasone-treated cells were 1.0 ± 0.1 and 2.5 ± 0.2, respectively (p < .01). The data represent the means ± SE from 5–6 dishes. a: p < .05 compared to control values.