Differential expression of polycytosine-binding protein isoforms in adrenal gland, locus coeruleus and midbrain.

Polycytosine-binding proteins (PCBPs) are RNA-binding proteins that participate in post-transcriptional control pathways. Among the diverse functions of these proteins is the interaction with a 27 nucleotide pyrimidine-rich domain within the 3'UTR of tyrosine hydroxylase (TH) mRNA. Mutations to this domain result in decreased stability of TH mRNA and loss of cAMP-mediated activation of TH mRNA translation. PCBPs are hypothesized to play key roles in these regulatory mechanisms. In order to further test this hypothesis, we examined the tissue distribution of PCBPs in catecholaminergic cells. Initial studies demonstrated that proteins from catecholaminergic tissues bind to TH mRNA 3'UTR sequences and these proteins have an apparent Mr of ∼ 44 kDa, which is close to the molecular sizes for PCBPs. Fluorescent immunohistochemistry and confocal microscopy was used to analyze the distribution of PCBP isoforms in TH-positive cells of the rat midbrain, locus coeruleus, and adrenal gland. Our results suggest that: (1) PCBP2 is the predominant isoform in TH-positive cells of the rat midbrain; (2) PCBP3 is the predominant isoform in TH-positive cells of the locus coeruleus; and (3) PCBP1 is the predominant isoform in the adrenal medulla. The localization of PCBP proteins to TH-positive cells in these catecholaminergic tissues is consistent with the hypothesis that PCBPs play a role in the regulation of TH expression.

Stress-induced catecholaminergic function: transcriptional and post-transcriptional control.

This review summarizes knowledge on the effects of stress on two catecholamine biosynthetic enzymes, tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT). Information is presented on differential responses of the enzymes to a variety of stressors as well as differential responses of the enzymes localized to the central nervous system vs. peripheral nervous system and tissues. Changes in mRNA and protein or activity are described, including species- and stressor-specific effects. While temporal changes in these parameters may differ for the particular stressor or enzyme, in general, maximal changes in mRNA and protein content occur at 6-8 and 24 h after stressor exposure, respectively. Elevation of TH and PNMT transcriptional activators prior to mRNA induction and nuclear run-on assays show that stress activates the genes encoding these enzymes. Yet, extents of induction of mRNA, protein and enzyme activity are often discordant depending on the stress, its duration and repetition of exposure. The extremes are concordant changes in mRNA and protein/activity vs. highly elevated mRNA with no change in protein/activity. Post-transcriptional and/or post-translational regulatory influences that may contribute to the complex effects of stress on TH, PNMT and the stress hormone epinephrine are explored.

The cAMP responsive element and CREB partially mediate the response of the tyrosine hydroxylase gene to phorbol ester.

Tyrosine hydroxylase (TH) gene promoter activity is increased in PC12 cells that are treated with the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA). Mutagenesis of either the cAMP responsive element (CRE) or the activator protein-1 element (AP1) within the TH gene proximal promoter leads to a dramatic inhibition of the TPA response. The TH CRE and TH AP1 sites are also independently responsive to TPA in minimal promoter constructs. TPA treatment results in phosphorylation of cAMP responsive element binding protein (CREB) and activation of cAMP-dependent protein kinase (PKA) in PC12 cells; hence, we tested whether CREB and/or PKA are essential for the TPA response. In CREB-deficient cells, the response of the full TH gene proximal promoter or the independent response of the TH CRE by itself to TPA is inhibited. The TPA-inducibility of TH mRNA is also blocked in CREB-deficient cells. Expression of the PKA inhibitor protein, PKI, also inhibits the independent response of the TH CRE to TPA. Our results support the hypothesis that TPA stimulates the TH gene promoter via signaling pathways that activate either the TH AP1 or TH CRE sites. Both signaling pathways are dependent on CREB and the TH CRE-mediated pathway is dependent on PKA.

Adrenal tyrosine hydroxylase activity and gene expression are increased by intraventricular administration of nicotine.

When nicotine is administered s.c. to rats, tyrosine hydroxylase (TH) enzyme activity and TH gene transcription rate are activated, and TH mRNA and TH protein are induced in adrenal medulla. In this report we test whether nicotine elicits these responses via trans-synaptic mechanisms initiated by the actions of the drug in the brain. Our results demonstrate that intraventricular (i.v.t.) administration of nicotine produces a dose-dependent activation of adrenal TH, which is blocked by i.v.t. administration of hexamethonium, but not by i.p. administration of this nicotinic acetylcholine receptor antagonist. We also show that surgical transection of the splanchnic nerve blocks the activation of adrenal TH by i.v.t.-administered nicotine. Repeated i.v.t. administration of nicotine over a 3-h period (injections spaced 30 min apart) leads to a sustained activation of adrenal TH, suggesting that this central response to nicotine does not readily desensitize. Intraventricular administration of nicotine also stimulates the TH gene transcription rate in rat adrenal medulla. When administered repeatedly i.v.t. or s.c. over 3 h, nicotine induces adrenal TH mRNA. This induction is dependent on innervation of the adrenal medulla, even when the drug is injected s.c. Our results demonstrate that the central effects of nicotine are sufficient to activate TH and induce TH gene expression in rat adrenal medulla. Furthermore, our results suggest that this centrally mediated response to nicotine is essential for the induction of adrenal TH mRNA.

The nigrostriatal dopaminergic system as a preferential target of repeated exposures to combined paraquat and maneb: implications for Parkinson's disease.

Experimental evidence supporting 1,1'-dimethyl-4,4'-bipyridinium [paraquat (PQ)] as a risk factor for Parkinson's disease (PD) is equivocal. Other agricultural chemicals, including dithiocarbamate fungicides such as manganese ethylenebisdithiocarbamate [maneb (MB)], are widely used in the same geographical regions as paraquat and also impact dopamine systems, suggesting that mixtures may be more relevant etiological models. This study therefore proposed that combined PQ and MB exposures would produce greater effects on dopamine (DA) systems than would either compound administered alone. Male C57BL/6 mice were treated twice a week for 6 weeks with intraperitoneal saline, 10 mg/kg paraquat, 30 mg/kg maneb, or their combination (PQ + MB). MB, but not PQ, reduced motor activity immediately after treatment, and this effect was potentiated by combined PQ + MB treatment. As treatments progressed, only the combined PQ + MB group evidenced a failure of motor activity levels to recover within 24 hr. Striatal DA and dihydroxyphenylacetic acid increased 1-3 d and decreased 7 d after injections. Only PQ + MB reduced tyrosine hydroxylase (TH) and DA transporter immunoreactivity and did so in dorsal striatum but not nucleus accumbens. Correspondingly, striatal TH protein levels were decreased only by combined PQ + MB 5 d after injection. Reactive gliosis occurred only in response to combined PQ + MB in dorsal-medial but not ventral striatum. TH immunoreactivity and cell counts were reduced only by PQ + MB and in the substantia nigra but not ventral tegmental area. These synergistic effects of combined PQ + MB, preferentially expressed in the nigrostriatal DA system, suggest that such mixtures could play a role in the etiology of PD.

Transient or sustained transcriptional activation of the genes encoding rat adrenomedullary catecholamine biosynthetic enzymes by different durations of immobilization stress.

The impact of stress on the transcription of rat adrenal tyrosine hydroxylase and dopamine beta-hydroxylase genes was examined. Nuclear run-on assays revealed that repeated immobilization stress elicited marked (about threefold) increases in the relative rates of transcription, being sustained for at least one day. Parallel changes in the steady-state messenger RNA levels for tyrosine hydroxylase and dopamine beta-hydroxylase were also observed. A single episode of stress triggered similar enhancements in their relative transcription rates. Depending on the duration of the stress signal, the effect on gene transcription varied in its persistence. After very short (5 min) immobilization, there was a marked transient rise in the transcription of both genes, without an accumulation of the corresponding mRNAs. Longer episodes of stress (30 min) increased the relative rate of tyrosine hydroxylase transcription for hours, causing elevations in the steady-state messenger RNA levels. In contrast, although dopamine beta-hydroxylase transcription was elevated to a similar extent by 30-min immobilization stress, the effect was transient and not reflected in significant accumulation of its messenger RNA. The results of our studies emphasize that the stress-evoked increases in the expression of the genes encoding adrenomedullary catecholamine biosynthetic enzymes involve transcriptional activation. Depending on the duration and reiteration of the stress signal, different transcriptional mechanisms may be employed.

Regulation of tyrosine hydroxylase gene expression by muscarinic agonists in rat adrenal medulla.

Tyrosine hydroxylase (TH) gene expression in the adrenal medulla is regulated by numerous stimuli via transsynaptic mechanisms. The adrenal chromaffin cell receptors that mediate this transsynaptic response remain unidentified. In this report we demonstrate that the muscarinic acetylcholine receptor agonist bethanechol stimulates the TH gene transcription rate in both innervated and denervated adrenal glands. Hence, this muscarinic response is not dependent on transsynaptic influences, suggesting that agonist occupation of adrenal chromaffin cell muscarinic receptors is sufficient to activate intracellular signaling pathways that stimulate the TH gene. When bethanechol is administered repeatedly over a 3-h interval (four injections spaced 1 h apart), TH mRNA levels are increased two- to threefold at 6 and 12 h after the initial injection of drug. It is surprising that this induction of TH mRNA does not lead to increases in TH activity or TH protein level. These results are consistent with the hypothesis that both transcriptional and posttranscriptional mechanisms must be regulated to induce TH protein and that muscarinic agonists activate only a subset of these mechanisms.

CREB mediates the cAMP-responsiveness of the tyrosine hydroxylase gene: use of an antisense RNA strategy to produce CREB-deficient PC12 cell lines.

cAMP initiates the PKA signaling cascade in rat pheochromocytoma PC12 cells, resulting in transcriptional activation of the tyrosine hydroxylase (TH) gene. This effect is mediated primarily through the cAMP responsive element (CRE), located at position -45 to -38 within the TH gene promoter. In this study, we applied an antisense RNA strategy to evaluate the role of the cAMP responsive element binding protein (CREB) in regulating TH gene expression. CREB antisense RNA expression vectors were stably introduced into PC12 cells to generate cell lines deficient in CREB. CREB protein and mRNA levels were diminished up to 90% in the stably transfected cell lines. Promoter analysis experiments demonstrated that cAMP-mediated inducibility of either TH gene proximal promoter activity or the activity of the TH CRE by itself fused upstream of a basal promoter was diminished in CREB-deficient cell lines. PKA activity in the CREB-deficient cell lines was comparable to the activity in control cell lines. In addition, neither ATF1, nor CREM proteins were significantly down-regulated in the CREB-deficient cells. Most significantly, the cAMP-inducibility of endogenous TH mRNA was completely blocked in the CREB-deficient cells, indicating that the response of the endogenous gene to cAMP was dependent on CREB. These results support the hypothesis that CREB (not other CRE-binding proteins) is the key transcription factor that is required for regulating TH gene expression in response to cAMP. Furthermore, our studies indicate that these CREB-deficient PC12 cells are excellent tools to study the participation of CREB in gene regulation.

Regulation of tyrosine hydroxylase activity by muscarinic agonists in rat adrenal medulla.

Regulation of tyrosine hydroxylase (TH) enzymatic activity in vivo by muscarinic receptor agonists in rat adrenal medulla was characterized in this study. Bethanechol and carbachol produce dose-dependent increases in rat adrenal TH activity. These increases are maximal (approximately 3-fold) using 10 mg/kg bethanechol or 0. 5 mg/kg carbachol and are totally inhibited by prior administration of 2 mg/kg atropine but not by 15 mg/kg hexamethonium. Transection of the splanchnic nerve innervating the adrenal gland leads to a loss in the activation of TH elicited by bethanechol, suggesting that transsynaptic influences are necessary for enzyme activation. When bethanechol is administered repeatedly once every hour for 3 hr (four injections), TH activity is not increased 20 min after the last injection, suggesting that the muscarinic receptor-mediated response desensitizes. In contrast, when nicotine is administered repeatedly once every hour for 3 hr, TH remains activated 20 min after the last injection. Cross-tolerance between the nicotine- and bethanechol-mediated effects on TH enzyme activity are not observed, when rats are injected repeatedly with nicotine and then administered bethanechol or vice versa. Coadministration of atropine and hexamethonium does not inhibit the nicotine-mediated activation of TH, suggesting that noncholinergic receptors participate in the transsynaptic activation of adrenal TH elicited by nicotine. Our results demonstrate that agonist occupation of muscarinic cholinergic receptors is associated with activation of TH enzyme in rat adrenal medulla. However, stimulation of the adrenal muscarinic receptor is not essential for the transsynaptic regulation of the enzyme.

Long-term effects of amphetamine neurotoxicity on tyrosine hydroxylase mRNA and protein in aged rats.

Four injections (intraperitoneal) of 3 mg/kg amphetamine (2 hr apart) produced pronounced hyperthermia and sustained decreases in dopamine levels and tyrosine hydroxylase (TH) protein levels in the striatum of 15-month-old male rats. A partial recovery of striatal dopamine levels was observed at 4 months after amphetamine. In contrast, TH mRNA and TH protein levels in the midbrain were unaffected at all time points tested up to 4 months after amphetamine treatment. The number of TH-immunopositive cells in the midbrain was also unchanged at 4 months after amphetamine, even though the number of TH-positive axons in the striatum remained dramatically decreased at this time point. Interestingly, TH-immunopositive cell bodies were observed 4 months after amphetamine in the lateral caudate/putamen, defined anteriorly by the genu of the corpus collosum and posteriorly by the junction of the anterior commissures; these striatal TH-positive cells were not observed in saline- or amphetamine-treated rats that did not become hyperthermic. In addition, low levels (orders of magnitude lower than that present in the midbrain) of TH mRNA were detected using reverse transcription-polymerase chain reaction in the striatum of these amphetamine-treated rats. Our results suggest that even though there is a partial recovery of striatal dopamine levels, which occurs within 4 months after amphetamine treatment, this recovery is not associated with increased TH gene expression in the midbrain. Furthermore, new TH-positive cells are generated in the striatum at this 4-month time point.

The THCRE2 site in the rat tyrosine hydroxylase gene promoter is responsive to phorbol ester.

Recently, our laboratory has identified a tyrosine hydroxylase (TH) gene promoter element, THCRE2 (located at -97 to -90), that is required for maximal response to cyclic AMP. In this study we test whether the THCRE2 is responsive to phorbol ester. Rat PC12 cells were transfected with a reporter gene construct, TH(-272/+27)-CAT, which is driven by the first 272 bp of the rat TH gene 5' flanking region. Treatment of transfected cells with 0.1 microM 12-O-tetradecanoylphorbol 13-acetate (TPA) elicited a 5-6-fold increase in TH gene proximal promoter activity. Mutagenesis of the THCRE2 sequence diminished TPA-responsiveness of the TH gene promoter by approximately 50%. Minimal promoter constructs driven by a single copy of the region of the TH gene that encodes the THCRE2 (from -117 to -59) were also responsive to TPA. Our results suggest that the THCRE2 is a phorbol ester-responsive element, as well as a cyclic AMP-responsive element.

Induction of tyrosine hydroxylase protein and a transgene containing tyrosine hydroxylase 5' flanking sequences by stress in mouse adrenal gland.

Prolonged stress is associated with the induction of tyrosine hydroxylase (TH) gene expression in rat adrenal medulla. We have used transgenic mice expressing a transgene encoding 4.5 kb of rat TH gene 5' flanking region fused upstream of the structural gene encoding chloramphenicol acetyltransferase (CAT) to test whether cold exposure or immobilization stress regulates TH gene expression in mouse adrenal gland. Exposure of mice to cold for 3 days increases adrenal TH protein and enzymatic activity. Cold exposure also increases adrenal TH-CAT expression two- to threefold. Immobilization stress induces mouse adrenal TH-CAT expression after either one immobilization or multiple immobilizations. TH-CAT expression increases transiently after a single immobilization, but after multiple immobilizations the induction of TH-CAT is sustained for at least 24 h. TH protein and TH enzymatic activity in mouse adrenal gland are elevated 2.8-fold and 1.5-fold, respectively, after seven immobilizations, but are not increased after either one, two, or three immobilizations. These results indicate that cold exposure and immobilization stress induce adrenal TH gene expression at least partially by stimulating the transcription rate of the TH gene. Furthermore, as observed in the rat, multiple mechanisms apparently regulate adrenal TH gene transcription rate and TH enzyme induction depending on whether mice are subjected to a single immobilization or multiple immobilizations. Our results indicate that these transgenic mice are an excellent model system to study the molecular mechanisms regulating TH gene expression in adrenal medulla.

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.

Membrane depolarization of PC12 cells using 50 mM KCl leads to induction of tyrosine hydroxylase (TH) mRNA. This induction of TH mRNA is apparently due to increased TH gene promoter activity mediated by the influx of Ca2+. In PC12 cells transiently transfected with a chimeric gene expressing chloramphenicol acetyltransferase (CAT) driven by the proximal TH gene 5'-flanking region, 50 mM KCl increases TH gene promoter activity 3-4-fold. Promoter analysis utilizing TH-CAT constructs containing mutagenized sequences indicates that this response to the depolarization-mediated influx of Ca2+ is primarily dependent on both the TH cAMP-responsive element (CRE) and TH activating protein-1 (AP1) site. Minimal promoter constructs that contain a single copy of either the TH CRE or TH AP1 site fused upstream of the TH gene basal promoter are only modestly responsive or nonresponsive, respectively, to depolarization. However, both these constructs are strongly responsive to the calcium ionophore, A23187. Gel shift assays indicate that TH AP1 complex formation is dramatically increased after treatment with either 50 mM KCl or A23187. Using antibodies to transcription factors of the Fos and Jun families, we show that the nuclear proteins comprising the inducible TH AP1 complex include c-Fos, c-Jun, JunB, and JunD. In cAMP-responsive element binding protein (CREB)-deficient cell lines that express antisense RNA complementary to CREB mRNA, the response of the TH gene promoter to cyclic AMP is dramatically inhibited, but the response to A23187 remains robust. This result indicates that transcription factors other than CREB can participate in the Ca2+-mediated regulation of the TH gene. In summary, our results support the hypothesis that regulation of the TH gene by Ca2+ is mediated by mechanisms involving both the TH CRE and TH AP1 sites and that transcription factors other than or in addition to CREB participate in this response.

Regulation of tyrosine hydroxylase gene expression by the m1 muscarinic acetylcholine receptor in rat pheochromocytoma cells.

Tyrosine hydroxylase (TH) gene transcription rate is increased in rat adrenal medulla after administration of muscarinic agonists. In order to study this muscarinic regulation of TH gene expression in more detail, we have generated a rat pheochromocytoma PC18 cell line that stably expresses the mouse m1 muscarinic acetylcholine receptor. Treatment of this cell line, designated PC18/m1-13, with carbachol leads to rapid increases in phosphatidylinositol turnover and intracellular [Ca2+]i; these increases are totally blocked by the muscarinic antagonist atropine. Carbachol produces no changes in cAMP levels or protein kinase A activity in PC18/m1-13 cells. TH mRNA levels in PC18/m1-13 cells increase approximately 3-fold after 6 h of treatment with carbachol. This induction of TH mRNA is also completely inhibited by simultaneous treatment with atropine. Transient transfection assays using a TH gene promoter-chloramphenicol acetyltransferase (TH-CAT) construct demonstrate that sequences within the most proximal 272 bp of the TH gene 5'-flanking region are responsive to carbachol in PC18/m1-13 cells. Studies using TH-CAT constructs with site-directed mutations within the TH gene promoter indicate that the responsiveness of the promoter to carbachol is mediated primarily by the cAMP response element; however, the AP1 site also participates to a lesser extent in this response. The carbachol-mediated stimulation of TH gene promoter activity is partially inhibited by down-regulation of protein kinase C (PKC) or by treatment with the Ca2+/calmodulin-dependent protein kinase inhibitor, KN62. These results are consistent with the hypothesis that agonist occupation of m1 muscarinic receptors stimulates the TH gene via signal transduction pathways that are initiated by activation of PKC and Ca2+/calmodulin-dependent protein kinase, leading to activation of transcription factors that interact with the TH CRE and AP1 sites.

The response of the tyrosine hydroxylase gene to cyclic AMP is mediated by two cyclic AMP-response elements.

Tyrosine hydroxylase (TH) gene transcription rate is stimulated by cyclic AMP in cultured rat pheochromocytoma cells. This effect is at least partially due to the interaction of transcription factors with the canonical cyclic AMP-response element (CRE) at position -45 to -38 within the TH gene promoter. In this study we test whether a region of the TH gene promoter, which is adjacent to and upstream of the canonical TH CRE, also participates in the response of the promoter to cyclic AMP. Using electrophoretic mobility shift assays, we demonstrate that nuclear proteins from rat pheochromocytoma cell lines bind to the region of the TH gene from -102 to -73. A comparison of promoter sequences indicates that sequences within this region of the TH gene are highly homologous to proenkephalin promoter sequences (between -110 and -80) designated ENKCRE-1 and ENKCRE-2. We designated the TH gene sequence homologous to ENKCRE-1 as TH E1 and the sequence homologous to ENKCRE-2 as TH E2. Competition displacement binding assays suggest that protein binding to the -102/-73 region of the TH gene is critically dependent on the TH E1 sequence. Transient transfection assays using minimal promoter constructs demonstrate that this region acts as a cyclic AMP-responsive element. Mutagenesis of the TH E1 sequence within the normal context of the TH gene proximal promoter leads to a 50% decrease in the cyclic AMP inducibility of the promoter. These results support the hypothesis that the full response of the TH gene to cyclic AMP requires both the canonical TH CRE and this newly discovered element, which we term TH CRE2.

Increased cell-cell contact stimulates the transcription rate of the tyrosine hydroxylase gene in rat pheochromocytoma PC18 cells.

Cell aggregation is one of several environmental cues that influence the expression of neurotransmitter phenotype during development. The expression of the catecholaminergic phenotype is increased in rat pheochromocytoma cells cultured at high density. In the present study we have investigated whether this cell density-mediated effect on the catecholaminergic phenotype is due to the stimulation of the tyrosine hydroxylase gene. When rat pheochromocytoma PC18 cells are cultured at high density (2 x 10(5) cells/cm2), tyrosine hydroxylase enzymatic activity and tyrosine hydroxylase protein increase two- to threefold over that observed in cells cultured at low density (1 x 10(4) cells/cm2). This increase in tyrosine hydroxylase protein observed in high-density cultures is fully accounted for by a preceding increase in tyrosine hydroxylase mRNA levels. The relative transcription rate of the tyrosine hydroxylase gene, measured using a nuclear run on assay, is two- to threefold greater in PC18 cells cultured at high density than in cells cultured at low density. Using flow cytometry, we have determined that in high-density cultures, there are approximately twice as many cells in the G0-G1 phases of the cell cycle compared with the number of G0-G1 cells observed in low-density cultures. However, when G0-G1 cells are isolated by cellular elutriation, tyrosine hydroxylase gene transcription rate remains two- to threefold greater in G0-G1 cells from high-density cultures than in G0-G1 cells from low-density cultures. These results indicate that increased cell-cell contact stimulates the transcription rate of the tyrosine hydroxylase gene, resulting in the subsequent increased expression of tyrosine hydroxylase mRNA and protein.

Regulation of tyrosine hydroxylase gene transcription rate and tyrosine hydroxylase mRNA stability by cyclic AMP and glucocorticoid.

Tyrosine hydroxylase mRNA is induced in rat pheochromocytoma PC18 cells by cAMP analogs and glucocorticoids. Previous studies have shown that these increases in tyrosine hydroxylase mRNA are due at least in part to stimulation of the tyrosine hydroxylase gene. However, the involvement of post-transcriptional mechanisms in the regulation of tyrosine hydroxylase mRNA by these inducing agents has not been investigated. In the present study, using nuclear run-on assays we show that the relative transcription rate of the tyrosine hydroxylase gene is stimulated 2-5-fold within 20 min after treatment of PC18 cells with cAMP analogs or dexamethasone and that the rate of transcription remains elevated 2-3-fold for at least 24 hr in the continual presence of these inducing agents. Pulse-labeling experiments using 4-thiouridine indicate that the rate of synthesis of tyrosine hydroxylase mRNA is increased approximately 3-fold or 10-fold after treatment with either a cyclic AMP analog or dexamethasone, respectively. These increases in rates of synthesis agree well with the fold increases in tyrosine hydroxylase mRNA levels after treatment with these inducers. Treatment of the cells with cycloheximide lowers the basal relative transcription rate of the tyrosine hydroxylase gene 2-3-fold; however, the relative transcription rate of the tyrosine hydroxylase gene is still elevated in cells treated with either dexamethasone or cAMP analogs in the presence of cycloheximide, compared with the transcription rate of the gene in cells treated with cycloheximide alone. These results indicate that protein synthesis is not required for the short term regulation of the gene by these inducing agents. The apparent t1/2 for tyrosine hydroxylase mRNA has been estimated by two different procedures, approach to steady state kinetics and pulse-chase analysis. Both procedures yield an estimated apparent t1/2 of approximately 6-9 hr for tyrosine hydroxylase mRNA under basal culture conditions. Dexamethasone does not substantially alter this apparent t1/2 value; however, cAMP appears to lower this apparent t1/2 value transiently. Our results suggest that cAMP and glucocorticoid regulate tyrosine hydroxylase mRNA levels primarily by stimulating the transcription rate of the tyrosine hydroxylase gene; however, cAMP may also regulate the stability of the mRNA for a short period of time, such that it is induced more rapidly in the cells.

Phosphorylation and activation of tyrosine hydroxylase in PC18 cells: a cell line derived from rat pheochromocytoma PC12 cells.

Treatment of rat pheochromocytoma PC18 cells (a variant subclone of PC12 cells) with forskolin produced increased activity and phosphorylation of tyrosine hydroxylase. In contrast, treatment of the PC18 cells with 56 mM K+, A23187, phorbol-12-myristate-13-acetate (PMA) or phorbol-12,13-dibutyrate (PDB) did not affect the activity and only slightly increased the phosphorylation of tyrosine hydroxylase. None of the treatments except forskolin increased cyclic AMP levels in PC18 cells. Furthermore, 45Ca2+ uptake into PC18 cells was not affected by 56 mM K+, PDB or forskolin; however, A23187 increased 45Ca2+ uptake 4-fold over basal uptake. Nevertheless, no activation and little increase in phosphorylation of tyrosine hydroxylase was observed in PC18 cells treated with A23187. When tyrosine hydroxylase levels in PC18 cells were elevated by treatment with dexamethasone, activation of tyrosine hydroxylase by 56 mM K+, PDB or A23187 was still not observed. Both purified Ca2+/calmodulin-dependent protein kinase and cyclic AMP-dependent protein kinase catalyzed the phosphorylation of tyrosine hydroxylase purified from PC18 cells in vitro. Furthermore, crude cell extracts from PC12 cells and PC18 cells possessed Ca2+/calmodulin-dependent protein kinase activity that catalyzed the phosphorylation of purified tyrosine hydroxylase. These results suggest that tyrosine hydroxylase activity in PC18 cells is regulated by a cyclic AMP-dependent mechanism. However, due to a number of abnormalities the Ca(2+)-dependent mechanisms do not result in the activation of tyrosine hydroxylase and only slightly increase the phosphorylation of the enzyme in PC18 cells.

Molecular aspects of the regulation of tyrosine hydroxylase by testosterone.

Previous studies have demonstrated that the sympathetic hypogastric ganglia (HG) are dependent upon the continued presence of testosterone for normal development and maintenance of tyrosine hydroxylase (TH) activity. The regulation of TH by testosterone has been examined further to determine whether the reduction in TH activity following castration is associated with changes in levels of TH protein and mRNA. TH protein was measured by immunotitration of HG homogenates using a TH-specific antibody, and TH-specific mRNA was detected by hybridization of dot blots of total RNA isolated from HG with a cDNA probe coding for TH. The results show that tyrosine hydroxylase activity, protein and mRNA are coordinately reduced in a graded fashion at 1, 2 and 4 weeks following castration. Testosterone replacement therapy immediately following castration prevents the decrease in TH levels. The results indicate that gonadal steroids regulate the biosynthesis of TH in the HG. Testosterone may control TH either directly by interacting with neurons of the HG, or indirectly by altering levels of trophic factors in the target tissues.