Signal transduction pathways and tyrosine hydroxylase regulation in the adrenal medulla following glucoprivation: an in vivo analysis.

The regulation of tyrosine hydroxylase (TH, the rate limiting enzyme involved in catecholamine synthesis) is critical for the acute and sustained release of catecholamines from adrenal medullary chromaffin cells, however the mechanisms involved have only ever been investigated under in vitro/in situ conditions. Here we explored the effects on, TH phosphorylation and synthesis, and upstream signalling pathways, in the adrenal medulla evoked by the glucoprivic stimulus, 2-deoxy-d-glucose (2DG) administered intraperitoneally to conscious rats. Our results show that 2DG evoked expected increases in plasma adrenaline and glucose at 20 and 60min. We demonstrated that protein kinase A (PKA) and cyclin dependent kinases (CDK) were activated 20min following 2DG, whereas mitogen activated protein kinase (MAPK) was activated later and PKC was not significantly activated. We demonstrated that phosphorylation of Ser40TH peaked after 20min whereas phosphorylation of Ser31TH was still increasing at 60min. Serine 19 was not phosphorylated in this time frame. TH phosphorylation also occurred on newly synthesized protein 24h after 2DG. Thus 2DG increases secretion of adrenaline into the plasma and the consequent rise in glucose levels. In the adrenal medulla 2DG activates PKA, CDK and MAPK, and evokes phosphorylation of Ser40 and Ser31 in the short term and induces TH synthesis in the longer term all of which most likely contribute to increased capacity for the synthesis of adrenaline.

Tyrosine hydroxylase, chromogranin A, and steroidogenic acute regulator as markers for successful separation of human adrenal medulla.

Progress in high throughput "-omic" techniques now allows the simultaneous measurement of expression levels of thousands of genes and promises the improved understanding of the molecular biology of diseases such as cancer. Detection of the dysfunction of molecular pathways in diseases requires healthy control tissue. This is difficult to obtain from pheochromocytomas (PHEOs), rare chromaffin tumors derived from adrenal medulla. The two options for obtaining adrenal tissue are: (1) whole organ removal post-mortem or during radical nephrectomy; (2) removal during PHEO surgery. Access to high quality normal adrenal tissue is limited. Removal of whole adrenals during nephrectomy is rare, because of improved surgical techniques. For adrenals removed post-mortem, the lag time to proper organ perfusion causes uncontrolled tissue degradation. Adjacent normal adrenal tissue can almost never be obtained from resected PHEOs, because they often replace the entire medulla or are well-encapsulated. If a margin of normal adrenal is attached to a resected PHEO, it seldom contains any medulla. The clean separation of medulla and cortex is further complicated, because their border is convoluted, and because adult adrenal consists of approximately 90% cortex. Thus, the quality of separation has to be evaluated with specific medullary and cortical markers. We describe the successful dissection of highly pure, medullary tissue from adrenals snap-frozen upon resection during radical nephrectomy or after brain death. Separation quality has been verified by quantitative reverse transcription with polymerase chain reaction for the medullary enzymes, tyrosine hydroxylase, and chromogranin A, and for the cortical enzyme, steroidogenic acute regulator.

Hypoxic stress-induced changes in adrenergic function: role of HIF1 alpha.

Sustaining epinephrine-elicited behavioral and physiological responses during stress requires replenishment of epinephrine stores. Egr-1 and Sp1 contribute by stimulating the gene encoding the epinephrine-synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT), as shown for immobilization stress in rats in adrenal medulla and for hypoxic stress in adrenal medulla-derived PC12 cells. Hypoxia (5% O(2)) also activates hypoxia inducible factor (HIF) 1alpha, increasing mRNA, nuclear protein and nuclear protein/hypoxia response element binding complex formation. Hypoxia and HIF1alpha over-expression also elevate PNMT promoter-driven luciferase activity in PC12 cells. Hypoxia may be limiting as HIF1alpha over-expression increases luciferase expression to no greater extent than oxygen reduction alone. HIF1alpha inducers CoCl(2) or deferoxamine elevate luciferase as well. PC12 cells harboring a HIF1alpha expression construct show markedly higher levels of Egr-1 and Sp1 mRNA and nuclear protein and PNMT mRNA and cytoplasmic protein. Inactivation of Egr-1 and Sp1 binding sites in the proximal -893 bp of PNMT promoter precludes HIF1alpha stimulation while a potential hypoxia response element (-282 bp) in the promoter shows weak HIF1alpha affinity at best. These findings are the first to suggest that hypoxia activates the proximal rat PNMT promoter primarily via HIF1alpha induction of Egr-1 and Sp1 rather than by co-activation by Egr-1, Sp1 and HIF1alpha. In addition, the rise in HIF1alpha protein leading to Egr-1 and Sp1 stimulation of PNMT appears to include HIF1alpha gene activation rather than simply prevention of HIF1alpha proteolytic degradation.

Cathepsin L participates in the production of neuropeptide Y in secretory vesicles, demonstrated by protease gene knockout and expression.

Neuropeptide Y (NPY) functions as a peptide neurotransmitter and as a neuroendocrine hormone. The active NPY peptide is generated in secretory vesicles by proteolytic processing of proNPY. Novel findings from this study show that cathepsin L participates as a key proteolytic enzyme for NPY production in secretory vesicles. Notably, NPY levels in cathepsin L knockout (KO) mice were substantially reduced in brain and adrenal medulla by 80% and 90%, respectively. Participation of cathepsin L in producing NPY predicts their colocalization in secretory vesicles, a primary site of NPY production. Indeed, cathepsin L was colocalized with NPY in brain cortical neurons and in chromaffin cells of adrenal medulla, demonstrated by immunofluorescence confocal microscopy. Immunoelectron microscopy confirmed the localization of cathepsin L with NPY in regulated secretory vesicles of chromaffin cells. Functional studies showed that coexpression of proNPY with cathepsin L in neuroendocrine PC12 cells resulted in increased production of NPY. Furthermore, in vitro processing indicated cathepsin L processing of proNPY at paired basic residues. These findings demonstrate a role for cathepsin L in the production of NPY from its proNPY precursor. These studies illustrate the novel biological role of cathepsin L in the production of NPY, a peptide neurotransmitter, and neuroendocrine hormone.

Post-transcriptional regulation of tyrosine hydroxylase expression in adrenal medulla and brain.

It is well established that long-term stress leads to induction of tyrosine hydroxylase (TH) mRNA and TH protein in adrenal medulla and brain. This induction is usually associated with stimulation of the TH gene transcription rate. However, a number of studies have reported major discrepancies between the stress-induced changes in TH gene transcription, TH mRNA, and TH protein. These discrepancies suggest that post-transcriptional mechanisms also play an important role in regulating TH expression in response to stress and other stimuli. In this report, we summarize some of our findings and literature reports that demonstrate these discrepancies in adrenal medulla, locus ceruleus, and midbrain dopamine neurons. We then describe our recent work investigating the molecular mechanisms that mediate this post-transcriptional regulation in adrenal medulla and midbrain. Our results suggest that trans-acting factors binding to the polypyrimidine-rich region of the 3' untranslated region of TH mRNA play a role in these post-transcriptional mechanisms. A hypothetical cellular model describing this post-transcriptional regulation is proposed.

Influence of polyphenolic compounds isolated from Rubus coreanum on catecholamine release in the rat adrenal medulla.

The aim of the present study was to investigate whether polyphenolic compounds isolated from wine brewed from Rubus coreanum MIQUEL (PCRC) may affect the release of catecholamine (CA) from the isolated perfused rat adrenal medulla, and to establish its mechanism of action. PCRC (20-180 microg/mL) perfused into an adrenal vein for 90 min dose- and time-dependently inhibited the CA secretory responses evoked by acetylcholine (ACh, 5.32 mM), high K+ (a direct membrane-depolarizer, 56 mM), DMPP (a selective neuronal nicotinic Nn receptor agonist, 100 microM) and McN-A-343 (a selective muscarinic M1 receptor agonist, 100 microM). Also, in the presence of PCRC (60 microg/mL), the secretory responses of CA evoked by Bay-K-8644 (a L-type dihydropyridine Ca2+ channel activator, 10 microM), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, 10 microM) were significantly reduced, respectively. In the simultaneous presence of PCRC (60 microg/mL) and L-NAME (an inhibitor of NO synthase, 30 microM), the inhibitory responses of PCRC on the CA secretion evoked by ACh, high K+, DMPP, and Bay-K-8644 were considerably recovered to the extent of the corresponding control secretion compared with the inhibitory effect of PCRC alone. Taken together, these results obtained from the present study demonstrate that PCRC inhibits the CA secretory responses from the isolated perfused adrenal gland of the normotensive rats evoked by stimulation of cholinergic (both muscarinic and nicotinic) receptors as well as by direct membrane-depolarization. It seems that this inhibitory effect of PCRC is exerted by inhibiting both the calcium influx into the rat adrenal medullary chromaffin cells and the uptake of Ca2+ into the cytoplasmic calcium store partly through the increased NO production due to the activation of nitric oxide synthase (NOS), which are at least relevant to the direct interaction with the nicotinic receptor itself. It is also thought that PCRC might be effective in prevention of cardiovascular disease.

Identification and developmental changes of aromatase and estrogen receptor expression in prepubertal and pubertal human adrenal tissues.

CONTEXT: The mechanisms of postnatal adrenal zonation remain unclear. OBJECTIVE: To provide a clue for a possible role of estrogens in adrenarche, we studied the expression of estrogen receptor (ER)alpha, ERbeta, G protein-coupled receptor (GPR)30, and cP450aromatase (cP450arom) in human adrenal tissue. DESIGN: Human adrenal tissue was collected from three postnatal age groups (Grs): Gr 1, younger than 3 months (n = 12), fetal zone involution; Gr 2, 3 months to 6 yr (n = 17), pre-adrenarche; and Gr 3, older than 6-20 yr (n = 12), post-adrenarche period. RESULTS: ERbeta mRNA in Grs 1 and 3 was higher than in Gr 2 (P < 0.05). By immunohistochemistry and laser capture microdissection followed by RT-PCR, ERbeta was expressed in zona reticularis and fetal zone, GPR30 in zona glomerulosa (ZG) and adrenal medulla, while ERalpha mRNA and protein were undetectable. cP450arom mRNA in Gr 3 was higher than in Grs 1 and 2 (P < 0.05), and localized to ZG and adrenal medulla by laser capture microdissection. cP450arom Immunoreactivity was observed in adrenal medulla in the three Grs and in subcapsular ZG of Gr 3. Double-immunofluorescence studies revealed that cP450arom and chromogranin A only colocalize in adrenal medulla of subjects younger than 18 months. In these samples, exon 1.b-derived transcript was 3.5-fold higher, while exon 1.a-, 1.c-, and 1.d-derived transcripts were 3.3-, 1.9-, and 1.7-fold lower, respectively, than in subjects older than 6 yr. CONCLUSIONS: Our results suggest that estrogens produced locally in adrenal medulla would play a role in zona reticularis functional differentiation through ERbeta. The cP450arom and GPR30 expression in subcapsular ZG, colocalizing with a high-cell proliferation index, previously reported, suggests a local GPR30-dependent estrogen action in proliferation and migration of progenitor adrenal cells.

Butyrate, a gut-derived environmental signal, regulates tyrosine hydroxylase gene expression via a novel promoter element.

Butyrate is a diet-derived, gut fermentation product with an array of effects on cultured mammalian cells including inhibition of proliferation, induction of differentiation and regulation of gene expression. We showed that physiological concentrations of butyrate can regulate transcription of tyrosine hydroxylase (TH) and preproenkephalin (ppEnk) gene in PC12 cells. In promoter deletion studies, electrophoretic mobility shift assays and by site-directed mutagenesis, we identified a novel butyrate response element (BRE) in the 5' upstream region of the rat TH gene, homologous to the previously mapped motif in the ppEnk promoter. No such enhancers were found in DBH or PNMT promoters, and both catecholamine system-related gene promoters were unaffected by butyrate. The BRE motif interacts with nuclear proteins in a sequence-specific manner, shows binding potentiation in butyrate-differentiated PC12 cells and bound protein(s) are competed away with TH-CRE oligonucleotides or by the addition of CREB-specific antibodies, suggesting involvement of CREB or CREB-related transcription factors. Moreover, single point mutation in the distal BRE abolished binding of transcription factors and reduced the response to butyrate in transient transfection studies. The canonical CRE motif of the TH promoter was also found necessary for transcriptional activation of the TH gene by butyrate. Our data identified a novel functional element in the promoter of both the TH and ppEnk genes mediating transcriptional responses to butyrate. Dietary butyrate may have an extended role in the control of catecholamine and endogenous opioid production at the level of TH and ppEnk gene transcription neuronal plasticity, cardiovascular functions, stress adaptation and behavior.

Inhibition of dopamine biosynthesis by tetrahydropapaveroline.

Tetrahydropapaveroline (THP) at 5-15 microM has been found to induce L-DOPA-induced oxidative apoptosis in PC12 cells. In this study, the inhibitory effects of THP on dopamine biosynthesis in PC12 cells and tyrosine hydroxylase (TH) activity in bovine adrenal were investigated. Treatment of PC12 cells with THP at 2.5-10 microM significantly decreased the intracellular dopamine content in a concentration-dependent manner (21.3% inhibition at THP 10 microM). The activity of TH was also inhibited by the treatment with THP at 2.5-10 microM (23.4% inhibition at 10 microM). In addition, THP had an inhibitory effect on bovine adrenal TH (IC50 value, 153.9 microM). THP exhibited uncompetitive inhibition on bovine adrenal TH with a substrate l-tyrosine with the Ki value with L-tyrosine of 0.30 mM. Treatment with L-DOPA at 20-50 microM increased the intracellular dopamine content in PC12 cells and the increase in dopamine content by L-DOPA was in part inhibited when L-DOPA (20 and 50 microM) was associated with THP at non-cytotoxic (5-10 microM) or cytotoxic (15 microM) concentration ranges. However, the reduction of dopamine content by THP (15 microM) or THP (15 microM) associated with L-DOPA (20 and 50 microM) in PC12 cells was inversed by the antioxidant N-acetyl-cysteine (0.1mM). These results indicate that THP at 5-10 microM decreases the basal dopamine content and reduces the increased dopamine content induced by L-DOPA in part by the inhibition of TH activity, and that THP at 15 microM does by oxidative stress in PC12 cells.

Expression of the noradrenaline transporter and phenylethanolamine N-methyltransferase in normal human adrenal gland and phaeochromocytoma.

Expression of the noradrenaline transporter (NAT) was examined in normal human adrenal medulla and phaeochromocytoma by using immunohistochemistry and confocal microscopy. The enzymes tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) were used as catecholamine biosynthetic markers and chromogranin A (CGA) as a marker for secretory granules. Catecholamine content was measured by using high performance liquid chromatography (HPLC). In normal human adrenal medulla (n=5), all chromaffin cells demonstrated strong TH, PNMT and NAT immunoreactivity. NAT was co-localized with PNMT and was located within the cytoplasm with a punctate appearance. Human phaeochromocytomas demonstrated strong TH expression (n=20 samples tested) but variable NAT and PNMT expression (n=24). NAT immunoreactivity ranged from absent (n=3) to weak (n=10) and strong (n=11) and, in some cases, occupied an apparent nuclear location. Unlike the expression seen in normal human adrenal medullary tissue, NAT expression was not consistently co-localized with PNMT. PNMT also showed highly variable expression that was poorly correlated with tumour adrenaline content. Immunoreactivity for CGA was colocalized with NAT within the cytoplasm of normal human chromaffin cells (n=4). This co-localization was not consistent in phaeochromocytoma tumour cells (n=7). The altered pattern of expression for both NAT and PNMT in phaeochromocytoma indicates a significant disruption in the regulation and possibly in the function of these proteins in adrenal medullary tumours.

Subunit composition and role of Na+,K+-ATPases in adrenal chromaffin cells.

Adrenal medullary (AM) cells are exposed to high concentrations of cortical hormones, one of which is a ouabain-like substance. Thus, the effects of ouabain on catecholamine secretion and distribution of Na+,K+-ATPase alpha and beta subunits in rat and guinea-pig AM cells were examined using amperometry and immunological techniques. While exposure to 1 microm ouabain did not have a marked effect on resting secretion, it induced an increase in secretion due to mobilization of Ca2+ ions that were stored during a 4 min interval between muscarine applications. Immunocytochemistry revealed that Na+,K+-ATPase alpha1 subunit-like and beta3 subunit-like immunoreactive (IR) materials were distributed ubiquitously at the cell periphery, whereas alpha2- and beta2-like IR materials were present in restricted parts of the cell periphery. The alpha1 and alpha2 subunits were mainly immunoprecipitated from AM preparations by anti-beta3 and anti-beta2 antisera, respectively. Peripheral BODIPY-FL-InsP3 binding sites were localized below membrane domains with alpha2- and beta2-like IR materials. The results indicate that in AM cells, alpha1beta3 isozymes of Na+,K+-ATPase were present ubiquitously in the plasma membrane, while alpha2beta2 isozymes were in the membrane domain closely associated with peripheral Ca2+ store sites. This close association of the alpha2beta2 isozyme with peripheral Ca2+ store sites may account for the facilitation of mobilization-dependent secretion in the presence of 1 microm ouabain.

Cyclooxygenase-2 is expressed in neuroblastoma, and nonsteroidal anti-inflammatory drugs induce apoptosis and inhibit tumor growth in vivo.

Neuroblastoma is the single most common and deadly tumor of childhood and is often associated with therapy resistance. Cyclooxygenases (COXs) catalyze the conversion of arachidonic acid to prostaglandins. COX-2 is up-regulated in several adult epithelial cancers and is linked to proliferation and resistance to apoptosis. We detected COX-2 expression in neuroblastoma primary tumors and cell lines but not in normal adrenal medullas from children. Treatment of neuroblastoma cells with nonsteroidal anti-inflammatory drugs, inhibitors of COX, induced caspase-dependent apoptosis via the intrinsic mitochondrial pathway. Treatment of established neuroblastoma xenografts in nude rats with the dual COX-1/COX-2 inhibitor diclofenac or the COX-2-specific inhibitor celecoxib significantly inhibited tumor growth in vivo (P < 0.001). In vitro, arachidonic acid and diclofenac synergistically induced neuroblastoma cell death. This effect was further pronounced when lipooxygenases were simultaneously inhibited. Proton magnetic resonance spectroscopy ((1)H MRS) of neuroblastoma cells treated with COX inhibitors demonstrated accumulation of polyunsaturated fatty acids and depletion of choline compounds. Thus, (1)H MRS, which can be performed with clinical magnetic resonance scanners, is likely to provide pharmacodynamic markers of neuroblastoma response to COX inhibition. Taken together, these data suggest the use of nonsteroidal anti-inflammatory drugs as a novel adjuvant therapy for children with neuroblastoma.

Angiotensin II AT1 receptor blockade prevents gastric ulcers during cold-restraint stress.

Cold-restraint stress reduces gastric blood flow and produces acute gastric ulcers. We studied the role of Angiotensin II (Ang II) on gastric blood flow and gastric ulceration during stress. Spontaneously hypertensive rats, a stress-sensitive strain, were pretreated for 14 days with the AT(1) receptor antagonist candesartan before cold-restraint stress. AT(1) blockade increased gastric blood flow 40% to 50%; prevented gastric ulcer formation by 70% to 80%; reduced the increase in adrenomedullary epinephrine and TH mRNA without preventing the stress-induced increase in adrenal corticosterone; decreased the stress-induced expression of tumor necrosis factor alpha (TNF-alpha) and adhesion protein ICAM-1 in arterial endothelium, and neutrophil infiltration in the gastric mucosa; and decreased PGE(2) content. AT(1) receptor blockers prevent stress-induced ulcerations by a combination of gastric blood flow protection, decreased sympathoadrenal activation, anti-inflammatory effects with reduction in TNF-alpha, and ICAM-1 expression, leading to reduced neutrophil infiltration while maintaining the protective glucocorticoid effects and PGE(2) release. Ang II has a crucial role, through stimulation of AT(1) receptors, in the production and progression of stress-induced gastric injury, and AT(1) receptor antagonists could be of therapeutic benefit.

Catecholamine synthesizing enzymes and their modulation by immobilization stress in knockout mice.

The c-fos knockout mice (c-fos KO) and corticotropin-releasing hormone knockout mice (CRH KO) can serve as interesting models for studying mechanisms involved in response of the organism to stress, focused mainly on the hypothalamic-pituitary-adrenal (HPA) axis and sympathoadrenal system (SAS). The present study focused on the investigation of changes in gene expression of catecholamine biosynthesizing enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT) in adrenal medulla of c-fos KO and CRH KO mice stressed by immobilization. Levels of TH, DBH, and PNMT mRNA were determined by reverse transcription-polymerase chain reaction (RT-PCR). Single immobilization for 2 h significantly increased adrenomedullary TH, DBH, and PNMT mRNA levels in both c-fos KO and wild-type (WT) mice compared to unstressed controls. In CRH KO mice, PNMT gene expression was not increased to the same extent after single, but especially after repeated immobilization as in WT mice, in contrast to TH and DBH mRNA levels. Thus, our data indicate that CRH deficiency can influence the PNMT mRNA level in adrenal medulla during stress, confirming the idea that the HPA axis plays the crucial role in PNMT gene regulation in mice. On the other hand, c-Fos protein probably does not play a crucial role in TH, DBH, and PNMT gene expression in adrenal medulla under stress conditions.

Adrenal medullary function and expression of catecholamine-synthesizing enzymes in mice with hypothalamic obesity.

The mechanisms underlying the onset of obesity are complex and not completely understood. An imbalance of autonomic nervous system has been proposed to be a major cause of great fat deposits accumulation in hypothalamic obesity models. In this work we therefore investigated the adrenal chromaffin cells in monosodium glutamate (MSG)-treated obese female mice. Newborn mice were injected daily with MSG (4 mg/g body weight) or saline (controls) during the first five days of life and studied at 90 days of age. The adrenal catecholamine content was 56.0% lower in the obese group when compared to lean controls (P < 0.0001). Using isolated adrenal medulla we observed no difference in basal catecholamine secretion percentile between obese and lean animals. However, the percentile of catecholamine secretion stimulated by high K+ concentration was lower in the obese group. There was a decrease in the tyrosine hydroxylase enzyme expression (57.3%, P < 0.004) in adrenal glands of obese mice. Interestingly, the expression of dopamine beta-hydroxylase was also reduced (47.0%, P < 0.005). Phenylethanolamine N-methyltransferase expression was not affected. Our results show that in the MSG model, obesity status is associated with a defective adrenal chromaffin cell function. We conclude that in MSG obesity the low total catecholamine content is directly related to a decrease of key catecholamine-synthesizing enzymes, which by its turn may lead to a defective catecholamine secretion.

The effect of adrenomedullin (ADM) on tyrosine hydroxylase (TH) enzyme activity and blood pressure in cold exposed rats.

It is known that, under stress conditions the hypothalamo-pituitary-adrenal (HPA) axis is stimulated and catecholamine production is increased. Adrenomedullin (ADM) is a novel peptide that elicits a long-vasorelaxation, and participates in blood pressure regulation via different mechanisms. In the present study, we investigated the administration of ADM on tyrosine hydroxylase (TH) enzyme activity in cold exposed rats. Four groups of Sprague-Dawley rats were studied for their TH enzyme activity in the adrenal medulla and hypothalamus. In addition to measuring blood pressure in these rats, TH enzyme activity in both the adrenal medulla and hypothalamus were examined in four groups of Sprague-Dawley rats: animals exposed to room temperature and cold stress (8 masculine C, 48 h), and rats injected with ADM (1.0 nmol/kg, i.v.) alone and/or together with cold stress. TH activity was shown to be increased in cold treated groups and decreased in ADM and ADM + cold stress group. Our findings appear to suggest that external ADM application caused an opposite effect on the same system in rats, decreasing the activity of tyrosine hydroxylase (TH) enzyme activity. Furthermore, externally applied ADM was shown to produce its expected hypotensive effect in cold-stressed rats. Our results suggest that a possible explanation for the effects of ADM is that, the uptake of ADM under cold stress may effect TH activity in studied tissues.

Chronic nicotine treatment leads to sustained stimulation of tyrosine hydroxylase gene transcription rate in rat adrenal medulla.

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.

Species differences in the expression of multiple tyrosine hydroxylase protein isoforms.

In subprimates, a single form of tyrosine hydroxylase (TH) is expressed, whereas two TH protein isoforms have been identified in monkeys and four isoforms have been demonstrated in humans. In order to establish the evolutionary pattern/emergence of these multiple TH isoforms, adrenal medullae from different mammalian species were analyzed by blot immunolabeling using pan-specific TH antibodies and antibodies specific to each of the four human TH isoforms. The expression of multiple TH isoforms was primate specific and restricted to anthropoids: only a single TH isoform was detected in adrenal medullae from several subprimate and prosimian species (six species from four families), while two TH isoforms were found in all of the anthropoid species studied. The presence of four TH isoforms could only be demonstrated in human specimens. Contrary to previous suggestions, only one TH protein isoform was found in rats and only four TH protein isoforms were found in humans.

Comparative tissue distribution of the processing enzymes "prohormone thiol protease," and prohormone convertases 1 and 2, in human PTHrP-producing cell lines and mammalian neuroendocrine tissues.

Peptide hormones are generated by proteolytic processing of their respective protein precursors by several prohormone processing proteases. The peptide hormone PTHrP is widely expressed in normal and malignant tissues, where proPTHrP undergoes proteolytic processing to generate PTHrP peptides with distinct biological actions. In this study, the tissue distribution of the prohormone processing enzymes PTP, PC1, and PC2 were compared by immunohistochemistry in human PTHrP-producing cancer cell lines, and in mammalian neuroendocrine and other tissues from rat and bovine that contain peptide hormones. PTP, PC1, and PC2 were prominently expressed in PTHrP-expressing human cancer cell lines originating from tumors of the breast, lung, prostate, as well as lymphoma. These processing enzymes also showed significant expression in normal mammalian neuroendocrine tissues from bovine and rat, including pituitary, hypothalamus, adrenal medulla, pancreas, and other tissues. Most neuroendocrine tissues contained prominent levels of at least two of the three processing enzymes examined, and all tissues contained at least one of these three enzymes. Differential expression of processing enzyme proteins was also demonstrated by Western blots. The differential expression of PTP, PC1, and PC2 observed in certain cancer and normal neuroendocrine cell types postulates selective roles for these processing enzymes in different tissues for generating biologically active peptide hormones. These results support the importance of these processing enzymes in their hypothesized roles in prohormone processing.