Variations in adrenal gland medulla and dopamine effects induced by the lack of Irs2.

The adrenomedullary chromaffin cells' hormonal pathway has been related to the pathophysiology of diabetes mellitus. In mice, the deletion of insulin receptor substrate type 2 (Irs2) causes peripheral insulin resistance and reduction in ß-cell mass, leading to overt diabetes, with gender differences on adrenergic signaling. To further unravel the relevance of Irs2 on glycemic control, we analyzed in adult Irs2 deficient (Irs2-/-) mice, of both sexes but still normoglycemic, dopamine effects on insulin secretion and glycerol release, as well as their adrenal medulla by an immunohistochemical and morphologic approach. In isolated islets, 10 µM dopamine significantly inhibited insulin release in wild-type (WT) and female Irs2-/- mice; however, male Irs2-/- islets were insensitive to that catecholamine. Similarly, on isolated adipocytes, gender differences were observed between WT and Irs2-/- mice in basal and evoked glycerol release with crescent concentrations of dopamine. By immunohistochemistry, reactivity to tyrosine hydroxylase (TH) in female mice was significantly higher in the adrenal medulla of Irs2-/- compared to WT; although no differences for TH-immunopositivity were observed between the male groups of mice. However, compared to their corresponding WT animals, adrenomedullary chromaffin cells of Irs2-/- mice showed a significant decrease in the cellular and nuclear areas, and even in their percentage of apoptosis. Therefore, our observations suggest that, together with gender differences on dopamine responses in Irs2-/- mice, disturbances in adrenomedullary chromaffin cells could be related to deficiency of Irs2. Accordingly, Irs2 could be necessary for adequate glucose homeostasis and maintenance of the population of the adrenomedullary chromaffin cells.

Cardiac sympathetic denervation in 6-OHDA-treated nonhuman primates.

Cardiac sympathetic neurodegeneration and dysautonomia affect patients with sporadic and familial Parkinson's disease (PD) and are currently proposed as prodromal signs of PD. We have recently developed a nonhuman primate model of cardiac dysautonomia by iv 6-hydroxydopamine (6-OHDA). Our in vivo findings included decreased cardiac uptake of a sympathetic radioligand and circulating catecholamines; here we report the postmortem characterization of the model. Ten adult rhesus monkeys (5-17 yrs old) were used in this study. Five animals received 6-OHDA (50 mg/kg i.v.) and five were age-matched controls. Three months post-neurotoxin the animals were euthanized; hearts and adrenal glands were processed for immunohistochemistry. Quantification of immunoreactivity (ir) of stainings was performed by an investigator blind to the treatment group using NIH ImageJ software (for cardiac bundles and adrenals, area above threshold and optical density) and MBF StereoInvestigator (for cardiac fibers, area fraction fractionator probe). Sympathetic cardiac nerve bundle analysis and fiber area density showed a significant reduction in global cardiac tyrosine hydroxylase-ir (TH; catecholaminergic marker) in 6-OHDA animals compared to controls. Quantification of protein gene protein 9.5 (pan-neuronal marker) positive cardiac fibers showed a significant deficit in 6-OHDA monkeys compared to controls and correlated with TH-ir fiber area. Semi-quantitative evaluation of human leukocyte antigen-ir (inflammatory marker) and nitrotyrosine-ir (oxidative stress marker) did not show significant changes 3 months post-neurotoxin. Cardiac nerve bundle α-synuclein-ir (presynaptic protein) was reduced (trend) in 6-OHDA treated monkeys; insoluble proteinase-K resistant α-synuclein (typical of PD pathology) was not observed. In the adrenal medulla, 6-OHDA monkeys had significantly reduced TH-ir and aminoacid decarboxylase-ir. Our results confirm that systemic 6-OHDA dosing to nonhuman primates induces cardiac sympathetic neurodegeneration and loss of catecholaminergic enzymes in the adrenal medulla, and suggests that this model can be used as a platform to evaluate disease-modifying strategies aiming to induce peripheral neuroprotection.

Neurobiological consequences of acute footshock stress: effects on tyrosine hydroxylase phosphorylation and activation in the rat brain and adrenal medulla.

Stress activates selected neuronal systems in the brain and this leads to activation of a range of effector systems. Our aim was to investigate some of the relationships between these systems under basal conditions and over a 40-min period in response to footshock stress. Specifically, we investigated catecholaminergic neurons in the locus coeruleus (LC), ventral tegmental area and medial prefrontal cortex (mPFC) in the brain, by measuring tyrosine hydroxylase (TH) protein, TH phosphorylation and TH activation. We also measured the effector responses by measuring plasma adrenocorticotrophic hormone, corticosterone, glucose and body temperature as well as activation of adrenal medulla protein kinases, TH protein, TH phosphorylation and TH activation. The LC, ventral tegmental area and adrenal medulla all had higher basal levels of Ser19 phosphorylation and lower basal levels of Ser31 phosphorylation than the mPFC, presumably because of their cell body versus nerve terminal location, while the adrenal medulla had the highest basal levels of Ser40 phosphorylation. Ser31 phosphorylation was increased in the LC at 20 and 40 min and in the mPFC at 40 min; TH activity was increased at 40 min in both tissues. There were significant increases in body temperature between 10 and 40 min, as well as increases in plasma adrenocorticotropic hormone at 20 min and corticosterone and glucose at 20 and 40 min. The adrenal medulla extracellular signal-regulated kinase 2 was increased between 10 and 40 min and Ser31 phosphorylation was increased at 20 min and 40 min. Protein kinase A and Ser40 phosphorylation were increased only at 40 min. TH activity was increased between 20 and 40 min. TH protein and Ser19 phosphorylation levels were not altered in any of the brain regions or adrenal medulla over the first 40 min. These findings indicate that acute footshock stress leads to activation of TH in the LC, pre-synaptic terminals in the mPFC and adrenal medullary chromaffin cells, as well as changes in activity of the hypothalamic-pituitary-adrenal axis.

Modulation of catecholamine-synthesizing enzymes in adrenal medulla and stellate ganglia by treadmill exercise of stressed rats.

The sympatho-adrenal system represents one of the main systems involved in the response to stressful events because its stress-induced activation results in an increased release of catecholamines. Exercise training acts as an important modulator of sympatho-adrenal system, adrenal medulla and stellate ganglia being two components of this system. This study aimed at investigating physical exercise-related changes in gene expression of catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine-ß-hydroxylase (DBH) and phenylethanolamine N-methyltransferase in the adrenal medulla and stellate ganglia of chronically psychosocially stressed adult rats exposed daily to 20-min treadmill exercise for 12 weeks, using TaqMan RT-PCR assay. Chronic psychosocial stress decreased gene expression of the examined enzymes in the adrenal medulla and treadmill exercise did not lead to further modulation of the corresponding gene expression. On the other hand, chronic psychosocial stress produced a significant increase of TH (about 51%) and DBH (about 103%) gene expression in stellate ganglia, while treadmill exercise decreased gene expression of these enzymes to control levels in psychosocially stressed rats. Our data indicate that treadmill exercise leads to a decreased gene transcription of catecholamine biosynthetic enzymes in stellate ganglia and attenuation of cardiac noradrenaline production in stressful situations. Reduction of catecholamine synthesis in stellate ganglia may be linked to the beneficial effects of treadmill exercise on cardiovascular system in stressed animals.

Lifelong caloric restriction prevents age-induced oxidative stress in the sympathoadrenal system of Fischer 344 x Brown Norway rats.

Aging is associated with oxidative damage and an imbalance in redox signaling in a variety of tissues, yet little is known about the extent of age-induced oxidative stress in the sympathoadrenal system. Lifelong caloric restriction has been shown to lower levels of oxidative stress and slow the aging process. Therefore, the aims of this study were twofold: (1) to investigate the effect of aging on oxidative stress in the adrenal medulla and hypothalamus and (2) determine if lifelong 40% caloric restriction (CR) reverses the adverse effects of age-induced oxidative stress in the sympathetic adrenomedullary system. Adult (18months) and very old (38months) male Fischer 344 x Brown Norway rats were divided into ad libitum or 40% CR groups and parameters of oxidative stress were analyzed in the adrenal medulla and the hypothalamus. A significant age-dependent increase in lipid peroxidation (+20%, P<0.05) and tyrosine nitration (+111%, P<0.001) were observed in the adrenal medulla while age resulted in a reduction in the protein expression of key antioxidant enzymes, CuZnSOD (-27%, P<0.01) and catalase (-27%, P<0.05) in the hypothalamus. Lifelong CR completely prevented the age-induced increase in lipid peroxidation in the adrenal medulla and restored the age-related decline in antioxidant enzymes in the hypothalamus. These data indicate that aging results in a significant increase in oxidative stress in the sympathoadrenal system. Importantly, lifelong CR restored the age-related changes in oxidative stress in the adrenal medulla and hypothalamus. Caloric restriction could be a potential non-pharmacological intervention to prevent increased oxidative stress in the sympathetic adrenomedullary system with age.

Heterogeneous levels of oxidative phosphorylation enzymes in rat adrenal glands.

Mitochondria are organelles that produce ATP and reactive oxygen species, which are thought to be responsible for a decline in physiological function with aging. In this study, we morphologically and biochemically examined mitochondria in the rat adrenal gland. Immunohistochemistry showed that the rank order for intensity of immunolabelling for complex IV was zona reticularis > zona fasciculata >> adrenal medulla, whereas for complex V α and ß subunits, it was zona fasciculata > zona reticularis and adrenal medulla. The immunolabelling for complex I was homogeneous in the adrenal gland. The difference in immunolabelling between complexes I and IV indicates that the ratio of levels of complex I to that of complex IV in the zona reticularis was smaller than that in the zona fasciculata and the adrenal medulla. Electron microscopy revealed that aging rats had zona reticularis cells with many lysosomes and irregular nuclei. The result suggests that the level of proteins involved in oxidative phosphorylation is coordinated within the complex, but differs between the complexes. This might be responsible for degeneration of zona reticularis cells with aging.

Effects of repeated maprotiline and fluoxetine treatment on gene expression of catecholamine synthesizing enzymes in adrenal medulla of unstressed and stressed rats.

1 Repeated maprotiline (a noradrenaline reuptake inhibitor) and fluoxetine (a serotonin reuptake inhibitor) treatment on gene expression of catecholamine biosynthetic enzymes were examined in adrenal medulla of unstressed control and chronic unpredictable mild stressed rats. 2 Maprotiline did not change gene expression of catecholamine biosynthetic enzymes in control and stressed rats. 3 Fluoxetine increased gene expression of tyrosine hydroxylase (TH) and dopamine-ß-hydroxylase (DBH), but did not phenylethanolamine N-methyltransferase in both unstressed and chronic unpredictable mild stressed animals. 4 In conclusion, we have demonstrated that repeated administration of fluoxetine enhanced gene transcription of TH and DBH and subsequently stimulates noradrenaline synthesis in adrenal medulla of control and stressed rats.

Immunohistochemical localization of catecholamine biosynthetic enzymes in the adrenal gland of the domestic fowl (Gallus domesticus).

The present study investigated the cellular localization of 3 catecholamine biosynthetic enzymes, tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT) to identify and analyze the localization of norepinephrine (NE) and epinephrine (E) cells in the adrenal gland in the chicken using peroxidase-antiperoxidase immunohistochemical techniques. Tyrosine hydroxylase immunoreactivity (IR) was observed in almost all adrenal medullary cells of the adult chicken. Dopamine beta-hydroxylase IR coincided with that of TH. Many medullary cells also exhibited PNMT IR, but PNMT-immunonegative cells were also observed. Tyrosine hydroxylase IR was localized in the E- and NE-containing cells, but PNMT IR was localized only in the E-containing cells. Approximately 69% of medullary cells were E-containing, and the remaining were NE-containing cells. The ratio of E- and NE-containing cells between the subcapsular and central zone was statistically significant (P < 0.01). Although cortical cells of the adrenal gland did not show TH-, DBH-, or PNMT-positive reactions, ganglia close to the adrenal gland showed TH, DBH, and PNMT immunoreactivities. These findings indicated the cellular localization of 3 catecholamine-biosynthesizing enzymes in chicken adrenal medulla and suggest that the majority of medullary cell are E-containing cells. The ratio of E cells to NE cells varies among the 3 zones in the adrenal glands of the chicken.

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.

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.

Adrenal GRK2 lowering is an underlying mechanism for the beneficial sympathetic effects of exercise training in heart failure.

Exercise training has been reported to exert beneficial effects on cardiac function and to reduce morbidity and mortality of chronic heart failure (HF). Augmented sympathetic nervous system (SNS) activity, leading to elevated circulating catecholamine (CA) levels, is a hallmark of chronic HF that significantly aggravates this disease. Exercise training has been shown to also reduce SNS overactivity in HF, but the underlying molecular mechanism(s) remain unidentified. We recently reported that adrenal G protein-coupled receptor kinase-2 (GRK2), an enzyme that regulates the sympathoinhibitory alpha(2)-adrenoceptors (alpha(2)-ARs) present in the CA-producing adrenal medulla, is upregulated in HF, contributing to the chronically elevated CA levels and SNS activity of the disease. In the present study, we tested whether exercise training can affect the adrenal GRK2-alpha(2)-AR-CA production system in the context of HF. For this purpose, a 10-wk-long exercise training regimen of adult male Sprague-Dawley rats starting at 4 wk postmyocardial infarction (post-MI) was employed, and examination at the end of this treatment period revealed significant amelioration of beta-AR-stimulated contractility in response to exercise training, accompanied by cardiac GRK2 reduction and restoration of circulating plasma CA levels. Importantly, adrenal GRK2 expression (72 + or - 5% reduction vs. post-MI untrained) and alpha(2)-AR number were also restored after exercise training in post-MI animals. These results suggest that exercise training restores the adrenal GRK2-alpha(2)-AR-CA production axis, and this might be part of the mechanism whereby this therapeutic modality normalizes sympathetic overdrive and impedes worsening of the failing heart.

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.

Effect of clonidine on tyrosine hydroxylase activity in the adrenal medulla and brain of spontaneously hypertensive rats.

In the present study, we evaluated the effect of the alpha(2)-adrenoceptor agonist clonidine on tyrosine hydroxylase activity in adrenal medulla and brain of spontaneously hypertensive rats and Wistar Kyoto rats. Six-week-old animals were treated with clonidine (100 microg/kg body weight, daily, i.p.) for 4 weeks. Treatment with clonidine significantly reduced mean arterial blood pressure in spontaneously hypertensive rats to values similar to normotensive Wistar Kyoto rats. In the adrenal medulla of spontaneously hypertensive rats, clonidine treatment produced a significant increase in tyrosine hydroxylase activity with higher V(max) values and no changes in K(M) values. This effect was accompanied by a significant increase in tyrosine hydroxylase protein expression and of noradrenaline and adrenaline levels. In the brain of spontaneously hypertensive rats, treatment with clonidine produced a significant decrease in tyrosine hydroxylase activity with lower V(max) values and no changes in K(M) values accompanied by a significant decrease in tyrosine hydroxylase protein expression and of dopamine and noradrenaline levels. In Wistar Kyoto rats, clonidine treatment had no effect on tyrosine hydroxylase activity and protein expression or catecholamine levels in adrenal medulla or brain. Clonidine treatment significantly reduced noradrenaline and adrenaline plasma levels in spontaneously hypertensive rats and Wistar Kyoto rats. In conclusion, treatment with the alpha(2)-adrenoceptor agonist clonidine prevented the increase in mean arterial blood pressure in young spontaneously hypertensive rats. This effect was accompanied by opposite effects on tyrosine hydroxylase activity in spontaneously hypertensive rat adrenal medulla and brain: an increase in adrenal medulla and a decrease in brain, bringing sympathetic function to a similar profile found in normotensive Wistar Kyoto rats.

Chronic isolation of adult rats decreases gene expression of catecholamine biosynthetic enzymes in adrenal medulla.

OBJECTIVE: Isolation of adult animals represents a form of psychsocial stress that produces sympatho-adrenomedullar activation. The aim of this work was to investigate the changes in gene expression and protein levels of catecholamine biosynthetic enzymes: tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in the adrenal medulla of naive control and chronically (12 weeks) socially isolated adult Wistar rat males and the response of these animals to additional immobilization stress (2 h). METHODS: TH, DBH and PNMT mRNA levels were quantified by quantitative real-time RT-PCR (qRT-PCR). TH, DBH and PNMT immunoproteins were assayed by Western Blot. RESULTS: In chronically isolated rats, gene expression levels of catecholamine biosynthetic enzymes in the adrenal medulla were decreased, but only TH mRNA was significantly decreased. However, protein levels of TH, DBH and PNMT of these animals were elevated by 55%, 20% and 18%, respectively, in relation to the corresponding control. Naive control and chronically socially isolated rats exposed to additional 2-h-immobilization showed increased gene expression of the examined enzymes, the increase being greater in socially isolated rats as compared to the controls. Additional immobilization of naive controls did not affect TH, DBH and PNMT protein levels. In contrast, this stress produced increased TH, DBH and PNMT protein levels in long-term socially isolated rats. CONCLUSION: We can conclude that psychosocial stress expressed a differential influence on gene expression and protein levels of catecholamine biosynthetic enzymes in the adrenal medulla of adult rats. The results indicate a possible adaptation of catecholamine-synthesizing system at the level of TH gene expression in adrenal medulla of chronically isolated animals.

Effect of age on angiotensin II-mediated downregulation of adrenomedullary catecholamine biosynthetic enzymes.

Expression of catecholamine biosynthesizing enzymes, tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DbetaH) increase with age in the adrenal medulla, however, the underlying mechanisms are unclear. In the present study, we examined the effect of peripheral angiotensin II (AngII) on the expression of TH and DbetaH, in the adrenal medulla of young (6 mo) and old (23 mo) Fischer-344 rats. Saline or AngII (230 ng/kg/min sc) was infused for 3 days using osmotic minipumps. Adrenomedullary TH and DbetaH mRNA levels increased significantly with age, and while AngII reduced the expression of these enzymes in young animals, it had no such effect in the old animals. Neuropeptide Y (NPY), which is co-released with catecholamines in the adrenal medulla and stimulates the synthesis of TH and DbetaH, was also upregulated with age and downregulated in response to AngII in young rats. However, in the old animals, the already elevated NPY expression remained unchanged following AngII treatment. This data indicate that the hypertensive effect of peripheral AngII is compensated by an inhibition of adrenomedullary catecholamine biosynthesis in young animals, but this mechanism is impaired in senescence, potentially contributing to the age-related increase in catecholamine biosynthesis.

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.

Increased secretory capacity of mouse adrenal chromaffin cells by chronic intermittent hypoxia: involvement of protein kinase C.

Previous studies have shown that catecholamine secretion from the adrenal medulla plays a critical role in chronic intermittent hypoxia (CIH)-induced alterations in cardiovascular function. In the present study we examined the cellular mechanisms associated with the effects of CIH on adrenal chromaffin cell catecholamine secretion. Experiments were performed on adult male mice (C57/BL6) that were exposed to 1-4 days of CIH or to normoxia. Perforated patch electrical capacitance recordings were performed on freshly prepared adrenal medullary slices that permit separating the chromaffin cell secretion from sympathetic input. CIH resulted in a significant increase in the readily releasable pool (RRP) of secretory granules, and decreased stimulus-evoked Ca(2+) influx. Continuous hypoxia (CH) either for 2.5 h (equivalent to hypoxic duration accumulated over 4 days of CIH) or for 4 days were ineffective in evoking changes in the RRP and Ca(2+) influx. CIH activated PKC in adrenal medullae as evidenced by increased phosphorylation of PKC at Thr(514) and PKC inhibitors prevented CIH-induced increases in the RRP and restored stimulus-evoked attenuation of Ca(2+) influx. CIH resulted in elevated thio-barbituric acid reactive substances (TBARSs, an index of oxidized proteins) and an antioxidant prevented CIH-induced changes in the RRP, suggesting the involvement of reactive oxygen species (ROS). These results demonstrate that CIH increases the RRP in adrenal chromaffin cells via ROS-mediated activation of PKC and suggest that CIH can directly affect the secretory capacity of chromaffin cells and contribute, in part, to elevated catecholamine levels.