Immunohistochemical and biochemical study on the development of the noradrenaline- and adrenaline-storing cells of the adrenal medulla of the rat

Summary The pre- and postnatal development of the adrenal medulla was examined in the rat by immunohistochemistry and by assay of catecholamines. Immunohistochemistry involved the use of antibodies to noradrenaline (NA), adrenaline (A) and the biosynthesizing enzymes dopamine -hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Adrenal glands were obtained from animals from the 16th day of gestation to the 7th postnatal day at daily intervals, and at the 14th postnatal day, and from adult rats. Tissues were fixed in ice-cold, 4% paraformaldehyde, buffered at pH 7.3. Cryostat sections (7 m) were stained with the indirect immunofluorescence technique. Adrenals from the same developmental stages were assayed for the presence of DA (dopamine), NA and A by ion-pair reversed-phase liquid chromatography with electrochemical detection.In adult adrenals the majority of the medullary cells (approximately 80%) were highly immunoreactive to A and moderately immunoreactive to NA. They also showed immunoreactivity to both DBH and PNMT, i.e., they are synthesizing and storing A. The remaining cell clusters were only stained by antibodies to DBH and NA (NA-synthesizing and -storing cells). These findings correlate well with the relative concentrations of A and NA as determined by assay.Three developmental phases could be distinguished. In the first phase, the 16th and 17th prenatal day, medullary cells were only immunoreactive to DBH and NA, and only very small amounts of A as compared to NA were found. During the second period, from the 18th prenatal day to 2 or 3 days after birth, all medullary cells were immunoreactive to DBH, NA, PNMT and A, and during this phase the adrenaline concentration increased daily and became the predominant amine on the 20th day of gestation. Adrenaline represented 75% of total catecholamine on the 1st to 3rd day after birth. The third phase started at the 2nd or 3rd postnatal day and was characterized by the presence of an increasing number of medullary cells solely immunoreactive to DBH and NA, hence synthesizing and storing NA. The remaining cells were immunoreactive to DBH, NA, PNMT and A. Postnatally, the relative concentration of A continued to rise reaching 79% by the 4th postnatal day. These results indicate that initially the adrenal medullary cells are synthesizing and storing almost exclusively NA. Probably, adrenaline synthesis begins at the 16th–17th day of gestation and the cells are then capable of synthesizing and storing both NA and A (mixed cell type) with A synthesis and storage rapidly becoming predominant. Finally, after birth, separate NA-synthesizing and -storing cell types are formed and the so-called A cells stored predominantly (probably >90%) adrenaline with a small proportion of noradrenaline.In the medullary blastema and in the sympathetic ganglia of prenatal animals two cell types, only immunoreactive to DBH and NA, were observed. Presumably, these cells represent developing sympathetic neurons and extra-adrenal chromaffin cells; the latter cell type occasionally invades the adrenal gland. Thus, prospective medullary cells are able to synthesize and store NA before they have made contact with the cortical blastema but A-synthesizing cells are found only within the adrenal gland.Low but significant amounts of DA were found in the adrenal before birth and during the first two postnatal weeks but in the adult animal this accounted for less than 0.1% of total catecholamine.Preliminary reports of this study were made to the American Association of Anatomists (Anat. Rec. 196; 196A, 1980), the Dutch Anatomical Society (Acta Morphol. Neerl. Scand. 19; 330, 1981, and the XIIIth Acta Endocrinologica Congress (Acta Endocrinol. 97: Suppl. 243, 285, 1981)     

Ganglion cells immunoreactive for catecholamine-synthesizing enzymes,neuropeptide Y and vasoactive intestinal polypeptide in the rat adrenal gland

Immunohistochemistry has been used to demonstrate tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactivities, and acetylcholinesterase (AChE) activity was demonstrated in rat adrenal glands. The TH, DBH, NPY and VIP immunoreactivities and AChE activity were observed in both the large ganglion cells and the small chromaffin cells whereas PNMT immunoreactivity was found only in chromaffin cells, and not in ganglion cells. Most intraadrenal ganglion cells showed NPY immunoreactivity and a few were VIP immunoreactive. Numerous NPY-immunoreactive ganglion cells were also immunoreactive for TH and DBH; these cells were localized as single cells or groups of several cells in the adrenal cortex and medulla. Use of serial sections, or double and triple staining techniques, showed that all TH- and DBH-immunoreactive ganglion cells also showed NPY immunoreactivity, whereas some NPY-immunoreactive ganglion cells were TH and DBH immunonegative. NPY-immunoreactive ganglion cells showed no VIP immunoreactivity. AChE activity was seen in VIP-immunopositive and VIP-immunonegative ganglion cells. These results suggest that ganglion cells containing noradrenaline and NPY, or NPY only, or VIP and acetylcholine occur in the rat adrenal gland; they may project within the adrenal gland or to other target organs. TH, DBH, NPY, and VIP were colocalized in numerous immunoreactive nerve fibres, which were distributed in the superficial adrenal cortex, while TH-, DBH- and NPY-immunoreactive ganglion cells and nerve fibres were different from VIP-immunoreactive ganglion cells and nerve fibres in the medulla. This suggests that the immunoreactive nerve fibres in the superficial cortex may be mainly extrinsic in origin and may be different from those in the medulla.     

EFFECTS OF DEXAMETHASONE AND NERVE GROWTH FACTOR ON PHENYLETHANOLAMINE N-METHYLTRANSFERASE AND ADRENALINE IN ORGAN CULTURES OF NEWBORN RAT SUPERIOR CERVICAL GANGLION

Abstract— Phenylethanolamine N -methyltransferase (PNMT) and adrenaline (A) have been studied in organ cultures of neonatal rat sympathetic ganglia. Organ culture for 2 days without added nerve growth factor (NGF) caused a fall in noradrenaline (NA) and PNMT contents but there was no change in dopamine (DA) or A contents compared to controls. However, in the presence of dexamethasone, there was a marked increase in both PNMT activity and A content, but no change in NA or DA content. Addition of NGF to cultures stimulated with dexamethasone caused no further significant change in PNMT activity or A content, whereas both NA and DA were increased. Prolonged culture without NGF, in the presence of dexamethasone resulted in reductions in both NA and DA content, but the high levels of PNMT activity and A content were sustained. The results are consistent with the hypothesis that both PNMT and A are not contained in the noradrenergic cell bodies but are located chiefly within the small intensely fluorescent cells in sympathetic ganglia.     

Immunocytochemical study of tyrosine hydroxylase and dopamine β-hydroxylase immunoreactivities in the rat pancreas

An immunohistochemical and immunoelectron microscopic study was used to demonstrate tyrosine hydroxylase (TH) and dopamine -hydroxylase (DBH) immunoreactivities in the rat pancreas. Small TH immunoreactive cells were found in close contact with large TH immunonegative ganglion cells among the exocrine glands and were occasionally found in some islets. Some of these TH immunoreactive cells were also DBH immunopositive. The immunoreaction product was seen diffusely in the cytoplasm and in the granule cores of TH immunoreactive cells. All intra-pancreatic ganglion cells were immunoreactive for DBH, but not for TH. The TH immunoreactive cells were identified as small intensely fluorescent (SIF) cells due to their localization and morphological characteristics and showed no insulin, glucagon, somatostatin or pancreatic polypeptide immunoreactivities. These results indicate that SIF cells may release dopamine or noradrenaline to adequate stimuli while the intra-pancreatic ganglion cells with only DBH may not synthesize catecholamines in a normal biosynthetic pathway. TH immunoreactive nerve bundles without varicosities and fibers with varicosities, associated or unassociated with blood vessels, were found in both the exocrine and endocrine pancreas. Close apposition of TH immunoreactive nerve fibers to the smooth muscle and endothelial cells of the blood vessels was observed. A close apposition between TH immunoreactive nerve fibers and exocrine acinar cells and islet endocrine cells was sometimes found in the pancreas. The immunoreaction product was seen diffusely in the axoplasm and in the granular vesicles of the immunoreactive nerve fibers. Since no TH immunoreactive ganglion cells were present in the rat pancreas, the present study suggests that noradrenergic nerve fibers in the pancreas may be extrinsic in origin, and may exert an effect on the regulation of blood flow and on the secretory acitivity of the acinar cells, duct cells and endocrine cells.     

Induction of Gene Expression of the Catecholamine-Synthesizing Enzymes by Insulin-Like Growth Factor-I

Abstract: The effects of insulin-like growth factor-I (IGF-I) on gene expression and the activities of the three enzymes specific for catecholamine biosynthesis, tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), and phenylethanolamine N -methyltransferase (PNMT), were determined in bovine adrenomedullary chromaffin cells primary cultured in serum-free medium. The mRNA level of TH was maximally elevated in the presence of IGF-I by 3.1 ± 0.4-fold after 48 h, DBH by 5.1 ± 0.3-fold in 24 h, and PNMT by 2.8 ± 0.5-fold in 72 h. In addition, the activity of TH was increased by 77%, DBH by 70%, and PNMT by 23% in IGF-I-exposed cultures. In the absence of the growth factor, the mRNA levels of TH and DBH were decreased to 45 ± 10% and 35 ± 12% of the time-zero control within 48 h while PNMT mRNA was decreased to 82 ± 5% only after 72 h. When the cells were cotreated with the protein tyrosine kinase inhibitor genistein, DBH induction by IGF-I was suppressed, confirming that the effect is mediated by tyrosine kinase. Cotreatment with the protein kinase A (PKA) inhibitor H89 caused complete reversal of the IGF-I-induced DBH increase and the effects of IGF-I treatment and PKA activation by forskolin were not additive, suggesting that PKA is involved in the signaling initiated by IGF-I in these cells.     

Effect of hydrocortisone on catecholamines and the enzymes synthesizing them in the developing sympathetic ganglion

Summary Newborn rats were daily injected with 0.2 mg hydrocortisone acetate for seven days. They were killed 1, 7 or 21 days after the last injection, together with untreated controls. Hydrocortisone caused a great increase in the number of the small, intensely fluorescent (SIF) cells and the appearance of similar small cells with intense immunohistochemical reactions for tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine (noradrenaline)N-methyltransferase (PNMT) in the superior cervical ganglion. At the same time, the adrenaline content and the PNMT activity of the ganglion greatly increased, while no significant changes were observed in the dopamine or noradrenaline content or TH or DBH activity. All these changes essentially disappeared after a recovery period of seven or 21 days.It is concluded that hydrocortisone causes a temporary increase in the number of SIF cells by causing a synthesis of TH, DBH and PNMT in previously existing small, non-fluorescent cells, which start to synthesize and store adrenaline, thus becoming intensely fluorescent SIF cells. These SIF cells are different from the normal SIF cells of the same ganglion, most of which appear at a later stage of postnatal development when response to hydrocortisone is lost, which contain TH but neither DBH nor PNMT, and which permanently remain in the ganglion.     

Immunocytochemical localization of tyrosine hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase in the adrenal glands of the frog and rat by a peroxidase-antiperoxidase method.

The subcellular localizations of tryrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) in the adrenal glands of the frog and rat have been examined by a peroxidase-antiperoxidase (PAP) method. TH was localized in the ground substance of the adrenaline-containing cells and noradrenaline-containing cells, but not in the nucleus or in the mitochondria. TH was also located on the outside of the membrane of the chromaffin granules. DBH was observed only inside the granules. PNMT was found not only in the ground substance but also on the membrane of some adrenaline-containing granules. Cortical lipid cells of the frog adrenals did not show TH-, DBH-, and PNMT-reactions. The negative reactions to TH-, DBH-, and PNMT-antiserum exhibited by the summer cells of the frog adrenals prove that they belong to the cortical cells.     

Immunoreactive atrial natriuretic polypeptides in the adrenal medulla and sympathetic ganglia

Atrial natriuretic polypeptide (ANP)-like immunoreactivity was found in the rat adrenal gland by using indirect immunofluorescence and peroxidase-antiperoxidase techniques. ANP-like immunostaining was present in most of chromaffin cells with varying degrees of immunoreactivity. The majority of medullary cells displayed very intense immunostaining, and several clusters revealed weaker immunostaining. No staining was found in the adrenal cortex or in the nerve fibers in this organ. In the consecutive sections treated for dopamine-beta-hydroxylase (DBH), apparently all medullary cells had intense immunofluorescence for DBH and its distribution pattern was very similar to that for ANP-like immunoreactivity. While phenylethanolamine N-methyltransferase (PNMT) immunoreactive cells largely corresponded to the intensely stained ANP-like immunoreactive cells, suggesting that adrenaline cells contained a large amount of ANP-like substance, noradrenaline cells contained a smaller amount of this substance than adrenaline cells. Ultrastructural study showed that end-products due to the immunoreaction with the ANP antiserum were primarily associating with chromaffin granules. In addition, the presence of ANP-like immunoreactivity was investigated in several sympathetic ganglia of the rat. No principal ganglion cells were ANP-positive, whereas a few small intensely fluorescent (SIF) cells were ANP-immunoreactive. The present findings suggest that catecholamines coexist with ANP which has a natriuretic and vasodilating effect, in adrenal medullary cells and SIF cells in several rat sympathetic ganglia, but not in principal ganglion cells.     

Immunohistochemical localization of the catecholamine-synthesizing enzymes,substance P and enkephalin in the human fetal sympathetic ganglion

Summary The human fetal sympathetic ganglia were studied using the indirect peroxidase-antiperoxidase PAP method for immunocytochemical demonstration of three catecholamine-synthesizing enzymes, tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) as well as the neuropeptides leucine (Leu⁵)-enkephalin and substance P. The neuroblasts of the ganglia showed intense peroxidase immunoreactivity for TH, moderate reaction to DBH, and no reaction to PNMT. The small intensely fluorescent (SIF) cells situated along the blood vessels also showed positive labelling for only two enzymes, TH and DBH. The immunocytochemical localization of these enzymes suggests that both neuroblasts and SIF cells synthesize noradrenalin. Neither the neuroblasts nor SIF cells showed a reaction to substance P, and only the SIF cells contained enkephalin-like immunoreactivity. The role of enkephalin in the noradrenalin-containing SIF cells is unknown, but may be related to neuromodulation of ganglionic transmission.     

Subcellular Site of Biosynthesis of the Catecholamine Biosynthetic Enzymes in Bovine Adrenal Medulla

The subcellular site of biosynthesis of the catecholamine biosynthetic enzymes was examined. Free and membrane-bound polysomes were prepared from bovine adrenal medulla and mRNA was isolated from these polysomes. Both were active in directing cell-free translations. Immunoprecipitation of cell-free products with specific antisera localized the biosynthesis of the subunits of tyrosine hydroxylase (TH) (apparent Mr = 61,000) and of phenylethanolamine N-methyltransferase (PNMT) (apparent Mr = 32,000) on free polysomes, compared with biosynthesis of subunits of dopamine beta-hydroxylase (DBH) (apparent Mr = 67,000) on membrane-bound polysomes. Cross-reactivity between translation products was observed. Antibodies for DBH recognized a polypeptide with electrophoretic mobility identical to newly synthesized PNMT. However increasing concentrations of antibodies to DBH recognized at most 1/20 of the PNMT formed. The results of this study show the subcellular distribution of the catecholamine synthesizing enzymes is determined by their site of biosynthesis.     

Tissue catecholamine concentrations in spontaneously hypertensive rats

Tissue concentrations of noradrenaline (NA), dopamine (DA) and adrenaline (A) were compared in spontaneously hypertensive (SHR) and normotensive (NCR) rats, aged 1, 3, 8, 14 and 24 weeks The organs analyzed included the brain, subdivided into prosencephalon and rhombencephalon, heart, adrenal glands and kidney. Brain catecholamines were significantly lower in SHR than in NCR, and the difference appeared already at the age of 3 weeks. Concomitant increase was found in the adrenal NA and A concentrations of the SHR. Concentration of NA in the heart decreased in the SHR following onset of hypertension. It is concluded that the diminished NA, DA and A concentrations in the brain as well as the augmented adrenal NA and A levels in the SHR may be causally related to the development of hypertension, while the heart NA level reflects the secondary, hypertension -- related changes.     

Characterization of adrenal medullary chromaffin cells by flow cytometry.

BACKGROUND: Adrenomedullary chromaffin cells are neural crest derivatives widely used as a model system to study neurosecretory mechanisms. Morphological, immunohistochemical, and functional data indicate that chromaffin cells are heterogeneous and support the distinction between adrenaline (A)- and noradrenaline (NA)-producing and secreting cells. The aim of this study was to characterize by flow cytometry the two main chromaffin cell subtypes in suspensions of cultured bovine chromaffin cells. METHODS: An indirect immunofluorescence method was used for the specific labeling of two intracellular enzymes, dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT), involved in the synthesis of NA and A, respectively. Flow cytometry analysis of fluorescence labeling was performed in two chromaffin cell fractions differentially enriched in A-containing cells by centrifugation through density gradients. PNMT and DBH-related fluorescence was also correlated with the A and NA content of the cells assayed by HPLC measurements. RESULTS: No significant differences were found in forward-side scatter plots between the two cell fractions (A-enriched cells and mixed cells); however, the degree of labeling of the enzymes and the corresponding PNMT/DBH-related fluorescence ratio was significantly greater in the A-enriched cell fraction. The existence of changes in DBH and PNMT content of chromaffin cells over time (1 week) in culture was also examined. No significant variation in enzyme related fluorescence values was detected in any of the two cell fractions, and this result correlated well with HPLC determinations of the catecholamine content (A and NA) of the cells. CONCLUSIONS: Flow cytometry appears to be a useful technique to characterize chromaffin cell subtypes and to follow their phenotypic changes in response to growth factors.     

Short and long term regulation of catecholamine biosynthetic enzymes by angiotensin in cultured adrenal medullary cells. Molecular mechanisms and nature of second messenger systems

The purpose of this study was to examine the effects of angiotensin on the enzyme activities and gene expression of two catecholamine synthesizing enzymes, tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT), in bovine adrenal medullary (AM) cells. Short term (15 min) incubation of cultured AM cells with 2 nM [Sar1]angiotensin II (s1-AII) did not increase basal secretion of catecholamines; however, longer incubations (3, 24, or 72 h) produced 4-10-fold increases. To determine whether angiotensin affects synthesis of catecholamines, the activities of TH and PNMT were examined. Incubation with s1-AII (15-30 min) decreased the Km of TH for its biopterine cofactor [6R)-5,6,7,8-tetrahydro-1-biopterin dihydrochloride (BH4] without affecting the Vmax, suggesting activation of TH. After long term incubation (72 h) the Km value was identical to that of control, while increases in the apparent Vmax were observed. PNMT activity was unaffected during a 30-min treatment with s1-AII; however, 2-fold increases occurred after a 48-72-h incubation. s1-AII (24 h) increased the relative abundance of TH and PNMT mRNAs, suggesting that the long term increase in enzyme activities reflected increased expression of TH and PNMT genes. Maximal increases were observed at 2 nM s1-AII and the changes were antagonized by saralasin. Induction of TH mRNA by s1-AII was additive to the effects of veratridine or forskolin indicating that effects of angiotensin were not due to membrane depolarization or increased cyclic AMP levels. Incubation with Ca2+ ionophore A23187 increased TH and PNMT mRNA levels in AM cells raising the possibility that the increase in cellular [Ca2+] could mediate effects of angiotensin. Angiotensin-induced increases in TH and PNMT mRNA were inhibited by nifedipine indicating involvement of voltage-dependent Ca2+ channels. In addition, the increases in TH, but not PNMT mRNA, were antagonized by dantrolene, which inhibits mobilization of Ca2+ from intracellular stores. Calmodulin involvement was suggested by the inhibition of s1-AII induced changes in mRNA with 1 microM calmidazolium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunocytochemical localization of tyrosine hydroxylase,dopamine-β-hydroxylase and phenylethanolamine-N-methyltransferase in the adrenal glands of the frog and rat by a peroxidase-antiperoxidase method

Summary The subcellular locilazations of tryrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) in the adrenal glands of the frog and rat have been examined by a peroxidase-antiperoxidase (PAP) method. TH was localized in the ground substance of the adrenaline-containing cells and noradrenaline-containing cells, but not in the nucleus or in the mitochondria. TH was also located on the outside of the membrane of the chromaffin granules. DBH was observed only inside the granules. PNMT was found not only in the ground substance but also on the membrane of some adrenaline-containing granules. Cortical lipid cells of the frog adrenals did not show TH-, DBH-, and PNMT-reactions. The negative reactions to TH-, DBH-, and PNMT-antiserum exhibited by the summer cells of the frog adrenals prove that they belong to the cortical cells.     

Differentiation of adrenomedullary catecholamine synthesizing enzyme responses to repeated immobilization in hybrid rats

The response of adrenomedullary catecholamine synthesizing enzymes to repeated immobilization was studied in hybrid (F₁) offspring of 2 inbred rat strains (LEW and F344). Immobilization-induced increases in tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyl-transferase (PNMT) activities in one of the parental strains (F344) previously were shown to be dependent upon intact adrenal gland innervation but independent of the pituitary gland, while responses in the other parental strain (LEW) were independent of adrenal innervation but dependent upon pituitary function. Factors determining immobilization-induced increases in adrenal enzymes of F₁ offspring were enzyme-specific. Increased PNMT activity was pituitary dependent in F₁ rats, whereas increased TH and DBH activities after immobilization were dependent upon an intact adrenal gland innervation. These results suggest that the factor(s) regulating PNMT responses are differentiable from those regulating TH and DBH responses. The results also indicate that analysis of PNMT responses to immobilization in backcross populations is feasible, and could indicate whether strain-specific response mechanisms are heritable.     

Dopamine-beta-hydroxylase inhibition acutely stimulates rats hypothalamic noradrenaline and dopamine neuronal activity as assessed from metabolic ratios and circulating glucose and ACTH responses

Because central noradrenaline neuronal activity is tonically inhibited by noradrenaline (NA) itself via an action at prejunctional alpha 2-adrenoceptors, it was hypothesised that the blockade of central NA synthesis following acute dopamine-beta -hydroxylase (DBH) inhibition might primarily deplete prejunctional NA levels and result in an increase in central NA neuronal activity through reduced NA autoinhibition. This hypothesis was tested in the rat following the acute administration of the DBH inhibitors diethyldithiocarbamate (DDC) and cysteamine (CSH). Computerised gas chromatography/mass spectrometry was used to precisely measure the hypothalamic levels of NA and dopamine (DA) together with those of their primary neuronal metabolites dihydroxyphenylethyleneglycol (DHPG) and dihydroxyphenylacetic acid (DOPAC), respectively. Both DDC (at 4 h) and CSH (at 30 min.) caused approximately a 50% reduction of hypothalamic NA concentrations. However this was associated with marked and highly significant increases in hypothalamic DHPG levels (by 50-100%) and in the hypothalamic ratio DHPG/NA. Also, when measured after CSH, the hypothalamic levels of the DHPG metabolite 3-methoxy-4-hydroxyphenylethyleneglycol were highly significantly increased. Consistent with increased DA neuronal activity, both DBH inhibitors raised DA and DOPAC levels and also the ratio DOPAC/DA in the hypothalami of treated rats and markedly suppressed serum prolactin levels (all p less than 0.01). The rise in hypothalamic concentrations of DHPG indicates that an increase in hypothalamic NA neuronal activity occurs following DBH inhibition. Significant elevations of blood glucose, corticosterone and ACTH were also observed after DBH inhibition. As we have previously demonstrated that increased central NA activity is associated with elevations of blood glucose, corticosterone and ACTH, these data provide further evidence for a functional increase in central NA activity caused by acute DBH inhibition. It is proposed that the increase in hypothalamic NA activity after DBH inhibition results from a primary depletion of the prejunctional alpha 2-active autoregulatory pool of NA.     

Catecholamine Metabolism in Paraganglioma and Pheochromocytoma: Similar Tumors in Different Sites?

Pheochromocytoma (PHEO) and paraganglioma (PGL) are catecholamine-producing neuroendocrine tumors that arise respectively inside or outside the adrenal medulla. Several reports have shown that adrenal glucocorticoids (GC) play an important regulatory role on the genes encoding the main enzymes involved in catecholamine (CAT) synthesis i.e. tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). To assess the influence of tumor location on CAT metabolism, 66 tissue samples (53 PHEO, 13 PGL) and 73 plasma samples (50 PHEO, 23 PGL) were studied. Western blot and qPCR were performed for TH, DBH and PNMT expression. We found a significantly lower intra-tumoral concentration of CAT and metanephrines (MNs) in PGL along with a downregulation of TH and PNMT at both mRNA and protein level compared with PHEO. However, when PHEO were partitioned into noradrenergic (NorAd) and mixed tumors based on an intra-tumoral CAT ratio (NE/E >90%), PGL and NorAd PHEO sustained similar TH, DBH and PNMT gene and protein expression. CAT concentration and composition were also similar between NorAd PHEO and PGL, excluding the use of CAT or MNs to discriminate between PGL and PHEO on the basis of biochemical tests. We observed an increase of TH mRNA concentration without correlation with TH protein expression in primary cell culture of PHEO and PGL incubated with dexamethasone during 24 hours; no changes were monitored for PNMT and DBH at both mRNA and protein level in PHEO and PGL. Altogether, these results indicate that long term CAT synthesis is not driven by the close environment where the tumor develops and suggest that GC alone is not sufficient to regulate CAT synthesis pathway in PHEO/PGL.     

Parallel Up-Regulation of Catecholamine Biosynthetic Enzymes by Dexamethasone in PC12 Cells

Abstract: We sought to investigate whether dexamethasone produces a coordinated, time-dependent effect on all enzymes in the catecholamine biosynthetic pathway in PC12 cells. The levels of mRNAs of tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), and dopamine γ-hydroxylase (DBH) were examined at 0, 6, 12, 24, and 48 h after dexamethasone (5 μ M ) treatment to PC12 cells. The levels of all enzyme mRNAs steadily increased for 24 h, although the increase of AADC mRNA content was slow. The increased mRNA levels of TH and AADC were maintained at 48 h, whereas the level of DBH mRNA was sharply decreased at 48 h. The maximally induced mRNA levels were ∼5.0-, 2.4-, and 7.0-fold higher than the control levels of TH, AADC, and DBH, respectively. The elevation of enzyme activities was detected later than the increase in levels of mRNAs. The maximal activities of TH, AADC, and DBH were reached between 48 and 72 h with 3.6-, 1.8-, and 8.0-fold increases, respectively. Low, but detectable, phenylethanolamine N -methyltransferase (PNMT) activity was observed in PC12 cells, and dexamethasone increased its activity 5.6-fold at 72 h. The PNMT mRNA was easily detected by northern blot analysis after exposure for 24 h to dexamethasone. The data suggest that, in PC12 cells, dexamethasone up-regulates all catecholamine biosynthetic enzyme genes in a parallel fashion.