Immunohistochemical colocalization of insulin,aromatic L-amino acid decarboxylase and dopamine beta-hydroxylase in islet B cells of chicken pancreas

Summary The identity of the monoamine which produces a very weak formaldehyde-induced fluorescence in some pancreatic islet cells was studied by fluorescence microscopy and immunohistochemistry either on the same tissue section or on serial tissue sections of tissue from male chickens. Pancreatic islet cells showing this very weak formaldehyde-induced fluorescence react immunohistochemically with antisera directed against insulin, aromatic L-amino acid decarboxylase and dopamine beta-hydroxylase and therefore appear to be islet B cells producing insulin and noradrenaline.     

A single gene codes for aromatic L-amino acid decarboxylase in both neuronal and non-neuronal tissues

We have sought to determine whether aromatic L-amino acid decarboxylase which functions as a neurotransmitter biosynthetic enzyme in neuronal cells can be distinguished from an enzyme with similar activity found in peripheral tissues where no neurotransmitters are synthesized. Aromatic L-amino acid decarboxylase was purified to electrophoretic homogeneity from bovine adrenal medulla, and highly specific antibodies were produced. In addition, a DNA clone complementary to aromatic L-amino acid decarboxylase mRNA was isolated by immunological screening of a lambda gt11 cDNA expression library. We have used these antibodies and cDNA probes for biochemical, immunochemical, and molecular analyses. A single form of aromatic L-amino acid decarboxylase is detected in rat and bovine tissue. Specifically, aromatic L-amino acid decarboxylase protein is biochemically and immunochemically indistinguishable in brain, liver, kidney, and adrenal medulla. Hybridization to aromatic L-amino acid decarboxylase cDNA identifies a single mRNA species of 2.3 kilobase pairs in rat tissue. Furthermore, Southern blot analysis reveals that a single gene codes for aromatic L-amino acid decarboxylase.     

Catecholamine-containing pancreatic islet cells of the domestic fowl

Summary In an attempt to identify pancreatic islet cells emitting formaldehyde-induced fluorescence (FIF), the pancreatic islets of the domestic fowl were studied by combined fluorescence, ultrastructural, silver-impregnation and immunohistochemical methods in the same section or in consecutive semi-thin and ultra-thin sections. The results indicate that islet cells emitting intense FIF exhibit a strongly argyrophil reaction with the Grimelius' silver method and also immunohistochemical reaction with anti-glucagon serum, but not with anti-5-HT serum. Therefore, the fowl islet A cell, a peptide hormone-producing cell, stores simultaneously catecholamine as biogenic amine. The islet B and D cells did not display any FIF, any argyrophil reaction with the Grimelius' silver method, or any immunoreactivity with anti-glucagon or anti-5-HT sera. The fluorescent but non-argyrophil cells dispersed in the exocrine acinus may well be PP cells.     

Evaluation of the holoenzyme content of aromatic L-amino acid decarboxylase in brain and liver tissues.

We have re-evaluated the content of the holo-form of aromatic L-amino acid decarboxylase in rat tissues. Aromatic L-amino acid decarboxylase was found to consume pyridoxal 5'-phosphate while it underwent decarboxylation-dependent transamination as a side reaction. We observed that the total dopamine formation was proportional to the amount of holoenzyme. Dopamine formation in a tissue extract, which was preincubated with pyridoxal 5'-phosphate, was compared with the same tissue sample but which was prepared without preincubation. Percentages of holo-form of aromatic L-amino acid decarboxylase obtained from such comparison were 78% for brain and 94% for liver tissues. These values were significantly higher than those reported earlier in which the decarboxylation-dependent transamination of the decarboxylase had been overlooked.     

Immunocytochemical localization of aromatic L-amino acid decarboxylase in human, rat, and mouse bronchopulmonary and gastrointestinal endocrine cells

Aromatic L-amino acid decarboxylase (AADC) catalyzes the cellular decarboxylation of L-aromatic amino acids and is therefore involved in the synthesis of several biogenic amines. Application of the indirect immunoperoxidase method on human, rat, and mouse tissues using specific antibodies to AADC revealed all AADC-containing cells. Besides mast cells and adrenergic nerve fibers, the following cells were immunostained: neuroendocrine cells in the tracheobronchial epithelium; neuroepithelial bodies in the bronchopulmonary epithelium; Kultschitzky cells in the small intestine and appendix as well as adrenal chromaffin cells. All the latter cells belong to the so-called APUD system, the "D" in the acronym standing for the activity of the enzyme aromatic L-amino acid decarboxylase. Immunocytochemistry for AADC may become an additional tool not only to highlight APUD cells in tissue sections but also to differentiate the sites of cellular amine synthesis from those of amine storage.     

The acute effects of 5HTP, fluoxetine and quipazine on insulin and glucagon release in the intact rat.

5-hydroxytryptophan (5HTP), the immediate precursor of serotonin, induces a release of insulin and glucagon in the intact rat. These effects of 5HTP, which have previously been shown to be blocked by L-aromatic amino acid decarboxylase inhibition, were also prevented by methysergide (a serotonin receptor antagonist). Quipazine (a serotonin receptor agonist) did not alter pancreatic hormone release. Fluoxetine, a serotonin neuronal reuptake blocker did not effect insulin secretion and had a slight glucagon stimulatory effect, however the effects of 5HTP on insulin and glucagon release were not potentiated by fluoxetine pretreatment. Alpha and beta-adrenergic receptor blockade did not alter the pancreatic effects of 5HTP.     

Expression of cell type-specific markers during pancreatic development in the mouse: implications for pancreatic cell lineages

Summary The islet cells of the mammalian pancreas are comprised of four different endocrine cell types, each containing a specific hormone. Islet cells also contain two enzymes of the catecholamine biosynthetic pathway: tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC). The cell lineage relationships of these different cell types have not been examined and it is not known whether, during development, they originate from the same or from different precursor populations. In this study we used immunocytochemical procedures to determine whether developing pancreatic cells express markers common to endocrine and exocrine cell types. We found that acinar cell precursors express AADC prior to the appearance of an exocrine marker and that the expression of AADC in acinar cells persists throughout embryogenesis to the first month of postnatal life. At this time, acinar cells do not contain AADC. We also found that exocrine cells containing AADC never express other islet-cell markers. These findings suggest that while acinar and islet cells both arise from precursor cells containing AADC, these progenitor cells do not express a combined endocrine-exocrine phenotype.     

Coexistence of pancreatic polypeptide (PP)-and glucagon-immunoreactivity in pancreatic endocrine cells of mouse

Immunocytochemical double staining techniques were used to study PP- and glucagon-like-immunoreactivity in pancreatic endocrine cells of mouse. An antiserum against FMRFamide appeared to react with all PP-immunoreactive endocrine cells. With fluorescence microscopy most PP/FMRFamide-immunoreactive cells also showed glucagon-immunoreactivity, but cells containing only PP- or glucagon-like substances were found as well. The proportion of cells containing PP-, glucagon, and both immunoreactivities varied strongly from islet to islet in all parts of the pancreas. Using an electron microscopical immunogold double staining procedure on Lowicryl-embedded pancreas, PP/FMRFamide- and glucagon-immunoreactivity appeared to be present in the majority of endocrine A cells; both immunoreactivities were randomly distributed within the granules of these cells. Cells containing only PP/FMRFamide- or glucagon-immunoreactivity were also found. Glucagon- and a faint FMRFamide-immunoreactivity was also observed in osmicated epon-embedded tissue. Independent of their immunoreactivity all positive cells showed the same round electron dense secretory granules.     

Coexistence of pancreatic polypeptide (PP)-and glucagon-immunoreactivity in pancreatic endocrine cells of mouse

Summary Immunocytochemical double staining techniques were used to study PP- and glucagon-like-immunoreactivity in pancreatic endocrine cells of mouse. An antiserum against FMRFamide appeared to react with all PP-immunoreactive endocrine cells. With fluorescence microscopy most PP/FMRFamide-immunoreactive cells also showed glucagon-immunoreactivity, but cells containing only PP-or glucagon-like substances were found as well. The proportion of cells containing PP-, glucagon, and both immunoreactivities varied strongly from islet to islet in all parts of the pancreas.Using an electron microscopical immunogold double staining procedure on Lowicryl-embedded pancreas, PP/FMRFamide-and glucagon-immunoreactivity appeared to be present in the majority of endocrine A cells; both immunoreactivities were randomly distributed within the granules of these cells. Cells containing only PP/FMRFamide-or glucagon-immunoreactivity were also found. Glucagon-and a faint FMRFamide-immunoreactivity was also observed in osmicated epon-embedded tissue. Independent of their immunoreactivity all positive cells showed the same round electron dense secretory granules.     

Decarboxylation of p-Tyrosine: A Potential Source of p-Tyramine in Mammalian Tissues

The question of the existence of a p-tyrosine decarboxylase pathway for the formation of p-tyramine in mammalian tissues remains unresolved. Development of a sensitive and specific assay for p-tyrosine decarboxylase has permitted demonstration of this activity in rat tissues and human kidney. Tyrosine decarboxylase was purified to electrophoretic homogeneity by pH 5.0 precipitation, ammonium sulfate precipitation, gel filtration, phenyl-Sepharose chromatography, DEAE-Sephacel chromatography, and preparative isoelectric focusing. A specific rabbit antiserum to tyrosine decarboxylase was also obtained. Purified tyrosine decarboxylase possessed a narrow pH dependency with an optimum at 8.0. Benzene and certain other organic solvents dramatically stimulated tyrosine decarboxylase activity of purified enzyme. Purified tyrosine decarboxylase activity also decarboxylated L-DOPA, 5-hydroxytryptophan, 3,4-dihydroxyphenylserine, o-tyrosine, m-tyrosine, phenylalanine, histidine, and tryptophan, which suggested that the purified enzyme was aromatic L-amino acid decarboxylase. This conclusion was supported by a constant ratio of 5-hydroxytryptophan decarboxylase to tyrosine decarboxylase throughout the purification scheme and by parallel immunoprecipitation of decarboxylase activities by the specific antityrosine decarboxylase antisera. Thus, we report that p-tyrosine is decarboxylated by aromatic L-amino acid decarboxylase and that this metabolic transformation may be an important source of p-tyramine in mammalian tissues. In conclusion, neuronal tissues that synthesize catecholamines or serotonin should now be considered capable of synthesizing p-tyramine and other biogenic amines.     

Effects of thyrotropin releasing hormone and its analogue, DN 1417, on biopterin concentration and tryptophan hydroxylation in rat raphe slices

The effects of subchronic administration of thyrotropin releasing hormone (TRH) and its analogue, gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-prolinamide citrate (DN 1417), on serotonin biosynthesis in situ were investigated in tissue slices of the midbrain raphe of rats. TRH or DN 1417 (10 mg/kg per day intraperitoneally) were administered to male Wistar rats for ten days. At twenty four hr after the last injection, tissue slices of the midbrain raphe were prepared and the rate of serotonin biosynthesis was estimated by measuring formation of 5-hydroxytryptophan (5-HTP) from tryptophan during inhibition of aromatic L-amino acid decarboxylase using high-performance liquid chromatography with fluorescence detection. Total biopterin content was determined by a specific radioimmunoassay. 5-HTP formation was decreased 22% and 29%, and total biopterin content 69% and 72%, in TRH- and DN 1417-treated rats, respectively. However, tryptophan concentration in raphe slices did not change. In contrast, the Vmax of tryptophan hydroxylase in the homogenate of the raphe nucleus in the presence of a saturating concentration of (6R)-L-erythro-tetrahydrobiopterin, the naturally occurring pterin cofactor, was significantly increased after repeated administration of TRH or DN 1417. These results indicate that reduction of in situ serotonin biosynthesis in tissue slices from the rats treated with TRH or DN 1417 subchronically contray to the increase in in vitro tryptophan hydroxylase may result from the decrease of the biopterin cofactor, and that changes in concentrations of the biopterin cofactor may play a regulatory role in serotonin biosynthesis in vivo under certain conditions.     

Disaggregation of brain polysomes by L-5-hydroxytryptophan: Mediation by serotonin

In rats, intraperitoneal administration of L-5-hydroxytryptophan (200 mg/kg) causes extensive disaggregation of whole brain polysomes after one hour. Polysome disaggregation is prevented if the conversion of L-5-hydroxytryptophan to serotonin is blocked by pretreatment with an aromatic L-amino acid decarboxylase inhibitor; disaggregation is potentiated by pretreatment with a monoamine oxidase inhibitor. The brain polysome disaggregation induced by L-phenylalanine administration (1 g/kg) is not blocked by decarboxylase inhibition.     

Histochemical characterization of the monoamine-containing cells of the adenohypophysis in the chinese quail

Summary The demonstration of biogenic amine-containing cells is classically performed by means of the formaldehyde-induced fluorescence technique after administration of the monoamine precursors, L-dopa or 5-hydroxytryptophan (5-HTP). The injection of these two compounds rapidly leads to the formation of large amounts of dopamine or serotonin. The mechanism and site of decarboxylation are therefore of great importance.The biochemical pathway evoked above is studied in the avian adenohypophysis by two different methods involving enzyme inhibition. In the first method, a seryl-derivative of hydrazine (benserazide, Ro 4-4602) is used for the inhibition of the aromatic L-amino acid decarboxylase. This experiment supports the hypothesis that a variable part of L-dopa or 5-HTP can be decarboxylated out of the gland, although we agree with the possibility that, in most cases, biogenic amine precursors may be taken up by endocrine cells. Interference between benserazide and precursor may readily be excluded.The second way involves monoamine oxidase (MAO) inhibition by nialamide. In is based on the differential breakdown of L-dopa on one side, and dopamine and serotonin on the other side. While L-dopa is catabolized by O-methylation, the amines undergo an oxidative deamination by means of MAO. Inhibition of this enzyme leads to the accumulation of fluorescent material in the PAS-positive cells of the adenohypophysis.Observations made under various experimental conditions suggest that, in the Chinese quail in particular, a variable part of dopamine and/or serotonin is synthetized out of the adenohypophysis, and that this substances may be subsequently taken up by the PAS-positive cells of the gland where they possibly play a rôle in synthesis, storage and/or secretion of the polypeptide hormone. It is highly probable that these PAS-positive cells are to be regarded as corticotrophs, but it is possible that -gonadotrophs, melanotrophs and/or somatotrophs must also be regarded as aminergic endocrine cells.A comparison between the metabolism of biogenic amines in a peripheral organ such as hypophysis and the situation in the central nuclei of the brain, as regards the blood-brain barrier, is made.     

多巴脱羧酶及其与神经类疾病的关联性研究进展

多巴脱羧酶(dopa decarboxylase,DDC)又称作芳香族L-氨基酸脱羧酶,是儿茶酚胺生物合成途径中重要的酶之一,具有多种生物学功能。多巴脱羧酶可分别催化L-3,4-二羟基苯丙氨酸(L-多巴)和L-5-羟色氨酸合成两种神经递质多巴胺和五羟色胺。多巴胺和五羟色胺在脊椎动物和无脊椎动物的生殖、发育、行为和免疫应答过程中均具有重要作用。此外,它还与多种神经类疾病和社会行为有关。多巴脱羧酶一般以二聚体的形式存在于哺乳类和昆虫的多种神经和非神经组织中。本文从多巴脱羧酶的结构、催化机制、与神经类疾病及其攻击性社会行为的关联性研究进展等方面进行了综述。     

Inhibitory effects of tetrahydropapaverine on serotonin biosynthesis in murine mastocytoma P815 cells

The inhibitory effects of tetrahydropapaverine on serotonin biosynthesis in serotonin-producing murine mastocytoma P815 cells were investigated. Tetrahydropapaverine at concentration ranges of 5-20 microM decreased serotonin content in a concentration-dependent manner in P815 cells and showed 42.1% inhibition of serotonin content at 5.0 microM at 24 hr. The value of 50% inhibitory concentration, IC50, of tetrahydropapaverine was 6.2 microM. Under these conditions, tryptophan hydroxylase (EC 1.14.16.4, TPH) was inhibited for 24-36 hr after treatment with tetrahydropapaverine in P815 cells (49.1% inhibition at 7.5 microM). However, aromatic L-amino acid decarboxylase activity was not affected by tetrahydropapaverine. In addition, tetrahydropapaverine inhibited the activity of TPH, prepared from the P815 cells (P815-TPH), with the IC50 value of 5.7 microM. Tetrahydropapaverine un-competitively inhibited P815-TPH with the substrate L-tryptophan, and non-competitively with the cofactor DL-6-methyl-5,6,7,8-tetrahydropteridin. The Ki value of tetrahydropapaverine with L-tryptophan was 10.1 microM. These data indicate that tetrahydropapaverine leads to a decrease in serotonin content by the inhibition of TPH activity in P815 cells.     

Cellular distribution of insulin, glucagon, pancreatic polypeptide hormone and somatostatin in the fetal and adult pancreas of the guinea pig: a comparative immunohistochemical study

Antibodies to insulin, glucagon, pancreatic polypeptide hormone and somatostatin were utilized to demonstrate the cellular localization of the hormones in pancreatic tissue of fetal guinea pig of advanced gestation by immunofluorescence histochemistry. The topographical distribution of the 4 endocrine cell types was compared with those of the adult pancreas and was found to be significantly different particularly for cells immunostaining for insulin, glucagon and somatostatin. These observations suggest changes in histogenesis of pancreatic endocrine cells during transition from fetal to postnatal and adult life. The presence of the 4 islet hormones in the fetal pancreas of this species implies that they may be important in fetal metabolism and growth.     

Disordered branched chain amino acid catabolism in pancreatic islets is associated with postprandial hypersecretion of glucagon in diabetic mice

Dysregulation of glucagon is associated with the pathophysiology of type 2 diabetes. We previously reported that postprandial hyperglucagonemia is more obvious than fasting hyperglucagonemia in type 2 diabetes patients. However, which nutrient stimulates glucagon secretion in the diabetic state and the underlying mechanism after nutrient intake are unclear. To answer these questions, we measured plasma glucagon levels in diabetic mice after oral administration of various nutrients. The effects of nutrients on glucagon secretion were assessed using islets isolated from diabetic mice and palmitate-treated islets. In addition, we analyzed the expression levels of branched chain amino acid (BCAA) catabolism-related enzymes and their metabolites in diabetic islets. We found that protein, but not carbohydrate or lipid, increased plasma glucagon levels in diabetic mice. Among amino acids, BCAAs, but not the other essential or nonessential amino acids, increased plasma glucagon levels. BCAAs also directly increased the intracellular calcium concentration in α cells. When BCAAs transport was suppressed by an inhibitor of system L-amino acid transporters, glucagon secretion was reduced even in the presence of BCAAs. We also found that the expression levels of BCAA catabolism-related enzymes and their metabolite contents were altered in diabetic islets and palmitate-treated islets compared to control islets, indicating disordered BCAA catabolism in diabetic islets. Furthermore, BCKDK inhibitor BT2 suppressed BCAA-induced hypersecretion of glucagon in diabetic islets and palmitate-treated islets. Taken together, postprandial hypersecretion of glucagon in the diabetic state is attributable to disordered BCAA catabolism in pancreatic islet cells.     

Immunohistochemical study on the endocrine pancreas of cattle with special reference to coexistence of serotonin and glucagon or bovine pancreatic polypeptide

Bovine pancreatic endocrine cells were investigated by light microscopic immunohistochemistry. Serotonin-immunoreactive cells as well as insulin-, glucagon-, somatostatin-, bovine pancreatic polypeptide (BPP)-immunoreactive cells were detected in the pancreatic islets. Generally, insulin-immunoreactive cells were distributed throughout the islet and the others took peripheral location. Since the distribution and shape of serotonin-immunoreactive cells were very similar to glucagon- and BPP-immunoreactive cells, serial sections were restained by using the elution method. All glucagon- and BPP-immunoreactive cells also showed serotonin immunoreactivity but glucagon and BPP immunoreactivities were never observed to be colocalized in the same cell. A small number of serotonin-immunoreactive cells were observed that showed serotonin immunoreactivity only.     

Structure of the Rat Aromatic L-Amino Acid Decarboxylase Gene: Evidence for an Alternative Promoter Usage

Abstract: Aromatic L-amino acid decarboxylase catalyzes the biosynthesis of the neurotransmitters dopamine and serotonin. This enzyme is also expressed in nonneuronal tissues. Two reported cDNA sequences show that the pheochromocytoma message differs from the liver message only at the 5'untranslated region. We present the complete exonal organization and promoter sequences of the rat gene encoding this enzyme. The rat aromatic L-amino acid decarboxylase gene is composed of two promoters and 16 exons spanning more than 80 kb in the genome. The first exon carries the majority of the 5'untranslated sequence of the liver cDNA, and the second exon carries that of the pheochromocytoma cDNA. In the third exon, there are two alternatively utilized splicing acceptors specific to the first and second exons. Therefore, both alternative promoter usage and alternative splicing are operative for the differential expression of this gene. The sequence of each promoter region shows putative binding sites for octamer factors and AP-2.