Immunohistochemical localization of three catecholamine synthesizing enzymes: aspects on methodology

Summary Three enzymes in the catecholamine synthesis—dopa decarboxylase (DDC), dopamine--oxidase (DBH) and phenylethanolamine-N-methyltransferase (PNMT)—have been isolated. Subsequently antibodies to these enzymes were prepared and used in immunohistochemical studies mainly with the aim to elucidate methodological problems. The indirect immunofluorescent technique was used throughout the study.It was found that cryostat sectioning followed by fixation in acetone or alcohol, a standard procedure in immunohistochemistry, was successful only with antibodies to the granule bound enzyme DBH. With antibodies to DDC and PNMT, two cytoplasmic enzymes, on the other hand, the results were hampered by diffusion artefacts. These drawbacks could be prevented by a brief aldehyde fixation, preferably by perfusion before cryostat sectioning. The best results were obtained with formalin followed by hydroxyadipaldehyde and acrolein. However, after glutaraldehyde fixation no specific fluorescence at all was observed.Freezing of fresh adrenals, followed by freeze-drying, treatment with formaldehyde vapours and paraffin embedding was tested but consistent results were only obtained with antibodies to PNMT.A new instrument, the Vibratome^®, which allows sectioning of unembedded fixed or unfixed tissue, was used and successful results were obtained with all three antibodies. Furthermore, the possibility with this instrument to combine the immunohistochemical technique e.g. with the formaldehyde fluorescence method for visualization of monoamines is demonstrated. It is emphasized that the Vibratome ^® technique may be a valuable tool for immunohistochemical studies on the central nervous system.     

Fluorescence of indolylethylamines condensed with formaldehyde

Summary The reaction between some indolylethylamines and formaldehyde has been studied in model protein layers. 6-Hydroxytryptamine and 5,6-dihydroxytryptamine give a specific formaldehyde-induced fluorescence similar to that of 5-hydroxytryptamine and tryptamine with an emission peak in the region of 490–525 m. Although each substance has its specific fluorescence spectrum, it is not possible to safely differentiate them visually. They can, however, be distinguished by the use of microspectrophotofluorimetry. The formaldehyde-induced fluorescence of 6-hydroxytryptamine has a fluorescence intensity about four times greater than that of 5-hydroxytryptamine, and is in the same order of magnitude as that of noradrenaline.Abbreviations Used 5-HT 5-hydroxytryptamine - -m-5-HT -methyl-5-hydroxy-tryptamine - 6-HT 6-hydroxytryptamine - 5,6-diHT 5,6-dihydroxytryptamine - NA noradrenaline - T tryptamine     

Immunohistochemical localization of tonin in rat salivary glands and kidney

Summary As a part of a microfluorometric investigation of the nucleoproteins of nuclei whose chromatin displays varying degrees of condensation, a comparison was made of mouse small thymocyte and hepatocyte nuclei stained with the acidic dye, brilliant sulfaflavine, at pH 2.8. These estimates of total protein content were compared with measurements obtained in similarly stained nuclei after extraction either with 0.4 N H₂SO₄ to remove all histones or with 0.35 M NaCl to remove nucleoplasmic proteins and some loosely bound non-histone chromosomal proteins. Treatment with 5% TCA at 60°C was used to remove nucleic acids and to reverse the effects of formaldehyde fixation. In all instances, the fluorescence of 2c hepatocyte nuclei greatly exceeded that of similarly treated thymocyte nuclei. While extraction with 0.4 N H₂SO₄ resulted in reductions of as much as 75% of the total fluorescence of small thymocyte nuclei, the losses of fluorescence in 2c hepatocyte nuclei amounted to only 20–30%. Nevertheless, the absolute values of fluorescence lost in both types of nuclei were very similar. After extraction in 0.35 M NaCl, thymocyte nuclei displayed slightly greater fluorescence than control thymocyte nuclei, while the total fluorescence of hepatocyte nuclei declined. In hepatocyte nuclei extracted with TCA, with and without treatment with 0.35 M NaCl, two populations of diploid nuclei were apparent: one corresponding to parenchymal cell nuclei and the other comprised of non-parenchymal cell nuclei. Only single diploid populations were visible in acid-extracted material. The ratios of 4c2c, 8c4c, and 8c2c hepatocyte nuclei in control, acid-extracted, and NaCl-extracted groups were generally lower than the expected 224 values. These results indicate that total nuclear histones may be estimated microfluorometrically by computing the difference between acid-extracted and unextracted preparations treated in otherwise equivalent ways. In addition, despite very similar absolute losses of fluorescence after removal of histones in thymocyte and 2c hepatocyte nuclei, the proportion of total protein ascribable to histones is much greater in thymocyte nuclei than in 2c hepatocyte nuclei — or, conversely, the percentage of total protein attributable to non-histone proteins is much greater in 2c hepatocyte nuclei than in thymocyte nuclei.     

Synthesis of peptides containing 5‐hydroxytryptophan,oxindolylalanine, N‐formylkynurenine and kynurenine

ROS, continuously produced in cells, can reversibly or irreversibly oxidize proteins, lipids, and DNA. At the protein level, cysteine, methionine, tryptophan, and tyrosine residues are particularly prone to oxidation. Here, we describe the solid phase synthesis of peptides containing four different oxidation products of tryptophan residues that can be formed by oxidation in proteins in vitro and in vivo: 5‐HTP, Oia, Kyn, and NFK. First, we synthesized Oia and NFK by selective oxidation of tryptophan and then protected the ${\bf \alpha}$ ‐amino group of both amino acids, and the commercially available 5‐HTP, with Fmoc‐succinimide. High yields of Fmoc‐Kyn were obtained by acid hydrolysis of Fmoc‐NFK. All four Fmoc derivatives were successfully incorporated, at high yields, into three different peptide sequences from skeletal muscle actin, creatin kinase (M‐type), and ${\bf \beta}$ ‐enolase. The correct structure of all modified peptides was confirmed by tandem mass spectrometry. Interestingly, isobaric peptides containing 5‐HTP and Oia were always well separated in an acetonitrile gradient with TFA as the ion‐pair reagent on a C₁₈‐phase. Such synthetic peptides should prove useful in future studies to distinguish isobaric oxidation products of tryptophan. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Optimization of reversed-phase microcapillary liquid chromatography for quantitative proteomics

A specific capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) method for the determination of serotonin (5HT) and its precursors tryptophan (Trp) and 5-hydroxytryptophan (5HTP) in human platelet rich plasma is described. The analytes were removed from the plasma samples and preconcentrated by solid phase extraction (SPE) on mixed mode cation-exchange sorbents. The SPE recoveries were 71.6 +/- 3.1 for 5HT, 91.0 +/- 2.8 for Trp, and 95.3 +/- 5.9% for 5HTP. Deuterated analogues of 5HT and Trp were used as internal standards for quantitation purposes. Submicromolar detection limits were obtained for standard mixtures of all compounds and their deuterated isotopes, except 5HTP, which had detection limits in the low micromolar range. The potential usefulness of this method in the clinical setting was demonstrated by analyzing plasma extracts from healthy volunteers as well as from pathological samples. While 5HTP was not present in any of the analyzed samples, the levels of 5HT and Trp in both normal and pathological plasma were determined.     

Responses of wheat plants to nutrient deprivation may involve the regulation of water-channel function

The sap flow (Jv) and the osmotic hydraulic conductance (L₀) of detached, exuding root systems from wheat (Triticum aestivum L. cv. Chinese Spring) plants deprived of nitrogen for 5 d (— N) or of phosphorus for 7 d (—P), were measured and compared with controls receiving a complete nutrient supply. In the roots of — N and — P plants, Jv and L₀ decreased markedly, but between 4 and 24 h after resupplying N to — N plants (NRS plants) and P to — P plants (PRS plants), Jv and Lo recovered to values similar to those of control plants. Values of Jv and L₀ were always greater during the light period than during the dark, due to the diurnal variation of these parameters. Reducing transpiration in the light had no effect on Jv and L₀ of — N and — P plants. Sap flow and L₀ were also determined using individual axes from plants which had been grown with their roots divided between nutrient-deficient (- N or- P) solution and a complete nutrient solution. Differences were observed in Jv and L₀ between axes of the same plant, but stomatal conductance (Gs), which was also measured, was not affected in these split-root experiments. In control plants, Jv and L₀ declined sharply to values similar to those of roots from — N and — P plants after HgCl₂ treatment (50 M), but were restored by treating with 5 mM dithiothreitol. In plasma membranes from — N and — P roots, the amount of stigmasterol increased relative to sitosterol compared with control roots. The degree of unsaturation of bound fatty acids also increased, compared with controls, as a result of a decline in the relative amounts of 160 and 180 and an increase in 182. Plasma-membrane fluidity, estimated by steady-state fluorescence polarisation using 1,6-diphenyl hexatriene, showed that the plasma membranes from nutrient-deprived plants were less fluid than those from control plants, measured during both the light and dark periods and in split-root experiments. In NRS plants, the relative abundance of sitosterol increased, so that the stigmasterol/sitosterol ratio returned to a value similar to that of controls. However, in PRS plants, the difference in stigmasterol/sitosterol ratio was maintained, compared with controls. The degree of unsaturation of bound fatty acids, membrane fluidity and the hydraulic conductivity of root systems also recovered in NRS and PRS plants to values similar to those of control plant plasma membranes. The results obtained suggested that — N and — P treatment decreased L₀, by reducing either the activity or the abundance of Hg-sensitive water channels. Also, there may be an interaction between the increase in membrane lipid ordering and the decrease in L₀.Abbreviations DTT dithiothreitol - Gs stomatal conductance - Js solute flow into the sap - Jv sap flow - L₀ hydraulic conductance - - N nitrogen-deprived for 5 d - NRS nitrogen resupplied after 5 d deprivation - - P phosphorus-deprived for 7 d - PRS phosphorus resupplied after 7 d deprivation M.C. was funded by a grant from CSIC, Spain. IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.     

Fluorescence histochemical demonstration of dopa thioethers by condensation with gaseous formaldehyde

Summary The usefulness of the formaldehyde (FA) and glyoxylic acid (GA) methods for the fluorescence histochemical demonstration of dopa thioethers has been tested using protein droplet models. Similar fluorescence intensities were recorded from these compounds after either FA or GA treatment. Cysteinyldopa gave a high fluorescence yield similar to that obtained from dopamine and dopa in the FA reaction, whereas glutationedopa showed a lower, although clearly visible fluorescence. Since the FA method seemed to be the most useful one for demonstration of catechol thioethers, the FA-induced fluorophores of these compounds were further characterized by microspectrofluorometry. The spectral characteristics of the thioether fluorophores (excitation maxima at 420 nm and emission maxima at 480–485 nm) distinguish these substances from dopa and other compounds fluorogenic in the Falck-Hillarp method. Dopa thioethers are proposed to form fluorophores with FA in a manner analogous to that of the primary catecholamines i.e. via low-fluorescent tetrahydroisoquinolines, along two different pathways, to strongly fluorescent 3,4-dihydroisoquinolines and 2-methyl-dihydroisoquinolinium compounds. These dihydroisoquinolines are in a pH-dependent tautomeric equilibrium with their quinoidal forms as reflected by a characteristic spectral shift upon acidification. The results of this study provide the guide-lines for the characterization of fluorogenic compounds in pigment-forming cells.     

Pharmacological profile of a model for central serotonin receptor activation

The head shake response in rats after systemic administration of the serotonin (5HT) precursor 5-hydroxytryptophan (5HTP) was pharmacologically characterized and shown to be a useful animal model to quantify brain 5HT receptor activation. The behavior occurred in a dose-dependent manner after injection of 5HTP and the 5HT agonist quipazine. Head shakes were also observed after injection of L-tryptophan, 5-methoxydimethyltryptamine and fenfluramine. The 5HT antagonists cyproheptadine and metergoline were potent blockers of the response. Xylamidine, a peripheral 5HT antagonist, had no effect on head shaking. Inhibition of 5HT uptake with fluoxetine potentiated the head shake response after 5HTP. Manipulation of central cholinergic or GABAergic mechanisms did not alter 5HTP-induced shakes. Alpha-noradrenergic receptor blockade had no significant effect on head shakes. However, desmethylimipramine was equipotent with methysergide as an antagonist of the behavior. Beta-noradrenergic receptor blockade had no specific effect on 5HTP head shakes. Concomitant dopamine receptor activation with SK&;F 38393 did not affect head shakes but the neuroleptics chlorpromazine and pimozide reduced the number of head shakes after 5HTP. The H₁ receptor antagonist pyrilamine had no effect on head shakes. It is concluded that 5HTP-induced head shakes in rats is a quantitative model of brain 5HT receptor activation which is particularly sensitive to 5HT antagonists.     

Microfluorometric investigations of chromatin structure

Summary The ability of the highly condensed chromatin of small thymocyte nuclei and the more loosely organized chromatin of hepatocyte nuclei to interact with nine DNA-specific fluorochromes was assessed by microfluorometry. Although the results obtained with five of the fluorochromes—mithramycin, 7-aminoactinomycin d, Hoechst 33258, DAPI, and propidium iodide—were found to be virtually unaffected by differences in the degree of condensation of the chromatin, the values obtained with the remaining fluorochromes—proflavine, quinacrine mustard, berberine sulfate, and pyronin Y—appeared to be affected significantly by organizational differences of the chromatin. All of the latter structural probes, except quinacrine mustard, produced fluorescence values which were higher in the 2c nuclei of hepatocytes than in the nuclei of small thymocytes. Quinacrine mustard yielded higher values in thymocyte nuclei; and in the hepatocyte polyploid series (2, 4, and 8c), it did not produce the expected multiples of the 2c value. Pretreatment of the two types of nuclei with RNase affected their total fluorescence in unpredictable ways. While RNase extraction lessened the differences between thymocyte and 2c hepatocyte nuclei stained with propidium iodide, Hoechst 33258, proflavine, and berberine sulfate, it increased the differences between nuclei stained with mithramycin, quinacrine mustard, pyronin Y, and 7-aminoactinomycin d. The ability of RNA-depleted chromatin to interact with various types of fluorochromes might be a useful parameter in subsequent studies of chromatin organization.     

Effects of Livagen and Epitalon, New Peptide Bioregulators, on Enkephalin-Degrading Enzymes from Human Serum

The effects of new peptide bioregulators—Livagen (Lys-Glu-Asp-Ala) and Epitalon (Ala-Glu-Asp-Gly)—on the endogenous opioid system was studied. In particular, attention was focused on their ability to change the activity of enkephalin-degrading enzymes of blood serum and interact with opioid receptors of the brain membrane fraction. Enkephalinase activity was assayed in vitro by the rate of ³H-Leu-enkephalin hydrolysis in the presence of Livagen and Epitalon. These peptides inhibited enkephalin-degrading enzymes of human serum. Livagen proved to be more efficient than some well-known peptidase inhibitors, such as puromycin, leupeptin, and D-PAM. The dose–inhibitory effect curves for Livagen and Epitalon were plotted; their IC₅₀ equaled 20 and 500 M, respectively. The interaction between the peptides and opioid receptors was estimated using a radioreceptor method with [³H][D-Ala², D-Leu⁵]-enkephalin. No interaction was observed between the peptides and - or -opioid receptors of the membrane fraction from the rat brain.     

The effects of the perinatal treatment with 5-hydroxytryptophan or tranylcypromine on the peripheral and central serotonin homeostasis in adult rats

Serotonin (5HT) is a biologically active amine present in mammals in the brain and the peripheral tissues. Autism is a neurodevelopmental disorder in which 5HT homeostasis is disturbed both centrally and peripherally, but the relationship between the 5HT disturbances in the two compartments is not understood. In an attempt to explore the relationship between the disturbed peripheral 5HT homeostasis and central 5HT functioning, we exposed the developing rat brain to increased 5HT concentrations, by treatment of rats with subcutaneous injections of the immediate 5HT precursor 5-hydroxy-l-tryptophan (5HTP, 25 mg/kg), or the non-selective MAO inhibitor tranylcypromine (TCP, 2 mg/kg), during the period of the most intensive development of 5HT neurons - from gestational day 13 to post-natal day 21. The effects of the mentioned treatments on peripheral and central 5HT levels were then studied in adult rats. Platelet and plasma 5HT concentrations (measured by ELISA), as well as cortical and midbrain 5HT, tryptophan and 5-hydroxyindoleacetic acid levels (measured by HPLC) were determined in twelve 5HTP treated and eight TCP treated rats, and compared with the values measured in 10 control, saline treated rats. Treatment with 5HTP significantly raised peripheral but not central 5HT concentrations. At adult age, peripheral 5HT homeostasis was re-established, while modest decrease in 5HT concentration was observed in frontal cortex, presumably due to hyperserotonemia-induced loss of 5HT terminals during brain development. Treatment with TCP induced significant 5HT elevations in both compartments. At adult age, permanent changes in 5HT homeostasis were observed, both peripherally (as hyperserotonemia) and centrally (as altered 5HT metabolism with decreased 5HT concentrations). Further studies are planned in order to explore the nature of the different disturbances of 5HT homeostasis induced by the two compounds, and their results are expected to shed some light on the role of hyperserotonemia in autism.     

Histochemically demonstrable monoamines in the pituitary gland and median eminence of the female rat during the postnatal development

Summary The postnatal development of formaldehyde induced fluorescence (FIF) was studied in the pituitary glands of female rats. The effects of 3,4-dihydroxyphenylalanine (L-dopa), D,L-5-hydroxytryptophan (DL-5-HTP) and dopamine (DA) treatments on the FIF were followed during the postnatal period.The appearance of specifically fluorescing monoamines into the cells of the pars intermedia occurred postnatally and the level of the adult fluorescence was reached at 4–5 weeks' age. The intensity of the fluorescence was independent on the density of the fluorescing nerves. Among the fluorescing nerves droplet fibres were regularly observed from the age of 3 weeks, which confirms the theory that these fibres are caused by toxic factors when the blood-brain barrier is not functioning.There was no change postnatally in the number of fluorescing cells in the pars distalis.The fluorescing innervation of the median eminence, developed most rapidly at the age of 1–3 weeks and the level of the adult fluorescence was reached at the age of 4–5 weeks.The first specifically fluorescing cells after L-dopa treatment were observed at 6 days age. A remarkable increase in the number of fluorescing cells was seen between 12 and 18 days. After DL-5-HTP treatment fluorescent cells were seen but at later stages. These observations suggest that the cells in the pituitary gland, which store amine-precursors and monoamines developmentally differ from the APUD-cells. The rapid increase of the fluorescing cells between 12 and 18 days and the simultaneous development of the fluorescing innervation of the median eminence suggest the following correlations: the development of dopaminergic innervation of the median eminence — the secretion of releasing hormones — the activity of PAS-positive cells (FSH, LH and TSH secretion) — the uptake of L-dopa and DL-5-HTP into the PAS-positive cells.Dopamine was not uptaken into the cells of pars distalis. The walls of the blood vessels began to show fluorescence suggesting a barrier mechanism, which prevents the DA-uptake into the PAS-positive cells.This work was supported by the Grant for Young Research Workers, University of Helsinki.     

High-performance liquid chromatographic determination of short-chain aliphatic aldehydes using 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole as a fluorescence reagent

High-performance liquid chromatographic determination of four short-chain aliphatic aldehydes using fluorescence detection was carried out with 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole (DBD-H). DBD-H derivatives with three aliphatic aldehydes — formaldehyde, acetaldehyde and propionaldehyde — were synthesized and their fluorescence properties were examined. Relative fluorescence intensities of these compounds in acetonitrile were ca. ten-fold larger than those in aqueous acetonitrile. DBD-hydrazones could be separated by reversed-phase chromatography using aqueous acetonitrile as eluent and detection at 560 nm with excitation at 445 nm. Submicromolar levels of formaldehyde, acetaldehyde, propionaldehyde and butylaldehyde could be determined. The HPLC procedure using propionaldehyde as internal standard was applied to the measurement of acetaldehyde levels in normal human plasma before and 30 min after ingestion of ethanol.     

New aspects on factors determining the sensitivity of the formaldehyde and glyoxylic acid fluorescence histochemical methods for monoamines

Summary The fluorophore and fluorescence yield from tryptamine and 3-methoxytyramine in histochemical protein models have been compared in the standard formaldehyde reaction, the acid-catalyzed formaldehyde reaction, the formaldehyde-ozone reaction, and the aluminum-formaldehyde reaction. In the standard formaldehyde reaction both the fluorophore and fluorescence yields are low. However, the other reactions give a dramatic increase in fluorescence intensity (18–20 times) from tryptamine and 3-methoxytyramine whereas only minor changes (up to 100% increase) in fluorophore yield are observed. It is concluded that the relative fluorescence intensity of each fluorophore molecule formed in the three modifications of the formaldehyde reaction is much higher than that of the molecules formed in the standard formaldehyde reaction. It has previously been demonstrated that the fluorophores formed from dopamine in the gaseous formaldehyde and glyoxylic acid reactions have a much higher (10 times) relative fluorescence intensity than the synthetic fluorophores. The present experiments show that if the histochemical models are dissolved in buffer after the reaction and new models are made from this solution, the fluorescence intensity of the fluorophores formed in the reaction is drastically reduced and becomes comparable to that of the synthetic ones. The results of this and our previous studies indicate that hitherto unknown fluorescence enhancing mechanisms play a major role for the fluorescence yield, i.e. the sensitivity, in the various formaldehyde and glyoxylic acid methods. One possible explanation to the high relative fluorescence intensity of the fluorophores formed in the histochemical reactions could be an energy transfer between, e.g. the non-fluorescent intermediary reaction products (the tetrahydro derivatives) and the fluorophores (the dihydroisoquinolines and dihydro--carbolines). Such an energy transfer is probably attenuated in the dissolved models, where the distances between and orientations of the various molecules have been changed.Abbreviations DA dopamine - FA formaldehyde - GA glyoxylic acid - 3-MT 3-methoxytyramine - 4-MT 3-hydroxy-4-methoxyphenylethylamine - T tryptamine - DHC dihydro--carbolines - THC tetrahydro--carboline - 2-Carb.Me-DHIQ 2-carboxymethyl-3,4-dihydroisoquinolinium compound - THIQ-1-COOH tetrahydroisoquinoline-1 carboxylic acid     

Immunogenicity of synthetic peptides related to the core peptide sequence encoded by the human MUC1 mucin gene: Effect of immunization on the growth of murine mammary adenocarcinoma cells transfected with the human MUC1 gene

The immune response of CAF1 mice to various synthetic peptides (SP) related to the amino acid sequence (PDTRPAPGSTAPPAHGVTSA) of the tandem repeat of the MUC1 human breast mucin core peptide was evaluated. The most immunogenic preparations of the synthetic peptides were those conjugated to keyhole limpet hemocyanin (KLH) or clustered in a dendritic multiple antigenic peptide (MAP-4) configuration. The mice were immunized subcutaneously with synthetic peptides emulsified in RIBI adjuvant, employing various immunization protocols. Equivalently high IgG responses were induced using SP-KLH conjugates (GVTSAPDTRPAPGSTA-KLH) or an SP — MAP-4 chimeric configuration (SP1-6), which also included a universal malarial CST-3 T-helper epitope (SP1-6 = SAPDTRPAEKKIAKMEKASSVFNVVNS — MAP-4). These IgG antibodies bound both the appropriate MUC1 synthetic peptides and the cell surface expressed MUC1 mucin on murine mammary cells that had been transfected with the human MUC1 gene and a human breast cancer cell line that expresses cell-surface MUC1. A MAP-4 molecule, which included the entire 20-aminoacid sequence of the MUC1 tandem repeat (SP1-5 = PDTRPAPGSTAPPAHGVTSA—MAP-4) induced a poor IgG response. In contrast, all three types of molecule: SP-KLH, SP1-6 and SP1-5, were found to be good immunogens for the induction of specific delayedtype hypersensitivity (DTH) reactions measured using either synthetic peptides or MUC1-transfected cells. In addition, immunization with irradiated MUC1-transfected cells induced strong DTH reactions measured using synthetic peptides that expressed the PDTRP sequence, which has been shown to be, or to overlap, a T cell epitope in humans and a B cell epitope in mice. Finally, it was demonstrated that synthetic MUC1 peptide vaccines could be used both prophylactically and therapeutically to inhibit the growth of MUC1-transfected tumor cells and prolong the survival of tumor-bearing mice.     

Uptake of norepinephrine by isolated neurons of rat pontine reticular formation

Summary Neurons isolated from the nucleus reticularis pontis caudalis and the lateral vestibular nucleus of the rat brain stem have been incubated in buffered sucrose containing norepinephrine. Norepinephrine accumulated intracellularly was then visualized by fluorescence microscopy after formaldehyde condensation.Incubation in sucrose containing 1.0 g/ml norepinephrine resulted in a 2-fold increase in fluorescence that was blocked by 1×10^–5M cocaine. Maximal enhancement of fluorescence, accomplished by incubation of isolated neurons with 40 g/ml norepinephrine, was unaffected by cocaine but effectively blocked by 1×10^–3M metanephrine. Accumulation of norepinephrine was unaffected by pretreatment of animals with reserpine and nialamide.These properties of norepinephrine uptake suggest a neuronal membrane transport system for norepinephrine in isolated neurons similar to Uptake 2 which occurs extraneuronally. Although the intracellular localization of exogenous norepinephrine accumulated is not known, some concentration of norepinephrine in the perikaryal cytoplasm is suggested.This work was supported by U.S. Public Health Service Research Grant NB 07044. The competent technical assistance of Mrs. Gisela Griffith and Miss Susan J. Decker is acknowledged with gratitude.     

Studies on uptake of intraventricularly administered tritiated noradrenaline and 5-hydroxytryptamine with combined fluorescence histochemical and autoradiographic techniques

Summary Tritiated noradrenaline (NA) and 5-hydroxytryptamine (5-HT) (1.5–30 C) have been injected intraventricularly into normal or reserpine-nialamide pretreated rats 1/2 to 2 hours before the killing. Various parts of the brains were freeze-dried, reacted with formaldehyde gas and embedded in paraffin or Araldite. Before application of the stripping film emulsion many sections were photographed in the fluorescence microscope in order to perform a combined histochemical and autoradiographic study of the monoamine neurons. By such an approach it was possible to demonstrate 1. that the accumulation of radioactivity in cell bodies after ³H-NA and ³H-5-HT injection is localized to catecholamine (CA) and 5-HT cell bodies respectively; 2. that injected ³H-NA and ³H-5-HT in the doses used relatively selectively are taken up into the NA and 5-HT nerve terminals respectively, since the distribution of grains in the sections follow that of the fluorescent terminals; 3. that the accumulation of silver grains only reaches the zone (200–400 ) close to the ventricles and the ventral part of the subarachnoidal space. By grain counting it was possible to estimate that the degree of concentration of radioactivity in the monoamine cell bodies was up to 4 times that in the immediate surroundings. — The Araldite sections consistently gave a better resolution in the autoradiographic picture than the paraffin sections. It is postulated that freeze-drying and plastic embedding for autoradiography will be a valuable method for the cellular demonstration of certain biogenic amines which are not easily demonstrated by the histochemical fluorescence method and of other biologically active water-soluble compounds, since diffusion will be restricted to a minimum.This work has been supported by grants from the Medical Research Council (14X-715-04A, B69-14X-530-04) and by grants from M. Bergvalls Stiftelse and E. och O. Ericssons Stiftelse.     

Serotonin-containing epithelial cells in rat duodenum. II. Quantitative study of the effect of 5HTP administration

Summary The effect of 5-hydroxytryptophan (5HTP) administration on serotonin (5HT)-containing epithelial cells in rat duodenum was investigated quantitatively using three-dimensional morphometry to determine cell density and HPLC to measure 5HT and 5HTP concentrations. The results are interpreted in terms of the amine precursor uptake and decarboxylation (APUD) capacity of the cells. After administration of 5HTP, no significant change was observed in the density of 5HT-fluorescent epithelial cells in the duodenal region examined. Moreover, no evidence could be obtained that the concentration of 5HT in duodenal villi was increased after 5HTP administration, despite a highly significant increase in serum 5HTP and 5HT levels. These results indicate that no cells in the duodenal epithelium have the ability to decarboxylate exogenously administered 5HTP and convert it to 5HT under physiological conditions.     

Serotonin-containing epithelial cells in rat duodenum. II. Quantitative study of the effect of 5HTP administration

The effect of 5-hydroxytryptophan (5HTP) administration on serotonin (5HT)-containing epithelial cells in rat duodenum was investigated quantitatively using three-dimensional morphometry to determine cell density and HPLC to measure 5HT and 5HTP concentrations. The results are interpreted in terms of the amine precursor uptake and decarboxylation (APUD) capacity of the cells. After administration of 5HTP, no significant change was observed in the density of 5HT-fluorescent epithelial cells in the duodenal region examined. Moreover, no evidence could be obtained that the concentration of 5HT in duodenal villi was increased after 5HTP administration, despite a highly significant increase in serum 5HTP and 5HT levels. These results indicate that no cells in the duodenal epithelium have the ability to decarboxylate exogenously administered 5HTP and convert it to 5HT under physiological conditions.     

Fluorescence methods for the histochemical demonstration of monoamines

Summary The histochemical fluorescence method of Falck and Hillarp for the demonstration of catecholamines and certain tryptamines, e.g. 5-hydroxytryptamine is based on the principle that these amines can be condensed with formaldehyde to yield strongly fluorescent 6,7-dihydroxy-3,4-dihydroisoquinolines and 6-hydroxy-3,4-dihydro--carbolines respectively. The investigation here reported presents the fluorescence characteristics and relative fluorescence yields for formaldehyde treated biogenic monoamines and certain related compounds enclosed in a dried protein layer. The fluorescence properties of some synthetic 6,7-substituted-3,4-dihydroisoquinolines and 3,4-dihydro--carbolines are given, and the fluorescence characteristics in relation to the molecular structure are discussed.Abbreviations used A adrenaline - DA dopamine - DOPA 3,4-dihydroxyphenylalanine - DOPS 3,4-dihydroxyphenyl-serine - 5-HT 5-hydroxytryptamine - 5-HTP 5-hydroxytryptophan - 5-MT 5-methoxytryptamine - -m-DA -methyl-dopamine - -m-DOPA -methyl-3,4-dihydroxyphenylalanine - -m-NA -methyl-noradrenahne - MTA 3-methoxy-tyramine - NA noradrenaline - NM normetanephrine - T Tryptamine - Try Tryptophan