Binding of catecholamines and related compounds to proteins after formaldehyde gas treatment

Summary Primary catecholamines e.g. dopamine and noradrenaline easily condense with formaldehyde in both tissues and model protein layers, yielding strongly fluorescent 3,4-dihydroisoquinolines. This reaction can be used for the histochemical demonstration of biogenic monoamines according to the method of Falck and Hillarp. The investigation reported presents data which suggest that the isoquinolines formed are mainly enclosed in or adsorbed on the network formed by the formaldehyde tanning of the protein. The products seem not to be chemically bound. No difference was found in the binding of the different biogenic catecholamines. The degree of binding can be reinforced by prolonged formaldehyde gas treatment and the use of more humid gas, which might prove useful when this binding method is employed for the autoradiography of catecholamines and 5-hydroxytryptamine. The possibilities of chemical binding are discussed.     

Demonstration of catecholamine and resorcinolamine derivatives as formaldehyde-induced fluorescence in protein models

We assessed in protein droplet models the potential use of the formaldehyde condensation method for histochemical demonstration of a wide range of catecholamines and resorcinolamines. The experiments showed that all of the amines tested, except salbutamol and carbuterol, formed fluorophores, and that the fluorescence was specific [i.e., there was no fluorescence in the absence of formaldehyde, the fluorescence was quenched by water, and the fluorophores were subject to photodecomposition by the exciting (405-nm) light]. Peak wavelengths of the emission spectra were 480-485 nm for fluorophores of resorcinolamine derivatives. The fluorescence intensity of the catecholamines was greater than that of the resorcinolamines. Fluorophore formation was not hindered by substitution of t-butyl, phenylisoprophyl, or p-hydroxyphenylisopropyl on the amino-N in catecholamines (t-butylnorepinephrine, Cc24, Cc25, respectively) or resorcinolamines (terbutaline, Th1161, fenoterol, respectively), and fluorophores also formed for catecholamines with the amino-N in a ring structure (rimiterol) or with a long alkyl chain substituted on the amino-N (hexoprenaline). Our study showed that fluorescence microphotometry can be used to detect a range of drugs that are catecholamines or resorcinolamines, and hence it should be possible to use this technique to study the properties of dissipation of these amines in tissues.     

Simultaneous immunohistochemistry and autoradiography of peptides and 5-hydroxytryptamine in bird retina

A method was developed for the simultaneous demonstration of two specific cellular constituents. Cryostat sections of formaldehyde fixed tissue from retinae treated with (³H)-5-hydroxytryptamine were subjected to immunohistochemistry and subsequent autoradiography. The method takes advantage of the very efficient and specific uptake mechanism that many types of neurons possess which makes it possible to label them with radioactivity.With this method a study was made on the possible co-occurrence in the avian retina of 5-hydroxytryptamine on one hand and somatostatin, glucagon and substance P on the other. Substance P and 5-hydroxytryptamine were investigated in pigeon retina whereas 5-hydroxytryptamine and somatostatin or glucagon were investigated in chicken retina. Though a large number of cell bodies were examined no co-occurrence of 5-hydroxytryptamine and any of the peptides was found. The sensitivity of the method allows an assertion that if present, double labelled neurons are likely to number less than 0.5–1% of the respective populations.     

Microspectrofluorometric characterization of the fluorescent derivatives of biogenic amines produced by aqueous aldehyde (Faglu) fixation

Summary The fluorescent derivatives of the reaction between an aqueous aldehyde (Faglu) solution and the biogenic amines (5-hydroxytryptamine, dopamine and noradrenaline) have been examined in order to determine the conditions required for maximal fluorescence yield. The fluorescence intensity and spectra of the final reaction products have been characterized and found to be highly dependent on the pH of the reaction mixture. Fluorophores derived from catecholamines have maximal yield and are most easily characterized when the reaction is performed at pH 7.3, whilst those derived from 5-hydroxytryptamine have maximal yield and are most readily characterized when the reaction is performed at pH 10.0. The addition of potassium ferricyanide to the Faglu further enhances the fluorescence yield of 5-hydroxytryptamine-containing models and tissues at both pH 10.0 and pH 7.3. Using the modified Faglu reaction mixture, it has been possible to demonstrate 5-hydroxytryptamine in the central nervous system without the need for pharmacological manipulation.     

New methods for the histochemical demonstration of catecholamines, tryptamines, histamine and other arylethylamines by acid- and aldehyde-induced fluorescence

Synopsis The histochemical demonstration of catecholamines, tyramines, tryptamines, and related substances depends upon condensation reactions leading to ring closure, ultimately forming a fluorescentisoquinoline, quinonoid or -carboline. At present, formaldehyde is widely used for such reactions. In this paper the possibilities of other histochemical reagents are considered, and preliminary results obtained with fixative vapours of some other aldehydes (acetaldehyde, glutaraldehyde) and carboxylic acids (formic and acetic acids) are described.     

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.     

Noradrenaline nerve terminals in the hippocampal region of the rat and the guinea pig

Summary The hippocampal region of the rat has been studied with the histochemical fluorescence method for demonstration of catecholamines and 5-hydroxytryptamine. Noradrenalin nerve terminals of characteristic appearance were observed throughout the region, but the density of the terminals in the various areas differed considerably. The highest amount of terminals were seen in the hilus of the area dentata of the rat. Otherwise, noradrenalin nerve terminals constitute a small percentage of the afferents to the hippocampal region. Noticeable differences existed between rat and guinea-pig. In both species there was a conspicuous lack of stratification and delineation of areas as compared with the pictures seen with most other methods, which have revealed precise architectonic patterns.Supported by a Research Grant (12X-715-02) from the Swedish Medical Research council and by a Grant from Stiftelsen M. Bergwalls Minne.     

The simultaneous demonstration of adrenergic fibres and enteric ganglion cells

Synopsis A method for the demonstration of adrenergic nerves and enteric neurons at the same time has been developed by combining the fluorescence histochemical technique for catecholamines and the histochemical technique for NADH:Nitro BT oxidoreductase.The method consists of a short incubation of the laminae from the wall of the intestine in an isotonic medium containing the substrate (NADH) and a tetrazolium salt (Nitro BT). After washing, the laminae are air dried, exposed to formaldehyde vapour and mounted.The adrenergic nerves in the myenteric plexus appear brightly fluorescent on excitation with u.v. light, whereas the neurons are heavily stained by deposits of formazan. Not all the neurons of the plexus are stained, but their morphology is well preserved. Differences in staining of the neurons reflect differences in penetration of the tetrazolium salt in the tissue and into the cells. The adrenergic axons do not establish exclusive connexions with individual neurons and some isolated neurons are not associated with any adrenergic fibres.     

Formaldehyde-induced fluorescence of peptides with N-terminal 3,4-dihydroxyphenylalanine or 5-hydroxytryptophan

Summary After formaldehyde condensation according to Falck and Hillarp, peptides with N-terminal (but not with C-terminal) dopa or 5HTP—enclosed in a dry protein matrix—emitted intense fluorescence with spectral characteristics similar to those of the formaldehyde induced fluorophores of catecholamines and 5HT respectively. The fluorescence of the peptides (as well as of the amines) could be abolished by treatment with sodium borohydride and regenerated by renewed formaldehyde condensation. Treatment with HCl vapor after condensation with formaldehyde caused a characteristic shift in the excitation maximum (from 410 to 375 m) of the dopyl-peptides, similar to that previously reported for dopamine.Abbreviations used dopa 3,4-Dihydroxyphenylalanine - 5 HTP 5-hydroxytryptophan - 5HT 5-hydroxytryptamine Peptides with N-terminal dopa or 5HTP are referred to as dopylpeptides and 5HTPyl-peptides, respectively.     

Histochemistry of the epimerases

Summary On the assumption that electron transfer is involved in the process of epimerization and by using tetrazolium salts as an indicator, a histochemical reaction for the demonstration of UDPGal-4-epimerase has been developed.By using UDPG or UDPGal as substrates it has been possible to ascertain the direction of the reaction catalysed by this enzyme in various tissues in normal physiological conditions.Biochemical tests support the concept that the histochemical reactions recorded were the result of UDPGal-4-epimerase activity.     

Liquid formaldehyde in catecholamine studies

Summary Based on the fluorescence technique for demonstration of catecholamines a method has been elaborated using liquid, 100% formaldehyde and embedding in Epon. This method has been applied for a comparative light-, fluorescence- and electron microscopical study of the adrenal medulla. In the supraoptic nucleus this fluorescence technique as well as conventional electron microscopy have been used for the localization of synaptic terminals.     

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     

Blood Concentrations of Adrenaline in Dogs after Intravenous Administration of 5-Hydroxytryptamine

THE release of catecholamines from the adrenal medulla of the cat by close intra-arterial injection of 5-hydroxytryptamine (5-HT) has been demonstrated by Reid¹. We have shown a similar release of catecholamines following intravenous injections of 5-HT in the dog, identified the catecholamine as adrenaline and demonstrated an enhancement of the release by treatment of the dog with hexamethonium.     

Water-stable fluorophores,produced by reaction with aldehyde solutions,for the histochemical localization of catechol- and indolethylamines

Summary The properties of a new fluorescence histochemical method for arylethylamines based on reaction with a mixture of 4% formaldehyde and 0.5% glutaraldehyde in aqueous solution are described. At room temperature the aldehyde mixture produced a well-localized fluorescence reaction in tissues, which, when examined microscopically in aqueous solution, was sufficiently intense for fine terminal noradrenergic axons to be seen. If the tissue was subsequently dried, the fluorescence intensity increased. At the same time as inducing the fluorophores, the aldehyde mixture fixed the tissue to a standard well suited for electron microscopy. It thus proved possible to locate amine containing cells in the fluorescence microscope and subsequently examine their ultrastructure. In aqueous models, the aldehyde mixture formed fluorescent products with adrenaline, noradrenaline, dopamine, dopa, 5-hydroxytryptamine and 5-hydroxytryptophan, but not with histamine or octopamine. The fluorescence induced in the aldehyde mixture remained stable if the tissue was subsequently transferred to saline or distilled water and when it was dehydrated in ethanol and cleared with xylene, benzene, chloroform or acetone.     

Water-stable fluorophores, produced by reaction with aldehyde solutions, for the histochemical localization of catechol- and indolethylamines.

The properties of a new fluorescence histochemical method for arylethylamines based on reaction with a mixture of 4% formaldehyde and 0.5% glutaraldehyde in aqueous solution are described. At room temperature the aldehyde mixture produced a well-localized fluorescence reaction in tissues, which, when examined microscopically in aqueous solution, was sufficiently intense for fine terminal noradrenergic axons to be seen. If the tissue was subsequently dried, the fluorescence intensity increased. At the same time as inducing the fluorophores, the aldehyde mixture fixed the tissue to a standard well suited for electron microscopy. It thus proved possible to locate amine containing cells in the fluorescence microscope and subsequently examine their ultrastructure. In aqueous models, the aldehyde mixture formed fluorescent products with adrenaline, noradrenaline, dopamine, dopa, 5-hydroxytryptamine and 5-hydroxytryptophan, but not with histamine or octopamine. The fluorescence induced in the aldehyde mixture remained stable if the tissue was subsequently transferred to saline or distilled water and when it was dehydrated in ethanol and cleared with xylene, benzene, chloroform or acetone.     

Simultaneous fluorescence histochemical demonstration of catecholamines and tryptophyl-peptides in endocrine cells.

Simple and efficient fluorescence histochemical methods for the concomitant demonstration of tryptophyl-peptide-containing cells and dopamine-containing cells have been developed in this study. Combined formaldehyde and chloral vapour or solution of 5% glyoxylic acid monohydrate in n-butanol induced concomitantly strong yellow fluorescence in the tryptophyl-peptide-containing cells and moderate green fluorescence in the dopamine-containing cells in the sections of the freeze-dried adenohypophysis.     

Cytofluorometric quantitation of 5-hydroxytryptamine in mast cells: an improved technique for the formaldehyde condensation reaction

A simple technique for the condensation of cellular 5-hydroxytryptamine (5-HT) with formaldehyde gas is described. The technique, which is especially suited for quantitative cytofluormetric studies, involves the generation of formaldehyde gas from dry paraformaldehyde in a closed reaction vessel with the addition of a measured quantity of water. The fluorescence yield of 5-HT was tested at various humidities. Optimal results were obtained with the addition of 100 mg water to a 1000 ml reaction vessel containing 6 g of dry paraformaldehyde. A major advantage of the method if the fact that the humidity during the reaction can be precisely controlled. The fluorescence yield of 5-HT, tested over a 50 day period showed excellent reproducibility. The stoichiometry of the reaction was tested by comparison of cytofluormetic data with that obtained by analysing the 5-HT content of pooled mast cells with an independent biochemical method. A highly satisfactory correlation (r = 0.96) was obtained within the range of 0.1 to 4 pg of 5-HT per cell. The limit of sensitivity of the cytofluorometric method was found to be of the order of 10(-13) g, and was determined by the fluorescence blank of the mast cells. This contributes to between 10 and 30 per cent of the total fluorescence emission from mast cells containing about 0.2 pg of 5-HT.     

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     

High-performance liquid chromatographic determination of indoleamines, dopamine, and norepinephrine in rat brain with fluorometric detection

A high-performance liquid chromatography-fluorescence procedure for the determination of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, tryptophan, dopamine, and norepinephrine has been developed. The method uses an ion-pairing system on an Ultrasphere ODS (5-microns) column with detector wavelength settings of excitation at 290 nm and emission at 330 nm. The procedure has been used to quantitate these indoleamines and catecholamines in rat brain tissue after homogenization in a perchloric acid solution; an aliquot of this solution is injected directly onto the HPLC column. Column sensitivities range from 6.1 pmol for tryptophan to 1.1 pmol for 5-hydroxytryptamine.     

Aspects of the arrangement of the adrenergic innervation in guinea-pigs as revealed by the fluorescence histochemical method applied to stretched,air-dried preparations

Summary The suitability of stretched, air-dried preparations for the fluorescence histochemical localization of catecholamines has been examined for a range of organs, including atrium, arteries, veins, digestive tract, urinogenital organs and sympathetic ganglia of the guinea-pig. Where possible, the tissue is divided into thin sheets under a dissecting microscope. It is then stretched over a glass slide to which it adheres as it dries. Stretch preparations can be easily and quickly prepared for the fluorescence localization of catecholamines and give clear and consistent results with many adrenergically innervated tissues. This technique allows the branching of adrenergic nerve trunks and of individual fibres to be readily followed. The relationships between fluorescent cells and other tissue elements can be traced quickly in situations where serial sections through the tissue would otherwise be necessary.