Differentiation of two types of endocrine cells which take up amine precursors using their capacity to take up the fluorescent dihydroisoquinoline derivative of dopamine.

A study was made of the accumulation of the strongly fluorescent 2-carboxymethyl-6,7-dihydroxy-3,4-dihydroisoquinolinium compound (2-Carb. Me-DIQ) derived from the condensation reaction of dopamine with glyoxylic acid in endocrine cells possessing the capacity to take up and store biogenic monoamine precursors. Thin-layer chromatographic studies of urine showed that 2-Carb. Me-DIQ was metabolized into two strongly fluorescent metabolites, possessing at least one hydroxyl group in the phenol moiety of the molecule, which were excreted in urine together with the parent compound. Histochemical observations, however, indicated that the tissue fluorescence showing maximal emission at 480 nm was due to 2-Carb. Me-DIQ. Generally, the injection of 2-Carb. Me-DIQ induced a strong fluorescence in those tissue components possessing the extraneuronal uptake mechanism of catecholamines. In the endocrine cells strong fluorescence was seen in the pineal glandular cells and in some cells of the pars distalis of the hypophysis, of which some cells also took up DL-5-HTP, as was seen following formaldehyde vapour treatment. No accumulation of 2-Carb. Me-DIQ was observed in the pancreatic islet cells, the C cells of the thyroid gland or the tracheal enterochromaffin-like cells. These findings lead to the conclusion that biogenic monoamines in the cells of the pars distalis of the hypophysis might use the phenolic moiety of the molecule to bind to some intracellular receptor. Thus, the pars distalis cells may have an intracellular binding mechanism for biogenic monoamines that is different from other endocrine cells showing the uptake and storage of biogenic monoamines. On the other hand, the findings gave further support to the suggestion that in the pancreatic islet cells, the thyroidal C cells and the tracheal enterochromaffin-like cells biogenic monoamines are stored by a mechanism in which the basic, positively charged amino group of biogenic monoamines is bound electrostatically to the anionic, negatively charged carboxyl group of a hormone storage granule. The pars distalis cells and the pineal glandular cells seemed to take up amines and amine derivatives in a similar manner. This suggests that in the pars distalis cells, too, biogenic monoamines have an active metabolism and possibly some regulative role in hormone synthesis and/or secretion.     

Differentiation of two types of endocrine cells which take up amine precursors using their capacity to take up the fluorescent dihydroisoquinoline derivative of dopamine

Summary A study was made of the accumulation of the strongly fluorescent 2-carboxymethyl-6,7-dihydroxy-3,4-dihydroisoquinolinium compound (2-Carb. Me-DIQ) derived from the condensation reaction of dopamine with glyoxylic acid in endocrine cells possessing the capacity to take up and store biogenic monoamine precursors. Thin-layer chromatographic studies of urine showed that 2-Carb. Me-DIQ was metabolized into two strongly fluorescent metabolites, possessing at least one hydroxyl group in the phenol moiety of the molecule, which were excreted in urine together with the parent compound. Histochemical observations, however, indicated that the tissue fluorescence showing maximal emission at 480 nm was due to 2-Carb. Me-DIQ. Generally, the injection of 2-Carb. Me-DIQ induced a strong fluorescence in those tissue components possessing the extraneuronal uptake mechanism of catecholamines. In the endocrine cells strong fluorescence was seen in the pineal glandular cells and in some cells of the pars distalis of the hypophysis, of which some cells also took up DL-5-HTP, as was seen following formaldehyde vapour treatment. No accumulation of 2-Carb. Me-DIQ was observed in the pancreatic islet cells, the C cells of the thyroid gland or the tracheal enterochromaffin-like cells. These findings lead to the conclusion that biogenic monoamines in the cells of the pars distalis of the hypophysis might use the phenolic moiety of the molecule to bind to some intracellular receptor. Thus, the pars distalis cells may have an intracellular binding mechanism for biogenic monoamines that is different from other endocrine cells showing the uptake and storage of biogenic monoamines On the other hand, the findings gave further support to the suggestion that in the pancreatic islet cells, the thyroidal C cells and the tracheal enterochromaffin-like cells biogenic monoamines are stored by a mechanism in which the basic, positively charged amino group of biogenic monoamines is bound electrostatically to the anionic, negatively charged carboxyl group of a hormone storage granule. The pars distalis cells and the pineal glandular cells seemed to take up amines and amine derivatives in a similar manner. This suggests that in the pars distalis cells, too, biogenic monoamines have an active metabolism and possibly some regulative role in hormone synthesis and/or secretion.This work was supported by grants from the Jalmari and Rauha Ahokas Foundation and the J.K. Paasikivi Foundation     

The distribution and endocrine nature of the abdominal paraganglia of adult man

The paraganglia of adult man were studied using the formaldehyde-induced fluorescence (FIF) method for histochemical characterization of biogenic monoamines. Microspectrofluorimetry was used to record the emission spectra and fluorescence intensities of the paraganglionic cells. The study of samples from six patients showed that well vascularized paraganglia were widely distributed throughout the retroperitoneal spaces. The paraganglia exhibited strong FIF with the spectral characteristics of monamines. Treatment with HC1 caused an increase in the fluorescence intensity of the paraganglia and a simultaneous shift of the emission maximum from 480--495 nm. This change suggests the presence of high concentrations of tryptophyl-containing peptides and is not due to monoamines. The possibility of a dual endocrine function for the paraganglia is discussed.     

Fluorescence microscopic differentiation of monoamines in the hypothalamus and spinal cord of the lamprey,using a new filter system

Summary Using a new filter system for fluorescence microscopy, in the hypothalamic area and spinal cord of the lamprey the yellow fluorescent cells and varicosities could clearly be differentiated from the blue-green fluorescent cells and varicosities. On the basis of the criteria for monoamines, the blue-green fluorescence and the yellow one were due to catecholamine and indolealkylamine (most probably 5-hydroxytryptamine), respectively. This filter system can specially be recommended for observations and color microphotography of monoamine fluorescence in trasmitted-light darkfield fluorescence microscopy.     

Indoleamines and the eccentric cells of the Limulus lateral eye

The lateral eye of the horseshoe crab, Limulus polyphemus, was studied by fluorescence microscopy according to Falck and Hillarp and microspectrofluorometry for identifying neuronal monoamines. After the formaldehyde treatment, the eccentric cells and their axons have a yellowish, rapidly fading fluorescence, such as is seen with 5-hydroxytryptamine. The microspectrofluorometric analysis was compatible with the fluorescence being caused by an indole, which could not be definitely identified, however. The eccentric cells have the ability to accumulate indoleamines such as 5-hydroxytryptamine, 6-hydroxytryptamine and 5,6-dihydroxytryptamine. Their axons were best demonstrated after being loaded with 6-hydroxytryptamine. Characteristic varicose terminals were seen in the neuropil, often arranged in clusters. Other terminals, possibly originating from the eccentric cells, were also seen among the pigment cells in the basal part of the ommatidia.     

A Catecholamine storage in the pancreatic A2-cells of owl monkey (Aotes trivirgatus)

Summary The fluorescence method ofFalck andHillarp was used to study the occurrence of biogenic monoamines in the islets of Langerhans of monkeys. A storage of a catecholamine was demonstrated in the A₂-cells of owl monkey, whereas no histochemically demonstrable amount of monoamines could be seen in the islet cells of marmoset,Rhesus monkey, squirrel monkey, andCebus monkey.Supported by grants from the Swedish Medical Research Council (No. B69-14x-712-04C) and by the National Institutes of Health (No. 06701-02).     

Distribution of monoamines in the diencephalon and pituitary of the dogfish,Scyliorhinus canicula L.

Summary The distribution of monoamines in the diencephalon and pituitary of the dogfish, Scyliorhinus canicula, has been investigated using the histochemical fluorescence technique of Falck and Hillarp (Falck and Owman, 1965). Terminals of monoamine-containing axons were found in the neurointermediate lobe of the pituitary and the axons were traced, by means of nialamide and L-dopa treatment and lesions, to the nucleus medius hypothalamicus. A separate hypothalamic system converging on the anterior median eminence and the occurrence of aminergic cells in the nuclei lobi inferiores and nucleus medius hypothalamicus were similarly demonstrated. Normal fish show a bilateral uncrossed tegmental tract and two areas of catecholamine-containing neurones in modified ependymal organs. The organum vasculosum hypothalami includes both primary catecholamine and 5-hydroxytryptamine-containing cell types whilst the organum vasculosum praeopticum has only the former type. Both organs contain cells which send club-like processes into the third ventricle. The subcommissural organ does not contain monoamines.The role of hypothalamic catecholamine systems in the regulation of pituitary function is discussed.     

Biogenic monoamines in ovum cells and preimplantation embryos of mice

Histofluorescence technique using glyoxylic acid revealed a specific fluorescence suggesting the presence of biogenic monoamines in early developmental stages of CBA x C57 Black mice. A yellow fluorescence observed in the blastomere surface from the stage of zygote up to that of four blastomere points to the presence of indole derivates. As development proceeds, the fluorescence increases and its colour becomes more and more green, which is characteristic of catecholamines. From the stage of eight blastomeres up to stage of blastocyst specific fluorescence is revealed in the cytoplasm. The inhibitors of monoamine oxidase, introduced into pregnant mice, markedly increased the specific fluorescence. An assumption is made of functional activity of biogenic monoamines in early mouse embryos.     

Effect of monoamines on the taste buds in the mouse

Summary Mouse taste buds were investigated following administration of monoamines and their precursors by fluorescence and electron microscopy. The appearance of fluorescent cells within the taste bud and the ultrastructural changes of vesicles in the gustatory cells were due to the treatment of 5-hydroxytryptophan. Small dense-cored vesicles (30–60 nm in diameter) appeared throughout the cytoplasm and accumulated especially at the presynaptic membranes of afferent synapses. Large dense-cored vesicles (80–100 nm) increased twice in number, and electron densities of their cores became more dense as compared with untreated mice. Fluorescent cells appeared in the taste bud of l-DOPA treated mice, whereas no ultrastructural changes were observed. These results suggest that the gustatory cells of the taste bud are capable of taking up and storing monoamines, which might act as neurotransmitters from the gustatory cells to the nerves.     

An electron microscopic study on the effects of reserpine on the subclavian glomera of the rabbit.

Young male and female New Zealand white rabbits were given a daily subcutaneous injection of reserpine (Serpasil, Ciba; 3 mg/kg) for two days and were sacrificed 24 hours after the last injection. The subclavian glomera (aortic bodies) were processed for electron microscopy to determine the effects of this biogenic amine depleting agent on the electron-opaque cytoplasmic granules of the parenchymal type I cells. Observations of glutaraldehyde-osmium tetroxide fixed glomera from reserpinized animals showed a slight decrease in granule density of the type I cells. Glomera fixed in glutaraldehyde and incubated in potassium dichromate (pH 4.1) demonstrated a reduction in granule opacity following reserpine treatment. Control glomera incubated in potassium dichromate displayed electron-opaque granules. These results indicate that reserpine does deplete the amines without granule disappearance or changes in granule population. The positive reaction of the control tissue granules to potassium dichromate incubation suggests that the predominant biogenic amines in the electron-opaque granules are unsubstituted monoamines. Persistence of the opaque granules following reserpinization and glutaraldehyde-osmium tetroxide double fixation, may be due to amine-binding protein within the granules. The mode of granule depletion could not be ascertained with certainty.     

Fluorescence studies on neural structures and endocrine cells in the pancreas of the cat

Summary With the use of the Falck-Hillarp histochemical technique for the detection of monoamines, nerve fibre fluorescence is observed throughout the tail of the pancreas of the cat and the arrangement and distribution of the nerve fibres can be studied in both the exocrine and endocrine tissue. In the exocrine pancreas, adrenergic nerve fibres innervate arterioles, larger veins and major pancreatic ducts. Adrenergic nerve fibres also appear to terminate on the non-adrenergic nerve cell bodies of the intrapancreatic ganglia. In the islets of Langerhans, adrenergic nerve fibres innervate both the endocrine cells and blood vessels. Some of the islet cells exhibit fluorescence with the Falck-Hillarp technique and these cells have been identified as alpha cells. In animals treated with reserpine, the fluorescence in nerve fibres and in alpha cells is absent.The author wishes to thank ProfessorG. C. Schofield and Dr.G. C. Smith for their encouragement and valuable criticism during the course of this study. The assistance of MissJ. Bennett and MissW. Kemp and the photographic help of Mr.J. S. Simmons, F.R.P.S., are gratefully acknowledged. The diagram was drawn by MissS. Flett.     

Histochemical distribution of monoamines in the hypothalamo-hypophysial region of the lamprey,Lampetra japonica

The distribution of monoamine fluorescence was studied in the hypothalamohypophysial region of the lamprey. Groups of intensely fluorescent cells were observed in the lateral walls of the caudal part of the third ventricle. The anterior part of the neurohypophysis which is situated over the pars distalis showed weak fluorescence. The posterior part of the neurohypophysis which is contiguous to the pars intermedia contained highly fluorescent material in its rostral part. The distribution of monoamines in the lamprey neurohypophysis is compared with that in the higher vertebrates and their functional significance is discussed.     

Detection of biogenic monoamines in the developing nervous system of Lymnaea stagnalis mollusk embryos]

Formaldehyde-induced fluorescence of intraneuronal monoamines can be demonstrated in the Lymnaea embryos from the "late veliger" stage on. Green specific fluorescence indicating the presence of a primary catecholamine occurs in two paired formations which contain a mass of fibres and varicosities. The formations are supposed to correspond to cerebral and pedal ganglia. Single fibres of the same type can be seen in the foot and other organs of the embryo.     

Monoamines in the liquor-contacting nerve cells in the hypothalamus of the lamprey,Lampetra fluviatilis L.

Summary The hypothalamus of adult lampreys (Lampetra fluviatilis L.) was studied by means of light and fluorescence microscopy (Falck's technique). Some single liquorcontacting nerve cells (LCNC) showing a weak green fluorescence were demonstrated in the ventral part of the third ventricle, above the preoptic recess. Caudally numerous fluorescent LCNC occur in the ventral part of the third ventricle, in the infundibular and in the posterior recess. The LCNC are to be observed between or below the ependymal cells lining the ventricular wall. These cells appear to be of the bipolar type. One process with a club-like protrusion is directed into the ventricular lumen, the other one into the opposite direction. Two types of fluorescent LCNC were distinguished: yellowish green cells, containing catecholamines, and yellowish orange cells, containing 5-hydroxytryptamine. Some similarity between the hypothalamic monoaminergic LCNC in lampreys and LCNC of the paraventricular organ of the other vertebrates was found. The localization, structure and monoaminergic nature of the hypothalamic LCNC in lampreys suggest the possibility, that their monoamines are released into the cerebrospinal fluid.I am very obliged to Prof. A.L. Polenov for his continuous help and advice. The skilful technical assistance of Mrs. G.N. Yakshina is gratefully acknowledged.     

Possible nature of the biogenic amine in the dumbbell-shaped cells of frog taste buds]

An attempt was made to identify specific monoamines contained in the dumb-bell shape cells of the frog taste bud by means of histochemical analysis. It was shown by fluorescent microscopy that preliminary administration of exogenous serotonin into the blood channel of frog tongue resulted in a sharp increase of specific fluorescence of the dumb-bell shape cells, whereas serotonin synthesis inhibition with p-chlorphenylalanine led to reduction and elimination of specific fluorescence. It was concluded that-specific monoamine of the dumb-bell shape cells was possibly of serotonin-like nature.     

Immunohistochemical localization of monoamine oxidase type B in pancreatic islets of the rat.

Monoamine oxidase (MAO) is regarded as a mitochondrial enzyme. This enzyme localizes on the outer membrane of mitochondria. There are two kinds of MAO isozymes, MAO type A (MAOA) and type B (MAOB). Previous studies have shown that MAOB activity is found in the pancreatic islets. This activity in the islets is increased by the fasting-induced decrease of plasma glucose level. Islet B cells contain monoamines in their secretory granules. These monoamines inhibit the secretion of insulin from the B cells. MAOB is active in degrading monoamines. Therefore, MAOB may influence the insulin-secretory process by regulating the stores of monoamines in the B cells. However, it has not been determined whether MAOB is localized on B cells or other cell types of the islets. In the present study, we used both double-labeling immunofluorescence histochemical and electron microscopic immunohistochemical methods to examine the subcellular localization of MAOB in rat pancreatic islets. MAOB was found in the mitochondrial outer membranes of glucagon-secreting cells (A cells), insulin-secreting cells (B cells), and some pancreatic polypeptide (PP)-secreting cells (PP cells), but no MAOB was found in somatostatin-secreting cells (D cells), nor in certain other PP cells. There were two kinds of mitochondria in pancreatic islet B cells: one contains MAOB on their outer membranes, but a substantial proportion of them lack this enzyme. Our findings indicate that pancreatic islet B cells contain MAOB on their mitochondrial outer membranes, and this enzyme may be involved in the regulation of monoamine levels and insulin secretion in the B cells.     

Cell size specific binding of the fluorescent dye calcofluor to budding yeast

The yeast Saccharomyces cerevisiae cell surface outside of the bud scars displayed an increasing fluorescence intensity with increasing cell size (volume), where fluorescence was due to irreversible binding of the fluorescent dye calcofluor. The increase in fluorescence intensity appeared to be due to an increase in the density of fluorescence per unit surface area of the cell. Exposure time measurements from a photomicroscope were used to quantitate fluorescence intensity on individual cells. The cell size dependent increase in fluorescence intensity was displayed by unbudded cells from stationary phase populations, and unbudded and parent cells from exponentially growing populations. Abnormally large cells generated during the arrest of cell division with alpha-factor or restrictive temperature for cdc3, 8, 13, 24, and 28 cell division cycle mutants, displayed significantly greater fluorescence intensity compared to the smaller cells generated during the arrest of division for cdc25, 33, and 35 mutant strains. Fluorescence intensity on newly emerging buds was broadly dependent on both the size of the bud, and the size of the parent cells on which the buds were growing.     

Hyperforin inhibits vesicular uptake of monoamines by dissipating pH gradient across synaptic vesicle membrane

Extracts of Hypericum perforatum (St. John's wort) have antidepressant properties in depressed patients and exert antidepressant-like action in laboratory animals. The phloroglucinol derivative hyperforin has become a topic of interest, as this Hypericum component is a potent inhibitor of monoamines reuptake. The molecular mechanism by which hyperforin inhibits monoamines uptake is yet unclear. In the present study we try to clarify the mechanism by which hyperforin inhibits the synaptic vesicle transport of monoamines. The pH gradient across the synaptic vesicle membrane, induced by vacuolar type H(+)-ATPase, is the major driving force for vesicular monoamines uptake and storage. We suggest that hyperforin, like the protonophore FCCP, dissipates an existing Delta pH generated by an efflux of inwardly pumped protons. Proton transport was measured by acridine orange fluorescence quenching. Adding Mg-ATP to a medium containing 130 mM KCl and synaptic vesicles caused an immediate decrease in fluorescence of acridine orange and the addition of 1 microM FCCP abolished this effect. H(+)-ATPase dependent proton pumping was inhibited by hyperforin in a dose dependent manner (IC(50) = 1.9 x 10(-7) M). Hyperforin acted similarly to the protonophore FCCP, abolishing the ATP induced fluorescence quenching (IC(50) = 4.3 x 10(-7) M). Hyperforin and FCCP had similar potencies for inhibiting rat brain synaptosomal uptake of [3H]monoamines as well as vesicular monoamine uptake. The efflux of [3H]5HT from synaptic vesicles was sensitive to both drugs, thus 50% of preloaded [3H]5HT was released in the presence of 2.1 x 10(-7) M FCCP and 4 x 10(-7) M hyperforin. The effect of hyperforin on the pH gradient in synaptic vesicle membrane may explain its inhibitory effect on monoamines uptake, but could only partially explain its antidepressant properties.     

Dual signal-responsive liposomes for temperature-controlled cytoplasmic delivery

For production of a new type of functional liposome whose destabilization can be triggered by a combination of a temperature signal and acidic pH signal, we prepared liposomes modified with hyperbranched poly(glycidol) derivatives having N-isopropylamide and carboxyl groups. HeLa cells incubated with the dual signal-responsive liposomes encapsulating a water-soluble fluorescent dye pyranine at 28 °C displayed punctate fluorescence of pyranine, indicating that the liposomes were trapped in endosome. However, after heating at 45 °C for 15 min, the same cells exhibited diffuse fluorescence of pyranine, indicating that destabilization of the liposomes in endosome with an acidic environment in combination with the brief heating caused efficient transfer of the contents into cytosol. The dual signal-responsive liposomes might have usefulness for site-specific delivery of membrane-impermeable molecules, which exhibit bioactivities in the intracellular spaces, such as siRNA and proteins.     

Role of the monoaminergic system in the transmission of the effect of androgens on neurons of separate limbic structures of the rat brain

By means of the histochemical method intensity of monoamines fluorescence has been studied in 3-, 5-, 7-, 10-, 20-, 30- and 60-day-old intact and neonatally androgenized female rats. The neonatal androgenization increases fluorescent intensity of monoamines in the neuropil of the adjoining nucleus of the septum, of the nucleus in the terminal stripe bed and the caudate nucleus. This is especially evident on the 3d, 7th and 30th days. On the 5th day of the postnatal life the difference in fluorescent intensity of monoamines in the brain of control and test animals is statistically insignificant. Possible mechanisms responsible for the fluorescent intensity of monoamines and the role of the latter in transmitting the sex hormones effect to the neurons of the forebrain structures investigated are discussed.