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 histochemical studies of the uptake of exogenous noradrenaline and dopamine by in vitro cultured cerebrocortex explants of the rat

Cerebrocortex of the neonatal rats were cultivated (--14 days). The cultures were studied living and with histological and fluorescence histochemical methods. A differentiation of neuronal cell- and fiber elements, oligodendro glial cells and astrocytes was found. The glyoxylic acid technique to estimate biogenic monoamines (Lindvall et al. 1974) was adapted up the cultivated explants. The normal cultures have only 24 h post cultivationem a specific fluorescence granularly in small concentration of the surface of the explant and in the explant self. Incubations with noradrenaline and dopamine demonstrated a various accumulation of the exogenous transmitters in the various parts of the cultivated explants. Uptake and releasing mechanisms in the cultivated material of the cerebrocortex were discussed with respect to the results of the sympathetic ganglia in vitro.     

The effect of biogenic monoamine antagonists on the development of preimplantation mouse embryos cultured in vitro

The effect of serotonin and adrenaline antagonists was tested on the early embryos of mice of three lines. All the substances tested produced an arrest or inhibition of cleavage division and the appearance of anomalies. Serotonin introduced in the incubation medium was effective against some serotoninolytics. We were unable to test the protective effect of adrenaline, as in the concentrations used it has its own effect on the development. From the data obtained, a conclusion is made of the existence in early mouse embryos of the structures sensitive to serotonin and adrenaline antagonists. The assumptions is made from the previously obtained data on the presence of biogenic monoamines in early mouse embryos, of functional activity of prospective mediators of the nervous system at the earliest stages of embryonic development of mammals.     

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.     

Localization and quantitative distribution of biogenic monoamines in the intestinal tract of locust, snail and carp

The localization and quantitative distribution of the biogenic monoamines of the intestinal tract has been studied in Locusta migratoria, Helix pomatia and Cyprinus carpio with morphological and biochemical methods.

Electron microscopically dense-core vesicles of aminergic character were found in the varicose nerve fibres located in the intestinal muscles of all three animal species. Intensive green fluorescence characteristic of catecholamines was detectable in both the varicose nerve fibres and perikarya.

Using a fluorimetric method the quantity of biogenic monoamines was measured in the gastrointestinal tract of all three species. The amount of adrenaline and noradrenaline was rather low: 0.03–0.29 μg/g. The dopamine content reached the value of 6 μg/g in the locust gut, and a significant amount of serotonin was present in the intestinal tract of all three species: 1.00–2.59 μg/g. Compared to the adrenaline and noradrenaline serotonin proved to be higher by more than one order in each case. On the basis of morphological and biochemical results the authors suggest that biogenic monoamines are involved in the regulation of the gut muscle functioning both in the form of transmitters as well as neurohormones.     

A and C particles in mouse embryos]

Electron microscopic examination of embryos of the BALB/c and AKR strains of mice from the 1-cell stage to 14 days of age revealed 2 types of intracisternal A particles, one type of C particle budding into the extracellular space and an endoplasmic reticulum-association "dense-cored veiscle". The occurrence of the particles showed a marked dependence on the developmental stage of the embryo. A small percentage of 1 blastomere embryos in both strains showed the presence of a small number of A particles. Embryos of 1 to 16 blastomeres of the AKR strain with A particles were on average more frequent than in the BALB/c strain. The percentage of 7 and 14 day embryos with C particles was much greater in AKR than in Balb/c mice.     

Sexing of mouse preimplantation embryos by detection of Y chromosome-specific sequences using polymerase chain reaction.

Detection of genes known to be present on the mammalian Y chromosome was adapted for sexing mouse early embryos using the polymerase chain reaction (PCR) method. Sry and Zfy genes located in the sex-determining region of the Y chromosome were chosen for Y-specific target sequences, and DXNds3 sequence on the X chromosome was chosen for control. The two-step PCR method using two pairs of primers for each of the target sequences was employed for detecting the sequences. When DNAs of male and female mice were amplified with these primers, male-specific fragments were detected even in DNAs that were equivalent in amount to two cells. Mouse embryos at the two-cell stage were separated into two individual blastomeres, and one blastomere was karyotyped at the second cleavage. The remaining blastomere was subjected to PCR amplification immediately or after having been cultured for 48 h up to the morula stage. The Sry and Zfy sequences were detected in about half the embryos; detection of the Sry and Zfy sequences corresponded exactly to the presence of the Y chromosome, except in one sample of male morula in which embryos may have been lost before the PCR amplification. It is concluded that the sex of mouse preimplantation embryos can be accurately determined through detection of the Y-specific sequences using the two-step PCR method, even with the single blastomeres separated at the two-cell stage.     

ROCK inhibition prevents early mouse embryo development

ROCK is a Rho-GTPase effector that is important for actin assembly and is involved in various cellular functions, including cell contraction, migration, motility, and tumor cell invasion. In this study, we investigated ROCK expression and function during early mouse embryo development. Inhibiting ROCK by Y-27632 treatment at the zygote stage resulted in first cleavage failure, and most embryos failed to develop to the 8-cell stage. When adding Y-27632 at the 8-cell stage, embryos failed to undergo compaction and could not develop into blastocysts. In addition, fluorescence staining intensity analysis indicated that actin expression at blastomere membranes was significantly reduced. After ROCK inhibition, two or more nuclei were observed in a cell, which indicated possible cytokinesis failure. Moreover, after ROCK inhibition with Y-27632, the phosphorylation levels of LIMK1/2, a downstream molecule of ROCK, were decreased at blastomere membranes. Thus, our results showed conserved roles for ROCK in this mammalian embryo model and indicated that a ROCK-LIMK1/2-actin pathway might regulate cleavage and blastocyst formation during early mouse embryo development.     

Cell proliferation in the ectoderm of the Xenopus embryo: development of substratum requirements for cytokinesis

The requirements for cell division in ectodermal blastomeres of the early Xenopus embryo were studied. Isolated blastomeres divide autonomously on nonadhesive agar in a simple salt solution up to the midblastula stage. After the midblastula transition, cell-cell contact is required for blastomere division. In isolated blastomeres of that stage, cytokinesis fails, but nuclear division continues normally for some time. Cell-cell contact as a prerequisite for blastomere division can be replaced by culturing blastomeres on an appropriate substratum. Clonal growth of isolated blastomeres is supported by a variety of protein substrata, indicating rather unspecific substratum requirements. Different substrata which do not support blastomere division can affect different steps in cytokinesis.     

Neuronal localization of dopamine and 5-Hydroxytryptamine in some mollusca

Summary The localization of biogenic monoamines in ganglionic tissues from Anodonta piscinalis, Helix pomatia, and Buccinum undatum has been studied by means of the histochemical fluorescence method of Falck and Hillarp.In cerebral, visceral, and pedal ganglia (besides nonfluorescent nerve cells) neurons emitting a green or yellow fluorescence were found. No other cell systems exhibiting a specific fluorescence were observed. An abundance of monoaminergic terminals were found in the central parts of these ganglia. Spectrophotofluorimetric determinations showed that there are large quantities of dopamine and 5-hydroxytryptamine in the tissues investigated. The amounts of dopamine and 5-hydroxytryptamine agree well with the distribution of green and yellow fluorescence, respectively, in the ganglia.There are many similarities between the vertebrate and the molluscan monoaminergic neurons. The morphology of the neurons is the same, the intraneuronal distribution of the monoamines is identical, depletion experiments with reserpine and denervation experiments give the same results, and the synaptic arrangement of monoaminergic fibres on non-adrenergic neurons has the same appearance. Apparently, however, dopamine and 5-hydroxytryptamine are the only monoamines acting as neuronal transmitters in the species investigated.The research reported in this document has been sponsored by the Air Force Office of Scientific Research under Grant AF EOAR 64-5 through the European Office of Aerospace Research (OAR), United States Air Force and by the Swedish Natural Science Research Council.     

The presence of gap junctions during early Patella embryogenesis: An electron microscopical study

The presence of gap junctions has been examined up to the sixth cleavage in the early Patella embryo. Gap junctions are located all over the blastomere borders. In 2-, 4-, and 8-cell embryos they were also observed at peripheral contacts. The frequency and size of the gap junctions increase at the 32-cell stage. The structure of gap junctions is similar in all stages investigated with hexagonally arranged equal-sized particles (11 nm) having a constant center-to-center spacing (13.0 nm). At the 32-cell stage formation plaques were observed, indicating an increase of gap junctions.     

Sex-chromosome constitution of postimplantation tetraploid mouse embryos

Tetraploid mouse embryos were produced at the two-cell stage by blastomere fusion induced by inactivated Sendai virus. The embryos were from chromosomally normal female mice that had been fertilised by homozygous Rb(1.3)1Bnr males carrying a pair of large metacentric marker chromosomes in their karyotype. These "reconstructed" one-cell tetraploid embryos were then transferred to the oviducts of pseudopregnant recipients, which were subsequently autopsied early on the 10th day of gestation. Two-cell stage embryos that did not undergo blastomere fusion after 4-5 h were transferred to a second group of recipients, which were also autopsied early on the 10th day of gestation. From a total of 153 tetraploid embryos transferred to females that subsequently became pregnant, 135 implanted. Sixty-eight implantation sites were found to contain resorptions, whereas 67 contained mostly headfold presomite-stage embryos. Four embryos possessed four to six pairs of somites. All 57 embryos that could be analysed cytogenetically were found to be tetraploid. G-banding analysis revealed that 30 of these embryos had an XXYY and 27 and XXXX sex-chromosome constitution. The presence of two marker chromosomes in all mitotic preparations from each of these tetraploid embryos confirmed that they had all been produced by duplication of their original XY or XX diploid chromosome constitution, respectively. The XXYY:XXXX sex ratio observed was not significantly different from unity. In the control series of transfers, all of the embryos recovered were at the forelimb bud stage and had a diploid chromosome constitution. The results reported here differ from human clinical findings, in which the XXYY:XXXX sex ratio of 120 human tetraploid spontaneous abortions recovered over the last 20 years is 45:75. Possible explanations for these differences are briefly discussed.     

Computer-operated microspectrofluorimetry to identify formaldehyde-induced fluorophores of biogenic monoamines and precursor substances in models and tissue sections

By means of a histochemical reaction using formaldehyde vapour (Falck and Owman 1965), biogenic monoamines and precursor substances, i.e., L-DOPA, dopamine, noradrenaline, adrenaline, 5-hydroxytryptophan and 5-hydroxytryptamine, may be converted into fluorophores with specific spectral characteristics, i.e., the emission spectrum, excitation spectrum and fading curve. The registration and correction of the spectral properties and changes induced by acidification with hydrochloric acid vapour or treatment with ammonia vapour, of these formaldehyde-induced fluorophores, are performed by an automated microspectrofluorimeter, developed by modification of a Leitz MPV 2 system. This work deals with the instrumental configuration and certain methodological features in order to identify the fluorogenic biogenic monoamines and precursor substances in models and tissue sections. Registrations of excitation peak values, for the first time extended to a wavelength range from 240-460 nm, are discussed, which enable the calculation of peak ratio values 410/260, 380/320, 320/260 or 385/315, suitable as identification parameters for formaldehyde-induced fluorophores of biogenic monoamines and precursor amino acids.     

Calciumm-dependent release of endogenous serotonin,dopamine and norepinephrine from nerve endings

The release of endogenous serotonin, dopamine, norepinephrine and 5-hydroxyindoleacetic acid was studied in static incubations of synaptosome (P₂) preparations from the telencephalon of the rat. Elevated potassium medium specifically stimulated the release of the biogenic monoamines while the deaminated metabolite of serotonin was not effected. The release of the monoamines was also sensitive in part to the presence of calcium in the incubation medium.     

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.     

Effect of some fragments of peptide hormones on the content of biogenic monoamines from mouse brain]

The effect of some tripeptides, which are fragments of peptide hormones, and their analogs on the content of biogenic monoamines (BM) from albino mice brain was studied. It was found that thyroliberin, melanostatin and the C-terminal tripeptide of gonadoliberin activate the dophaminergic (DA-ergic) system in the forebrain of mice treated with reserpine or haloperidol, whereas the C-terminal tripeptide of gastrin acts as a synergic blocker of the DA receptors. The N-terminal tripeptides (with and without the amido group) do not affect the content of BM. No effect of the tripeptides was observed in intact animals. It is assumed that the agonistic or antagonistic effect of the tripeptides on BM is due to certain structural peculiarities of the tripeptides, e.g. the presence of the C-terminal amido group and their endogenous nature.     

The occurrence of the cell type containing a specific monoamine in the taste bud of the rabbit's foliate papila.

The fluorescence histochemical examination on biogenic amines of the rabbit's foliate papilla revealed that a specific monoamine exhibiting an yellow fluorescence was present in a certain cell type of taste buds. The fluorescence had the emission maximum at 520 mmu and faded rapidly under the influence of the UV-irradiation. The green fluorescence of adrenergic nerve had the emission maximum at 480 mmu and was fairly stable upon the UV-irradiation. The yellow fluorescence disappeared completely following reserpine treatment, while it was markedly enhanced by nialamide treatment. From the observations, it is suggested that certain taste bud cells of the foliate papilla contain a biogenic monoamine, probably 5-hydroxytryptamine (serotonin).