Precursors of monoamine-storage organelles in developing megakaryocytes of the rat

Summary Identification and distribution of the precursors of aminestorage organelles in rat megakaryocytes during cell maturation were studied, using the uranaffin reaction for adenine nucleotide. The precursors of the amine-storage organelles appeared as 200–300 nm vesicles having an uranaffin electron dense granule, whereas they appeared as empty vesicles by conventional glutaraldehyde-OsO₄ fixation. X-ray probe microanalysis confirmed the existence of U and P in the uranaffin reaction positive vesicles. The precursors appeared in the immature megakaryocytes, especially at the trans(mature) face of the Golgi apparatus, and rapidly increased in number in the maturing cells. The size of the uranaffin granules in the precursory organelles increased gradually during cell maturation and became almost equivalent to the dense body of blood platelets in the final stage of cell maturation.     

Uptake of 3H-dopamine in megakaryocytes and blood platelets measured by quantitative electron-microscope autoradiography

We studied the uptake of dopamine by mature megakaryocytes and blood platelets in mouse spleen after a single intraperitoneal injection of 3H-dopamine. In order to compare the uptake of 3H-dopamine in mature megakaryocytes and blood platelets, we used quantitative autoradiography at the electron-microscope level. Dense accumulations of silver grains were observed on both mature megakaryocytes and blood platelets; all other tissue elements of the spleen exhibited considerably less dense labeling. No significant differences with regard to dopamine uptake were observed in megakaryocytes and blood platelets. This is in contrast to the previous finding of very different patterns of 3H-5-hydroxytryptamine labeling in mature megakaryocytes and blood platelets (Daimon and Uchida 1985). The results of the present study provide new evidence in favor of the hypothesis that the active uptake mechanism of dopamine through the plasma membrane is different from the uptake mechanism of 5-hydroxytryptamine.     

Uptake of 3H-dopamine in megakaryocytes and blood platelets measured by quantitative electron-microscope autoradiography

Summary We studied the uptake of dopamine by mature megakaryocytes and blood platelets in mouse spleen after a single intraperitoneal injection of ³H-dopamine. In order to compare the uptake of ³H-dopamine in mature megakaryocytes and blood platelets, we used quantitative autoradiography at the electron-microscope level. Dense accumulations of silver grains were observed on both mature megakaryocytes and blood platelets; all other tissue elements of the spleen exhibited considerably less dense labeling. No sigificant differences with regard to dopamine uptake were observed in megakaryocytes and blood platelets. This is in contrast to the previous finding of very different patterns of ³H-5-hydroxytryptamine labeling in mature megakaryocytes and blood platelets (Daimon and Uchida 1985). The results of the present study provide new evidence in favor of the hypothesis that the active uptake mechanism of dopamine through the plasma membrane is different from the uptake mechanism of 5-hydroxytryptamine.     

Dopamine: Localization of uptake in the pedal ganglion of Quadrula pustulosa (Pelecypoda)

Uptake of ³H-dopamine in the pedal ganglion of Quadrula pustulosa was localized using combined fluorescence and optical light microscopy, in addition to electron microscope autoradiography. Light microscope autoradiograms of the same sections previously prepared for fluorescence microscopy indicated most radioactivity occurred over green, long-lasting fluorescent fiber tracts and nerve cell bodies. Ultrastructural autoradiographic results showed the majority of the radioactivity was localized over synaptic vesicles. Nerve fibers containing neurotubules, and also a limited number of nerve cell bodies were labelled with ³H-dopamine. These results suggest uptake of dopamine to be specific for dopaminergic structures.     

Uptake of tritium-labelled biogenic amines by the prostomium of the polychaete Nereis virens (Sars) (Annelida)

The uptake of tritium-labelled 5-HT, noradrenaline, 5-hydroxytrytophan, DOPA and dopamine by the cerebral ganglion and prostomial nervous system of the polychaete Nereis virens has been examined using radioautography at the level of the light microscope. Pronounced uptake of (3)H-5HT occurred in the antennal, palpal, tegumentary and nuchal nerves as well as in ganglionic nuclei 13, 14, 15, 16, 17, 20, 24 and 25, the mid-brain neuropile, the neurosecretory neuropil and the infracerebral organ; (3)H-NA uptake was observed in small cells in the prostomial epidermis, and the infracerebral organ; (3)H-dopamine only in one of two common types of epidermal mucus cells. Prostomial muscles labelled generally with (3)H-NA and at specific sites with (3)H-5HT. These observations support the concept of an efferent serotonergic system originating in several cerebral ganglionic nuclei and serving prostomial muscle and epidermis. Evidence for an afferent adrenergic system is less substantial. The role of dopamine remains obscure.     

Dopaminergic neurones in various retinas and the postnatal development of tyrosine-hydroxylase immunoreactivity in the rabbit retina

The localisation of tyrosine-hydroxylase immunoreactive neurones in retinas of a variety of animals were examined. Immunoreactivity was associated with specific populations of amacrine neurones in all species examined, viz; rabbit, guinea pig, monkey, cow, frog, pigeon and goldfish. Only in the goldfish was immunoreactivity also associated with processes situated in the outer plexiform layer showing that in this species catecholamine interplexiform cells exist. The development of tyrosine-hydroxylase immunoreactive neurones in the rabbit retina was also analysed. The first immunoreactive positive cells were observed by the third postnatal day. The immunoreactive positive neurones at this stage are weak and lack processes. The intensity of the immunoreactivity increases with development, but processes are lacking, until the 10th postnatal day. The immunoreactive neurones only appear fully developed by the 22nd to 28th postnatal day. Autoradiographical analysis of 3H-dopamine uptake strongly suggests that neurones containing tyrosine-hydroxylase immunoreactivity in the different retinas have the capacity to take up exogenous dopamine. It is therefore concluded that localisation of either 3H-dopamine uptake or tyrosine-hydroxylase provides a means of locating catecholamine neurones.     

Amperozide, a putative anti-psychotic drug: uptake inhibition and release of dopamine in vitro in the rat brain

The effects of amperozide (a diphenylbutylpiperazinecarboxamide derivative) on the uptake and release of 3H-dopamine in vitro were investigated. Amperozide inhibited the amphetamine-stimulated release of dopamine from perfused rat striatal tissue in a dose-dependent manner. With 1 and 10 microM amperozide there was significant inhibition of the amphetamine-stimulated release of dopamine, to 44 and 36% of control. In contrast, 10 microM amperozide significantly strengthened the electrically stimulated release of dopamine from perfused striatal slices. Amperozide 1-10 microM had no significant effect on the potassium-stimulated release of dopamine. 10 microM amperozide also slightly increased the basal release of 3H-dopamine from perfused striatal tissue. These effects on various types of release are similar to those reported for uptake inhibitors (Bowyer et al, 1984). The uptake of dopamine in striatal tissue was inhibited by amperozide with IC50 values of 18 microM for uptake in chopped tissue and 1.0 microM for uptake in synaptosomes. Amperozide also inhibited the uptake of serotonin in synaptosomes from frontal cortex, IC50 = 0.32 microM and the uptake of noradrenaline in cortical synaptosomes, IC50 = 0.78 microM. In conclusion, amperozide shows uptake-inhibiting properties in both release and uptake studies done in vitro on the rat. In the in vivo studies, however, amperozide differs from dopamine uptake inhibitors.     

Dopaminergic neurones in various retinas and the postnatal development of tyrosine-hydroxylase immunoreactivity in the rabbit retina

Summary The localisation of tyrosine-hydroxylase immunoreactive neurones in retinas of a variety of animals were examined. Immunoreactivity was associated with specific populations of amacrine neurones in all species examined, viz; rabbit, guinea pig, monkey, cow, frog, pigeon and goldfish. Only in the goldfish was immunoreactivity also associated with processes situated in the outer plexiform layer showing that in this species catecholamine interplexiform cells exist.The development of tyrosine-hydroxylase immunoreactive neurones in the rabbit retina was also analysed. The first immunoreactive positive cells were observed by the third postnatal day. The immunoreactive positive neurones at this stage are weak and lack processes. The intensity of the immunoreactivity increases with development, but processes are lacking, until the 10th postnatal day. The immunoreactive neurones only appear fully developed by the 22nd to 28th postnatal day.Autoradiographical analysis of ³H-dopamine uptake strongly suggests that neurones containing tyrosine-hydroxylase immunoreactivity in the different retinas have the capacity to take up exogenous dopamine. It is therefore concluded that localisation of either ³H-dopamine uptake or tyrosine-hydroxylase provides a means of locating catecholamine neurones.     

RADIOAUTOGRAPHIC DEMONSTRATION OF 5-HYDROXYTRYPTAMINE-3H UPTAKE BY PULMONARY ENDOTHELIAL CELLS

The lung is able to rapidly remove 5-hydroxytryptamme (5-HT) from the circulation by a Na⁺-dependent transport mechanism. In order to identify the sites of uptake, radioautographic studies were done on rat lungs which had been isolated and perfused with 5-HT-³H and 0 5 mM iproniazid, a monoamine oxidase inhibitor. In control experiments 10^-4 M imipramine was added to the perfusate to inhibit the membrane transport of 5-HT At the light microscope level, silver grains were seen concentrated near capillaries and in the endothelium of large vessels From electron microscope radioautographs a semiquantitative grain count was made and 90% of the silver grains were observed over capillary endothelial cells. The grains were found over the nucleus and cytoplasm of the cell and shewed no preferential association with any particular cytoplasmic inclusion bodies, organelles, or vesicles Other cell types were unlabeled except for a few mast cells, certain vascular smooth muscle cells, and one nerve ending. This radioautographic demonstration of the cell type responsible for the rapid removal of 5-HT from the lung circulation clearly establishes the existence of a new metabolic role for pulmonary endothelial cells.     

Microcolonies of hemopoietic cells in the rat choroid plexus during the neonatal period

Microcolonies of hemopoietic cells have occasionally been found in the choroidal stroma of the rat myelencephalic choroid plexus during neonatal life. These hemopoietic foci are mixed colonies mainly composed of erythroblasts and maturing megakaryocytes; granulocyte precursors were not identified. The morphological data indicate that both erythro- and magakaryopoiesis occur in these microcolonies. With respect to their origin, we suggest that circulating pluripotential stem cells may colonize the choroidal stroma and produce erythro- and megakaryocyte cell lines.     

Lipid composition and metabolism in megakaryocytes at different stages of maturation

The lipid composition and metabolism of isolated guinea pig megakaryocyte subgroups at various stages of maturation were investigated. Three groups were studied: 1) 67% of megakaryocytes in Group A were immature; 2) Group B was heterogeneous and contained both immature and mature subgroups of megakaryocytes; 3) 92% of megakaryocytes in Group C were mature. Lipid composition was determined by thin-layer chromatography, lipid-phosphorus, and gas-liquid chromatography. Cholesterol, ceramide, and de novo fatty acid synthesis were evaluated with [14C]acetate. [14C]Glycerol was used to assess de novo phospholipid synthesis. 14C-Labeled fatty acids were used to evaluate fatty acid uptake. The phospholipid and cholesterol content was found to be four times greater in mature megakaryocytes than that in immature megakaryocytes, which paralleled the protein content and volume of mature and immature cells. The cholesterol-phospholipid ratio was similar and there were no differences in the phospholipid species in the three groups. Phospholipid and cholesterol synthesis were established in immature megakaryocytes and persisted at about the same level in mature megakaryocytes. The uptake of arachidonic and palmitic acids also occurred primarily in immature cells, while the de novo synthesis of palmitic acid occurs predominantly in mature megakaryocytes. There was an inverse relationship between the uptake of exogenous palmitic acid and fatty acid synthesis, but the uptake of palmitic acid primarily inhibited fatty acid synthesis in mature megakaryocytes. There were differences in the acylation of phospholipid species with arachidonic acid in megakaryocytes at different stages of maturation since the acylation of phosphatidylcholine occurred primarily in immature megakaryocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induced neurotransmitter release from primary cultures of rat brain neurons

Neural cells from fetal rat brain were grown in tissue culture in the absence of serum and maintained for 4–5 weeks without medium renewal. Over 80% of the embryonic cells in the culture had a neuronal appearance and formed intercellular synaptic connections. When mature, a definite population of the neuronal cells accumulated ³H-dopamine in a sodium-dependent, benztropine inhibited process. The mature cells were also able to release ³H-dopamine in a potassium evoked, calcium-dependent process, with half maximal dopamine release achieved at a Ca²⁺ concentration of 120μM. In the maturing cells the capacity for potassium evoked, calcium-dependent dopamine release increased from an undetectable level in the first three days to a plateau level after 10–11 days $\text{in vitro}$. The fully expressed release capacity (20–30% of the neurotransmitter retained in the cells) was maintained thereafter. These results demonstrate that primary brain neurons develop a functional neurosecretion apparatus in a chemically defined medium in the absence of animal serum. This extends the utility of primary cultures of brain neurons for developmental structural and biochemical studies of neurotransmission.     

Anion transport during maturation of erythroblastic cells

Bromide uptake was measured in single maturing erythroblastic cells of rabbits by means of X-ray microanalysis. Increase in bromide uptake as the cells matured was observed. The order of cells from low to high bromide uptake was: early erythroblast less than late erythroblast less than marrow red cells less than peripheral red blood cells. The transition from low to high bromide uptake is correlated to the accumulation of iron which begins in the late erythroblast. A decrease in rubidium uptake also occurs as iron accumulates in the cell. These results indicate that the anion and cation transport changes during maturation are parallel in time course but opposite in direction. In addition, the increase in bromide uptake can be accounted for by the increase in surface-to-volume ratios of the cells. Surface-to-volume ratios were estimated by morphometric techniques.     

3H-dopamine binding to rat striatal D-2 and D-3 sites: Enhancement by magnesium and inhibition by guanine nucleotides and sodium

Previous studies have demonstrated high affinity ³H-dopamine binding sites on mammalian striatal membranes. These putative dopamine receptors of unknown physiological significance have been termed D-3 sites. Such studies have failed, however, to demonstrate high affinity ³H-dopamine binding to D-2 sites, which can be labeled by ³H-butyrophenones, and which represent the putative dopamine receptors most stronly implicated in the behavioral correlates of dopaminergic CNS activity. We now report that preincubation of membrane homogenates with Mg⁺⁺ and inclusion of Mg⁺⁺ (1–10mM) or other divalent metal cations during binding allows high affinity D-2 specific ³H-dopamine binding in rat striatal membranes, and that these ions also increase the Bmax of D-3 specific ³H-dopamine binding. GTP, GDP, and GppNHp can completely abolish all D-2 specific ³H-dopamine binding, while only a magnesium-dependent portion of D-3 sites appears to be GTP sensitive. These data are consistent with the hypothesis that the striatal D-2 receptor exists in two agonist affinity states whose interconversion is effected by guanine nucleotides and divalent metal cations. The GTP sensitive/magnesium dependent nature of ³H-dopamine binding to so-called D-3 sites suggests that some such sites may in fact represent a high agonist-affinity state of the D-1 adenylate cyclase stimulating dopamine receptor also found in this tissue.     

Mast cells express tyrosine hydroxylase and store dopamine in a serglycin-dependent manner

Here we show that mast cells contain dopamine and that mast cell activation causes dopamine depletion, indicating its presence within secretory granules. Dopamine storage increased during mast cell maturation from bone marrow precursors, and was dependent on the presence of serglycin. Moreover, the expression of tyrosine hydroxylase, the key enzyme in dopamine biosynthesis, was induced during mast cell maturation; histidine decarboxylase and tryptophan hydroxylase 1 were also induced. Mast cell activation caused a robust induction of histidine decarboxylase, but no stimulation of tyrosine hydroxylase or tryptophan hydroxylase 1 expression. The present study points toward a possible role of dopamine in mast cell function.     

Functional comparison of full-length and N-terminal-truncated octopamine transporters from Lepidoptera

We have cloned two new lepidopteran octopamine transporters (OATs), members of the solute-linked carrier family 6 (SLC6) of nutrient transporters, from the CNS of the European corn borer Ostrinia nubilalis and the cabbage white Pieris rapae. Comparison of these sequences with the previously cloned OAT from the cabbage looper Trichoplusia ni showed that the T. ni OAT sequence previously reported was truncated by 74 amino acids at the N-terminus. The cytoplasmic N-termini deduced here are considerably longer than the N-termini of other monoamine transporters in the SLC6 family and contain many more high-probability serine- and threonine-phosphorylation sites. Monoamine uptake and competitive inhibition studies on baculovirus-infected Sf9 cells expressing these three cloned OATs indicate that they are able to transport tyramine, octopamine and dopamine with high affinity (K(m) and K(i) range, 0.4 microM-2.7 microM) and capacity ((3)H-dopamine uptake by TrnOAT, 2.5 pmol/well/min). We aimed to examine the role of the N-terminus of OAT by comparing the properties of the full-length T. ni OAT with those of the previously reported N-truncated version. Results for the new full-length T. ni OAT showed no difference in the protein's affinity for octopamine or dopamine, although at low levels of viral infection it did show slightly higher transport activity ((3)H-dopamine uptake by truncated TrnOAT, 1.5 pmol/well/min). Treatment of Sf9 cells expressing full-length or truncated TrnOAT with a variety of protein kinase activators and inhibitors, however, did not change transporter activity. Neither an intact N-terminus, nor apparently a particular phosphorylation state of this extended N-terminus, is required for OAT to transport monoamines.     

Distribution of the Golgi apparatus in the mitosis of cultured tobacco cells as revealed by DiOC6 fluorescence microscopy

Summary We developed a new method for distinguishing the Golgi apparatus from the other membranous organelles which contain DNA, such as mitochondria and chloroplasts, under a fluorescence microscope. Thin sections of cells embedded in Technovit 8100 resin were stained with both 3,3-dihexyloxacarbocyanine iodide (DiOC₆) and 4,6-diamidino-3-phenylindole (DAPI), and those three membranous organelles were observed under an epifluorescence microscope. The Golgi apparatus, which do not contain DNA, were easily recognized when the two images stained with DiOC₆ and DAPI were superimposed using an image processor. Using this method, we investigated the dynamics of cellular membranes and organelles during the mitotic cycle of synchronized cultured tobacco cells BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2). The Golgi apparatus did not accumulate in the rim of the formating early cell plate at anaphase, while it accumulated near the maturing cell plate at telophase, and this accumulation seemed to be related to the maturation of cell plates. To confirm this hypothesis, synchronized BY-2 cells were treated with caffeine, which is known to inhibit the cell plate formation. Most of the cells treated with caffeine remained in a phase in which Golgi vesicles were accumulated at the equatorial plate, but the cell plate was only partially maturing. The Golgi apparatus accumulated only near the partially maturing cell plate, but not by the equatorial plate where the Golgi vesicles had accumulated.Abbreviations DiOC₆ 3,3-dihexyloxacarbocyanine iodide - DAPI 4,6-diamidino-3-phenylindole - LSD a modified Linsmaier and Skoog's medium containing 2,4-D     

Dopamine uptake by platelets is selective, temperature dependent and not influenced by the dopamine-D1 or dopamine-D2 receptor

The human platelet, which takes up and releases dopamine, has been proposed as a peripheral model for the study of dopaminergic neurons in the central nervous system (CNS). In addition, the platelet has been shown to possess membrane components with pharmacological properties similar to the dopamine-D1 (DA-D1) and D2 (DA-D2) receptor on dopaminergic neurons. We have therefore studied the specificity of the platelet uptake system for dopamine and, as dopamine uptake comprises both internalised and membrane bound dopamine, the contribution of the DA-D1 and DA-D2 receptor to the uptake of dopamine has been assessed. Significant uptake of 3H-dopamine by platelet rich plasma (PRP) occurred after 10 min incubation at 37 degrees C, uptake being maximal after 90 min. In contrast, at 4 degrees C no uptake of 3H-dopamine occurred up to 60 mins incubation but at 20 degrees C was approximately 8% of the 60 min uptake at 37 degrees C. The neurotransmitters serotonin and dopamine inhibited dopamine uptake by platelets in a dose dependent manner. Uptake of dopamine appeared to be via two systems, one of high affinity with low capacity and the other of lower affinity but high capacity. In contrast, noradrenaline, adrenaline, acetylcholine, gamma-aminobutyric acid and histamine (10 microM) had no effect on dopamine uptake by platelets. The DA-D1 receptor antagonist SCH 23390 (10 microns) and the DA-D2 receptor antagonists (10 microM) spiperone, domperidone and (+)-butaclamol did not significantly affect dopamine uptake by platelets. In addition, ouabain and desipramine (100 microM) inhibited dopamine uptake by 21% and 24% respectively whilst reserpine and imipramine (100 microM) increased uptake by 14% and 15%. We therefore conclude that platelets take up dopamine via a selective, temperature dependent mechanism. Our data also suggest that dopamine uptake by platelets does not involve the DA-D1 or DA-D2 receptor.     

Ultrastructure of chronic megakaryocytic-granulocytic myelosis.

To study chronic megakaryocytic-granulocytic myelosis, bone marrow biopsies from 5 patients were obtained. Ultrastructural quantitative and qualitative assessments demonstrate proliferation of both the megakaryocytic and granulocytic cell lines. Factors indicative of malignant growth in megakaryocytes included atypical maturation, nuclear-cytoplasmic asynchrony, nuclear inclusions and production of micromegakaryocytes. Abnormal thrombocyte delineation provoked giant platelet production. The neutrophil series also presented atypia as generally observed in chronic myelogenous leukemia. Even in cases without evidence of myelofibrosis under the light microscope, megakaryoblasts were associated with fibrillar structures. These cells may be responsible for the initial step in fibrillogenesis by providing a medium conductive to the collagen formation found in later stages of this disease.     

Phospholipid composition of rat megakaryocytes and its rearrangement in platelets

Rat platelets and their megakaryocyte precursors were examined for phospholipid composition. (1) The phospholipid composition of rat megakaryocytes, which were enriched and prepared from bone marrow cells, was almost identical to that of platelets. (2) The subclass composition of choline-containing glycerophospholipids (CGP) of rat megakaryocytes differed significantly from that of platelets: 1-alkenyl-2-acyl glycerophosphocholine (GPC) in megakaryocytes accounted for 29% of the total, whereas that in platelets was only 7%. (3) Rat platelets contained a larger amount of arachidonic acid than megakaryocytes, especially in ethanolamine-containing glycerophospholipids (EGP). (4) [32P]Phosphoric acid was significantly incorporated into megakaryocytes, whereas platelets showed little incorporation. On the other hand, the uptake of [3H]arachidonic acid into platelet phospholipids was about 15-times higher than that observed with megakaryocytes. (5) As reported previously for other blood cells, such as neutrophils and macrophages, the radioactivity of labeled arachidonic acid incorporated into CGP of platelets decreased, whereas that incorporated into EGP increased during a subsequent chase period. Hardly any such change was observed with megakaryocytes. These results suggest that the phospholipid composition of rat platelets is mainly determined at the time of thrombopoiesis, whereas the composition of molecular species is remodeled during circulation after thrombopoiesis.