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.     

Location of sites of dopamine storage in megakaryocytes by autoradiography

We have used quantitative electron microscope autoradiography to study the subcellular sites of 3H-dopamine uptake in mouse megakaryocytes after a single intraperitoneal injection. Autoradiographic grains were found to be associated almost exclusively with the vesicles of precursors of monoamine-storage organelles. The labeling intensity (radioactivity) of the demarcation membrane system which is continuous with the plasmalemma was also significantly greater than would be expected. On the other hand, radioactivity associated with the remaining sites in the cytoplasm was not significantly different from that expected of a random distribution. In order to compare 3H-dopamine uptake during cell maturation, light microscope autoradiographic studies were also done. Immature megakaryocytes were labeled slightly, but the number of silver grains increased significantly in maturing cells. Mature megakaryocytes were 2.7 times more radioactive than the maturing cells. Our results suggest that the megakaryocytes were able to accumulate dopamine from the early stages of cell maturation and to store dopamine in precursors of monoamine-storage organelles.     

Regional differences in the high affinity uptake or 3H-dopamine and 3H-norepinephrine in synaptosome rich homogenates of cat brain.

The uptake of ³H-dopamine and ³H-norepinephrine into synaptosome rich fractions of cat brain was studied. Following kinetic analysis of the high affinity uptake mechanism, the five areas investigated could be divided into two distinct groups: (1) the dorsal caudate nucleus and the septal region which showed a greater affinity for dopamine than for norepinephrine and (2) the hypothalamus, prefrontal and cerebellar cortex which showed equal affinities for both dopamine and norepinephrine. The distribution of monoamine oxidase activity did not correspond with the affinities for norepinephrine or dopamine but choline acetyltransferase activity was higher in areas which showed a preference for dopamine. A high affinity uptake system for 5-hydroxytryptamine was also observed in the septal region.     

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.     

One-step purification of the serotonin transporter located at the human platelet plasma membrane.

A 68-kDa glycoprotein bearing the biological activity of the plasma membrane serotonin (5-hydroxytryptamine, 5-HT) transporter has been purified from human blood platelets, a classical cell model for the study of 5-HT uptake. After treatment of the whole platelet population or its plasma membrane fraction by sulfhydryl-dependent bacterial protein toxins or by digitonin, purification was reproducibly obtained by a one-step affinity chromatography using two different columns with 5-HT or 6-fluorotryptamine as ligands and elution by 5-HT or Na(+)-free buffer. The purified fraction migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band with an apparent molecular mass of 68 kDa and exhibited an apparent isoelectric point of 5.6-6.2. Two sialic acid residues were detected in the purified material. The purified glycoprotein bound the 5-HT uptake blocker [3H]paroxetine with a Kd (0.25 nM) similar to the one observed for intact human platelets. It also bound [3H] 5-HT but neither [3H]hydroxytetrabenazine nor [3H] ouabain, the respective markers of the granular monoamine transporter and of the Na+,K(+)-ATPase associated to the plasma membrane 5-HT transporter. 5-HT derivatives and 5-HT uptake inhibitors exhibited similar Ki values for 5-HT uptake and paroxetine binding in intact human platelets and in the purified glycoprotein. Under laser UV irradiation, 40% of this purified glycoprotein could be labeled by either [3H]paroxetine or [3H]cyanoimipramine. No labeling was detected with either [3H] gamma-aminobutyric acid or [3H]GBR 12783, the respective markers of gamma-aminobutyric acid and dopamine carriers. The purified 68-kDa protein is therefore likely to correspond at least to the binding domain of the 5-HT transporter located at the human platelet plasma membrane.     

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.     

Light and Electron microscopic autoradiography of 3H-dopamine uptake in the ganglia of the freshwater mussel, Anodonta cygnea L

In the central nervous system of the freshwater mussel, Anodonta cygnea, uptake of 3H-dopamine has been investigated by light and electron microscopic autoradiography following in vitro incubation and topical application, respectively. The autoradiographic reaction observed over the ganglia can be taken as a morphological evidence of the existence of a specific uptake mechanism for dopamine. It has been found that labeled dopamine was taken up by the axon profiles and terminals, while perikarya remained free of activity, indicating a basic difference between the perkaryal and axonal membrane in respect of the 3H-dopamine uptake. According to the electron microscopic autoradiography, labeled terminals and axon profiles contained both normal and accentric dense-core vesicles and they proved to be similar to those seen after 3H-serotonin in vitro incubation. Therefore, it seems possible that certain Anodonta central neurons contain both dopamine and serotonin.     

Specific binding sites for 5-hydroxytryptamine on rat blood platelets.

5-Hydroxytryptamine changes the shape of rat blood platelets by combination with a cinanserin-sensitive receptor which is not associated with the active uptake of 5-hydroxytryptamine. Binding of 5-hydroxy[3H]tryptamine to platelets at 4 degrees C demonstrates the presence of three saturable sites, and the highest-affinity site is apparently this 5-hydroxytryptamine receptor.     

Thrombin Stimulates Glucose Transport in Human Platelets via the Translocation of the Glucose Transporter GLUT-3 from α-Granules to the Cell Surface

Increased energy metabolism in the circulating blood platelet plays an essential role in platelet plug formation and clot retraction. This increased energy consumption is mainly due to enhanced anaerobic consumption of glucose via the glycolytic pathway. The aim of the present study was to determine the role of glucose transport as a potential rate-limiting step for human platelet glucose metabolism. We measured in isolated platelet preparations the effect of thrombin and ADP activation, on glucose transport (2-deoxyglucose uptake), and the cellular distribution of the platelet glucose transporter (GLUT), GLUT-3. Thrombin (0.5 U/ml) caused a pronounced shape change and secretion of most α-granules within 10 min. During that time glucose transport increased approximately threefold, concomitant with a similar increase in expression of GLUT-3 on the plasma membrane as observed by immunocytochemistry. A major shift in GLUT-3 labeling was observed from the α-granule membranes in resting platelets to the plasma membrane after thrombin treatment. ADP induced shape change but no significant α-granule secretion. Accordingly, ADP-treated platelets showed no increased glucose transport and no increased GLUT-3 labeling on the plasma membrane. These studies suggest that, in human blood platelets, increased energy metabolism may be precisely coupled to the platelet activation response by means of the translocation of GLUT-3 by regulated secretion of α-granules. Observations in megakaryocytes and platelets freshly fixed from blood confirmed the predominant GLUT-3 localization in α-granules in the isolated cells, except that even less GLUT-3 is present at the plasma membrane in the circulating cells (~15%), indicating that glucose uptake may be upregulated five to six times during in vivo activation of platelets.     

An assay to measure the simultaneous uptake of [3H]dopamine and [14C]serotonin by the human platelet reveals an imipramine-insensitive [3H]dopamine uptake mechanism.

To determine whether a specific dopamine uptake mechanism is present on the human platelet the simultaneous uptake of [3H]dopamine and [14C]serotonin by platelets was measured. Utilising a dual radiolabel uptake technique, platelets have been shown to take up serotonin more rapidly and to a greater extent than they take up dopamine. Furthermore, at high concentrations serotonin was able to reduce dopamine uptake by platelets by 60% whereas dopamine had no effect on serotonin uptake. Similarly, imipramine and reserpine reduced (97% and 74% respectively) serotonin uptake by platelets in a dose-dependent manner, but did not affect the uptake of dopamine. Our data show that platelets take up dopamine by a mechanism independent of the imipramine-sensitive serotonin uptake mechanism. Furthermore, the increased capacity of platelets to store serotonin is because serotonin, unlike dopamine, is transported into the dense granules of the platelet.     

The first luminal domain of vesicular monoamine transporters mediates G-protein-dependent regulation of transmitter uptake

The activity of vesicular monoamine transporters (VMATs) is down-regulated by the G-protein alpha-subunits of G(o2) and G(q), but the signaling pathways are not known. We show here that no such regulation is observed when VMAT1 or VMAT2 are expressed in Chinese hamster ovary (CHO) cells. However, when the intracellular compartments of VMAT-expressing CHO cells are preloaded with different monoamines, transport becomes susceptible to G-protein-dependent regulation, with differences between the two transporter isoforms. Epinephrine induces G-protein-mediated inhibition of transmitter uptake in CHOVMAT1 cells but prevents inhibition induced by dopamine in CHOVMAT2 cells. Epinephrine also antagonizes G-protein-mediated inhibition of monoamine uptake by VMAT2 expressing platelets or synaptic vesicles. In CHOVMAT2 cells G-protein-mediated inhibition of monoamine uptake can be induced by 5-hydroxytryptamine (serotonin) 1B receptor agonists, whereas alpha1 receptor agonists modulate uptake into CHOVMAT1 cells. Accordingly, 5-hydroxytryptamine 1B receptor antagonists prevent G-protein-mediated inhibition of uptake in partially filled platelets and synaptic vesicles expressing VMAT2. CHO cells expressing VMAT mutants with a shortened first vesicular loop transport monoamines. However, no or a reduced G-protein regulation of uptake can be initiated. In conclusion, vesicular content is involved in the activation of vesicle associated G-proteins via a structure sensing the luminal monoamine content. The first luminal loop of VMATs may represent a G-protein-coupled receptor that adapts vesicular filling.     

Dopamine sulfate formation and phenol sulfotransferase activity in dog and human platelets

High performance liquid chromatography with radioactive flow detection was used to examine the accumulation and sulfoconjugation of dopamine by human and dog platelets. Platelets from both species accumulated similar amounts of dopamine from the incubation medium, but only human platelets were found to convert 3H-dopamine to 3-H-dopamine sulfate. This difference between the two species was associated with a relative absence of phenol sulfotransferase activity in dog platelets as compared to human platelets. Dog platelets did not appear to contain an inhibitor of phenol sulfotransferase activity. Despite the apparent difference in the ability of platelets to form dopamine sulfate, conc concentrations of dopamine-3-O-sulfate and dopamine-4-O-sulfate were similar in dog and human plasma. These data suggest that platelets may represent a potential source of at least some of the dopamine sulfate found in human plasma, but not in dog plasma.     

Mechanism of imipramine inhibition of platelet 5-hydroxytryptamine transport.

Plasma membrane vesicles isolated from porcine blood platelets take up approximately 8 to 15 pmol of [3H]imipramine per mg of membrane protein. This apparent binding requires Na+ in the external medium and is reversed by 5-hydroxytryptamine and fluoxetine. The apparent KD for imipramine uptake is 23 nM, which agrees well with the KI for competitive inhibition of 5-hydroxytryptamine transport by imipramine. In contrast to 5-hydroxytryptamine transport, imipramine uptake is not dependent on transmembrane Na+ and K+ gradients and is insensitive to ionophores such as nigericin and gramicidin which dissipate these gradients. Although 5-hydroxytryptamine rapidly and competitively displaces imipramine from membrane vesicles, imipramine does not cause 5-hydroxytryptamine efflux and inhibits 5-hydroxytryptamine exchange. These results are consistent with the proposal that imipramine binds to the substrate site of the 5-hydroxytryptamine transporter but cannot be transported.     

Composition and synthesis of glycolipids in megakaryocytes and platelets: differences in synthesis in megakaryocytes at different stages of maturation

The composition and synthesis of megakaryocyte and platelet glycolipids were compared since these lipids are thought to be important for biologic activities such as adhesion and maturation. Highly purified guinea pig megakaryocytes at different stages of maturation and platelets were studied. Glycolipids and gangliosides were extracted, separated by thin-layer chromatography, and the carbohydrate content was analyzed by gas-liquid chromatography (GLC). Synthesis of ceramides and glycolipids was determined by the incubation of megakaryocytes with [14C]acetate, [3H]palmitic acid, and [3H]galactose. A major neutral glycolipid present in guinea pig megakaryocytes and platelets was identified as asialoGM2 by selective enzymatic hydrolysis with beta-N-acetylhexosaminidase, alpha-galactosidase and endo-beta-galactosidase, and carbohydrate analysis by GLC. Trace amounts of asialoGM1 were detected immunologically. The cells also contained glucosyl ceramide and lactosyl ceramide. Several ganglosides were detected of which one was identified as GM1 by its reaction with the beta-subunit of cholera toxin and by the identification of an asialoGM1 core with anti-asialoGM1 antibody after desialylation. The synthesis of ceramides from palmitic acid and acetate was 5 and 10 times greater, respectively, in megakaryocytes than in platelets. Ceramide and glycolipid synthesis from palmitic acid occurred primarily in immature megakaryocytes while synthesis from acetate occurred primarily in more mature megakaryocytes. The glycosylation of ceramides from galactose was 42 times greater in megakaryocytes than in platelets. Thus, ceramides and glycolipids are primarily synthesized in megakaryocytes, but platelets retain the capacity to synthesize significant amounts of free ceramides. The glycosylation of free ceramides occurs almost exclusively in megakaryocytes and only in trace amounts in platelets. These data indicate that megakaryocytes determine the composition of glycolipids in platelets and that there is considerable compartmentalization of glycolipid synthesis and membrane assembly at various stages of megakaryocytes development.     

Subcellular distribution of [14C]5-hydroxytryptamine in the blood platelets of rabbits: effects of imipramine and and haloperidol

Long-term (10 days) administration of imipramine [20 mg/(kg X d)] to rabbits significantly increases the Km value (4.0 micron) of 5-hydroxytryptamine uptake in their platelets compared to those of saline- (0.7 micron) or haloperidol- (0.4 micron) treated rabbits. Administration of haloperidol inhibits the 5-hydroxytryptamine uptake non-competitively, and in vitro it had an ID50 value of 22 micron. Intravenous injections of [14C]5-hydroxytryptamine were given to the animals 1 h before blood collection. After isolation of platelets, their sonicates were subjected to 30-60% continuous sucrose gradient centrifugation. The subcellular distribution of [14C]5-hydroxytryptamine indicates that imipramine treatment, in contrast to the control and haloperidol treatment, led to a shift in the exogenous 5-hydroxytryptamine peak from within the granular zone (d 1.18) to the extragranular cytoplasm (d 1.15). Compared to control values, the imipramine treatment caused 63% inhibition in the platelet Na-K-ATPase activity.     

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)     

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.     

Escaping the nuclear confines: signal-dependent pre-mRNA splicing in anucleate platelets

Platelets are specialized hemostatic cells that circulate in the blood as anucleate cytoplasts. We report that platelets unexpectedly possess a functional spliceosome, a complex that processes pre-mRNAs in the nuclei of other cell types. Spliceosome components are present in the cytoplasm of human megakaryocytes and in proplatelets that extend from megakaryocytes. Primary human platelets also contain essential spliceosome factors including small nuclear RNAs, splicing proteins, and endogenous pre-mRNAs. In response to integrin engagement and surface receptor activation, platelets precisely excise introns from interleukin-1beta pre-mRNA, yielding a mature message that is translated into protein. Signal-dependent splicing is a novel function of platelets that demonstrates remarkable specialization in the regulatory repertoire of this anucleate cell. While this mechanism may be unique to platelets, it also suggests previously unrecognized diversity regarding the functional roles of the spliceosome in eukaryotic cells.     

Kinetics of serotonin in platelets in essential hypertension.

In a study of the kinetics of 5-hydroxytryptamine (5-HT) in platelets in 26 hypertensive subjects with a mean systolic and diastolic blood pressure of 153.9 +/- 26.9 and 106.9 +/- 9.1 mm Hg respectively, it has been observed that in hypertensive platelets there was a marked decrease in 5-HT uptake and content, an increase in 5-HT efflux and an accompanying increase in Plasma 5-HT and 5-HIAA levels. Regression analysis of the data indicated a significant correlation between rise in diastolic blood pressure and these changes in 5-HT kinetics.     

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.