The Vesicular Monoamine Content Regulates VMAT2 Activity through Galpha q in Mouse Platelets

EVIDENCE FOR AUTOREGULATION OF VESICULAR TRANSMITTER UPTAKE*

Markus HöltjeDagger §, Sandra WinterDagger §, Diego Walther, Ingrid PahnerDagger ||, Heide Hörtnagl**, Ole Petter Ottersen||, Michael Bader, and Gudrun Ahnert-HilgerDagger DaggerDagger

From the Dagger  Institut für Anatomie der Charité, Humboldt Universität zu Berlin, Philippstrasse 12, 10115 Berlin, Germany, the  Max-Delbrück-Centrum für Molekulare Medizin, 13092 Berlin Buch, Germany, the || Centre for Molecular Biology and Neuroscience and Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, P. O. Box 1105, Blindern, N-0317 Oslo, Norway, and the ** Institut für Pharmakologie der Charité, Humboldt Universität zu Berlin, Dorotheenstr. 94, 10117 Berlin, Germany

Received for publication, December 17, 2002, and in revised form, February 6, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Variations in the neurotransmitter content of secretory vesicles enable neurons to adapt to network changes. Vesicular content may be modulated by vesicle-associated Go2, which down-regulates the activity of the vesicular monoamine transmitter transporters VMAT1 in neuroendocrine cells and VMAT2 in neurons. Blood platelets resemble serotonergic neurons with respect to transmitter storage and release. In streptolysin O-permeabilized platelets, VMAT2 activity is also down-regulated by the G protein activator guanosine 5'-(beta igamma -imido)triphosphate (GMppNp). Using serotonin-depleted platelets from peripheral tryptophan hydroxylase knockout (Tph1-/-) mice, we show here that the vesicular filling initiates the G protein-mediated down-regulation of VMAT2 activity. GMppNp did not influence VMAT2 activity in naive platelets from Tph1-/- mice. GMppNp-mediated inhibition could be reconstituted, however, when preloading Tph1-/- platelets with serotonin or noradrenaline. Galpha q mediates the down-regulation of VMAT2 activity as revealed from uptake studies performed with platelets from Galpha q deletion mutants. Serotonergic, noradrenergic, as well as thromboxane A2 receptors are not directly involved in the down-regulation of VMAT2 activity. It is concluded that in platelets the vesicle itself regulates transmitter transporter activity via its content and vesicle-associated Galpha q.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transport of monoamines like serotonin, catecholamines, or histamin into the secretory vesicles of a variety of cells is mediated by vesicular monoamine transporters (VMATs).1 In mammals two closely related transporters, VMAT1 and VMAT2, are known (1, 2). These two subtypes differ in their substrate specificity, pharmacological properties, and tissue distribution (3, 4). Both transporters represent proteins with 12 predicted transmembrane domains of different vesicle types like large dense core vesicles, small synaptic vesicles (SSVs), and synaptic-like microvesicles (2, 5). In contrast to the transporters of the plasma membrane, which mainly use the Na+ gradient across the plasma membrane to drive transport, activity of vesicular transporters relies on the proton electrochemical gradient (Delta µH+) generated by a vacuolar H+-ATPase (6). The variability of quantal size has been established for monoaminergic cells like PC12 cells (7, 8) or leech Retzius cells (9) using carbon fiber amperometry. These studies suggest that secretory vesicles vary in their transmitter content and that this variability may result from changes in vesicular volume rather than from changes in transmitter concentration.

Recently, we have identified functional subsets of heterotrimeric G proteins on secretory vesicle membranes (10, 11) and have shown that the activity of both VMAT1 and VMAT2 is regulated by the alpha -subunit of Go2 (11-13). By this mechanism neurons and other secretory cells might vary their vesicular transmitter content and therefore alter the amount of transmitter released during exocytosis. The major goal of the present study was to elucidate the upstream signals for the G protein activation leading to the observed inhibition of VMAT activity. In general, heterotrimeric G proteins are activated by heptahelical receptors of the plasma membrane. Exceptions to this rule are the growth cone associated protein GAP 43 (14) as well as the amyloid precursor protein (15) that also activate G protein signaling. Another question concerned G proteins probably involved in regulating transporter activity in cells that store monoamines but do not express Go2. To address these issues we examined mouse blood platelets. Platelets are the major storage sites of serotonin apart from the central nervous system. They take up serotonin by the serotonin plasma membrane transporter (SERT) and, as has been shown for human platelets, package it by VMAT2 activity (16, 17) into large dense bodies together with other substances like ADP (for review see Ref. 18). Once activated by various substances at sites of endothelial injury, these cell fragments become spherical, acquire surface stickiness, aggregate, and release mediators like serotonin that act at endothelial cells and smooth muscles. This activation cascade repairs damaged blood vessels, maintains hemostasis, and may even lead to diseases like thrombosis. We took advantage of mice deficient of peripheral tryptophan hydroxylase activity (Tph1-/-), the key enzyme in serotonin synthesis. Platelets from Tph1-/- mice contain only very small amounts of serotonin compared with wild type ones (19). We used these platelets as an accessible model system for monoamine transmitter-depleted vesicles that allowed us to study the impact of the transmitter content on G protein-mediated VMAT regulation. Using mice deficient for the alpha -subunit of Gq, we could identify this G protein subunit as a regulator of VMAT2 activity in platelets.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Antibodies-- Mouse monoclonal antibodies against Galpha o2 (clone 101.1) and synaptobrevin II (clone 69.1) (20) as well as a rabbit polyclonal antiserum against VMAT2 (13) were kindly provided by R. Jahn (Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany). The following G protein subtype-specific polyclonal antisera were used: Galpha o2 (AS 371) (21), Galpha q/11 (AS 370) (22), Gbeta 2 (AS 36) (23), and Gbeta 5 (AS 422) (24). All of the antisera were characterized with respect to specificity and cross-reactivity (11, 24). A monospecific antiserum against Galpha q was obtained from Calbiochem. Horseradish peroxidase-labeled anti-rabbit or anti-mouse IgG was obtained from Vector Laboratories (Burlingham, CA).

Transmitters and Receptor Ligands-- 5-Hydroxy-[3H]tryptamine trifluoroacetate (serotonin; specific activity, 3260 Bq/mmol) and [3H]noradrenaline (specific activity, 444 Bq/mmol) were obtained from Amersham Biosciences. The 5HT2A receptor antagonist spiperone, the alpha 2A receptor antagonist BRL 44408, the D3 receptor antagonist GR 103691, as well as the TXA2 agonist U46619 were obtained from Tocris.

Toxins-- Streptolysin O from beta -hemolytic strepococci (SLO) (25) and alpha -toxin from Staphylococcus aureus were kindly supplied by U. Weller (Institut Ray-Rocky-Weller, Baden-Baden, Germany).

Mice-- Wild type and peripheral tryptophan hydroxylase knockout (Tph1-/-) mice were bred as given (19). Wild type and Galpha q-/- mice were kindly supplied by S. Offermanns (Institut für Pharmakologie, Heidelberg, Germany) and bred as previously described (26).

Preparation of Blood Platelets-- Genotyped mice of either sex were anesthetized by intraperitoneally injecting a mixture of 50 µl of Ketavet (Curamed Pharma, Karlsruhe, Germany) and 50 µl of Rompun (Bayer, Leverkusen, Germany) per 10 g of body weight. Blood was taken from the inferior caval vein in the presence of heparine (30 IU/ml). The blood was mixed with 0.5 volume of Tyrode-Hepes (TH) buffer (134 mM NaCl, 0.34 mM Na2HPO4, 2.9 mM KCl, 12 mM NaHCO3, 20 mM HEPES, 5 mM glucose, 1 mM MgCl2, pH 7.3) and centrifuged for 7 min at 210 × g. The platelet-rich plasma supernatant was taken and centrifuged for 5 min at 1090 × g. The resulting pellet was resuspended in TH buffer, and the platelets were counted in a Neubauer chamber. Platelets from wild type and Tph1-/- or Galpha q-/- mice were adjusted to an equal number of platelets/ml prior to the experiments.

Serotonin Uptake-- Sedimented platelets (see above) were washed two times with TH buffer. For vesicular uptake, platelets were suspended in potassium glutamate buffer (KG buffer) containing 150 mM potassium glutamate, 20 mM 1,4-piperazine diethanesulfonic acid, 4 mM EGTA, 1 mM MgCl2, 1 mM dithiothreitol, adjusted to pH 7.0 with KOH supplemented with SLO. Incubation was performed for 5 min on ice. Unbound toxin was removed by centrifugation (5 min, 1090 × g). The pellets were resuspended in KG buffer with 2 mM ATP added. The platelets were then divided into individual reaction cups, and uptake was started by adding 100 µl of KG-ATP buffer supplemented with 1 mM ascorbic acid and 80 nM [3H]serotonin. Additives such as the nonhydrolyzable GTP-analogon GMppNp or tetrabenazine were applied during this step. Incubation was performed for 15 min at 37 °C and stopped by the addition of 1 ml of ice-cold KG buffer followed by rapid centrifugation. The pellets were then lysed in 0.4% Triton-X-100 to determine radioactivity by liquid scintillation counting and protein content using the bicinchoninic acid method (BCA kit; Pierce).

Serotonin uptake by intact platelets was performed in TH buffer supplemented with 80 nM [3H]serotonin and 1 mM ascorbic acid for 30 min at 37 °C. Incubation was stopped by diluting with ice-cold TH buffer, and the samples were processed as in the case of permeabilized platelets.

For reconstitution experiments intact blood platelets were preloaded with 15 µM serotonin dissolved in TH buffer for 30 min at room temperature before they were subjected to the permeabilization and vesicular uptake procedure or analyzed by HPLC. In general, the samples were analyzed in triplicate.

Monoamine Determination by HPLC-- The monoamine content of wild type Tph1-/- or Galpha q-/- platelets was quantitated by HPLC using either a fluorescence detection system or an electrochemical detection system yielding similar results. The platelets were resuspended in 0.1 N perchloric acid dissolved in PBS and further processed with a fluorescence detection system (27) or electrochemical detection system (19) as described previously. The amounts of serotonin calculated from the standard curve were given in nanograms and normalized to protein content or given per platelet (see above).

Preparation of Synaptic Vesicles-- Crude synaptic vesicles (lysis pellet 2 fraction) were prepared from mice whole brain homogenates according to a protocol described (28). Serotonin uptake was performed as described recently (13).

Electron Microscopy-- Sedimented platelets (see above) were suspended in 4% paraformaldehyde, 0.1% glutaraldehyde dissolved in PBS, and fixed for 15 min at room temperature. After fixation the platelets were spun down again at 2000 × g for 5 min, resuspended, and incubated for 10 min at room temperature in PBS containing 50 mM glycine and 0.1% sodium borhydride as quenching solution. After another PBS washing/centrifugation step, the platelets were cryoprotected in glycerol/PBS for the following freeze substitution and embedded in Lowicryl HM 20 (Electron Microscopy Sciences, Fort Washington, PA) as described (29). Ultrathin sections (70 nm) were cut on a Leica Ultratome and mounted onto Formvar-covered (Serva, Heidelberg, Germany; 0.8% in chloroform) nickel grids. The sections were subjected to postembedding immunogold procedure as described (30). Briefly, the sections were etched with 1% H2O2 diluted in H2O for 30 min followed by immunocytochemistry. Unspecific binding sites were blocked with 2% bovine serum albumin and 5% normal goat serum in Tris-buffered saline containing 0.01% Triton X-100 (pH 7.6) for 1 h at room temperature. Then the sections were incubated with the respective primary antiserum diluted in the blocking solution for 3 h at room temperature. After a further blocking step with 2% bovine serum albumin in Tris-buffered saline containing 0.01% Triton X-100 (pH 7.6), the sections were incubated with goat anti-rabbit Fab fragments coupled to 5- or 10-nm gold particles (British BioCell International, Cardiff, UK) diluted 1:20 in blocking solution for 1.5 h at room temperature. The sections were stained with uranyl acetate and lead citrate. The micrographs were taken on a Zeiss EM 900 or a Philipps CM 10.

Immunoreplica Analysis-- Sedimented platelets or synaptic vesicles from wild type or deletion mutants were dissolved in Laemmli buffer, and equal amounts of protein were separated by SDS-PAGE. After transfer to nitrocellulose the respective proteins were detected by the indicated antibodies using the ECL detection system (Amersham Biosciences).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Comparison of the Serotonin Uptake by Wild Type and Tph1-/- Platelets-- As a first approach we compared vesicular monoamine uptake into platelets from wild type and Tph1-/- mutants permeabilized by SLO. In wild type platelets a serotonin uptake of about 50 pmol/mg protein could be detected, which was less compared with the 65 pmol/mg protein found for Tph1-/- platelets (Fig. 1A). In both preparations serotonin uptake was completely abolished when reserpine or tetrabenazine was added, indicating that the observed uptake was mediated by VMAT2 (4). A significant inhibition of uptake was observed, when GMppNp (100 µM), an activator of trimeric G proteins, was added to wild type platelets. In contrast, the addition of GMppNp displayed no effect on uptake into permeabilized Tph1-/- platelets. A summary of the data obtained from five individual experiments revealed that the vesicular serotonin uptake into mutant platelets was 40% higher compared with the wild type preparations (Fig. 1A, inset). When comparing the GMppNp-induced inhibition of vesicular [3H]serotonin uptake in wild type and mutant platelets (combined data from five individual experiments, given as % of control), the uptake in the former was inhibited by 40%, whereas no effect was seen in mutant platelets (Fig. 1A, inset).


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Fig. 1.   [3H]Serotonin uptake into blood platelets and brain SSV from wild type and Tph1-/- mice: Effects of GMppNp. A, SLO-permeabilized platelets were resuspended in KG-ATP buffer containing 80 nM [3H]serotonin with additions as indicated and incubated for 15 min at 37 °C. The observed uptake was reserpine- and tetrabenazine-sensitive. Both inhibitors of VMAT2 (final concentration, 5 and 10 µM, respectively) completely blocked uptake activity in either platelet preparation. The addition of the nonhydrolyzable GTP analogue GMppNp (100 µM) inhibited the vesicular serotonin uptake to about 35% in wild type but not in Tph1-/- platelets. The values represent the means of three determinations ± S.D. Statistical significance (p < 0.05) was verified using Student's t test. Inset, summary of the data obtained from five individual experiments comparing vesicular uptake (upper graph, wild type given as 100%) and GMppNp-induced inhibition (lower graph, GMppNp-mediated inhibition given as a percentage of control uptake) between wild type and Tph1-/- platelets. B, [3H]serotonin uptake into brain synaptic vesicles from wild type and Tph1-/- mice. A crude synaptic vesicle preparation from a whole brain homogenate was incubated for 10 min with 80 nM [3H]serotonin. In both wild type and knockout animals, the addition of GMppNp inhibited the vesicular serotonin uptake to 25%. The significance of the GMppNp effects was p = 0.0098 for wt and p = 0.025 for Tph1-/-. Unspecific uptake performed in duplicates in the presence of tetrabenazine (0.85 and 0.99 pmol/mg protein for wild type and Tph1-/-, respectively) was subtracted. wt, wild type.

In Tph1-/- mice the activity of the peripheral tryptophan hydroxylase is abolished, whereas the respective enzyme of central neurons is maintained, resulting in normal serotonin levels in brain. So the GMppNp-mediated down-regulation of serotonin uptake into a crude vesicular preparation from wild type and mutant brains was analyzed for comparison. The tetrabenazine-sensitive uptake was unchanged in Tph1-/- compared with wild type mice, and more importantly, the addition of GMppNp inhibited the uptake to about 25% in either group (Fig. 1B).

To analyze how transport is affected by G protein activation, a Michaelis-Menten kinetic was performed using wild type platelets. The presence of GMppNp decreased Vmax from 18.37 pmol/mg/min in control to 13.9 pmol/mg protein/min (values in a second experiment were 26.11 and 20.05 pmol/mg protein/min, respectively) and increased Km from 0.47 µM (control) to 0.57 µM (second experiment, 0.74 to 0.91 µM) (Fig. 2). The obtained kinetic values for VMAT2 activity in mouse platelets are comparable with kinetic data published for human VMAT2 (4).


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Fig. 2.   Kinetic analysis of GMppNp-mediated regulation of VMAT2 in wild type platelets. SLO-permeabilized platelets were resuspended in KG-ATP buffer containing 40 nM [3H]serotonin and various concentrations of unlabeled serotonin to yield the final concentration of serotonin (abscissa) in the absence or presence of 100 µM GMppNp. Uptake was performed for 15 min at 37 °C. Michaelis-Menten curve yields Vmax of 18.37 or 13.9 pmol/mg of protein/min in the absence or presence of GMppNp, respectively. Lineweaver-Burk analysis (inset) showed a slight increase in Km from 0.47 to 0.57 µM in the presence of GMppNp. The values (nonlinear regression) represent the means of triplicate experiments corrected for the unspecific uptake in the presence of tetrabenazine and are calculated using Graph Pad Prism software.

Thus, GMppNp affected both Vmax and Km of VMAT2 in platelets. Deletion of peripheral tryptophan hydroxylase, which reduces serotonin content in the periphery, abolished the G protein-mediated down-regulation of VMAT2 only in secretory vesicles of platelets but not in SSV.

Reconstitution of GMppNp-mediated Down-regulation of [3H]Serotonin Uptake by Preloading Tph1-/- Platelets with Serotonin-- Having seen that the GMppNp-mediated down-regulation of serotonin uptake is functional in Tph1-/- brain SSV but completely abolished in the respective platelets, we preloaded Tph1-/- platelets with serotonin to shift the vesicular serotonin content to a level that closely resembled the one found in platelets from wild type animals. When treating Tph1-/- platelets with 15 µM serotonin prior to the permeabilization and uptake procedure, GMppNp strongly inhibited uptake by about 50%, whereas uptake in similarly treated controls was unaffected (Fig. 3A). Preincubation with increasing serotonin concentrations between 0 and 150 µM showed a dose-dependent reconstitution of the GMppNp-induced down-regulation of vesicular [3H]serotonin uptake with a maximal effect at 15 µM serotonin that caused an inhibition by 40% (Fig. 3B). At higher serotonin concentrations the inhibition as well as the overall uptake decreased, which might be attributed to enhanced platelet clotting.


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Fig. 3.   Preincubation of Tph1-/- platelets with serotonin reconstitutes down-regulation of vesicular [3H]serotonin uptake by GMppNp. A, intact Tph1-/- platelets were incubated for 30 min with buffer alone or supplemented with 15 µM nonlabeled serotonin. After preincubation and following SLO permeabilization, the addition of GMppNp inhibited the vesicular [3H]serotonin uptake by 50%. No GMppNp-induced inhibition was observed in platelets receiving just buffer during the preincubation. The values given in pmol/mg protein represent the means of three determinations ± S.D. Statistical significance (p = 0.022) was verified using Student's t test. B, platelets from Tph1-/- mice were preincubated with buffer or buffer supplemented with increasing amounts of serotonin (concentrations given in µM on the abscissa). A down-regulation of vesicular [3H]serotonin uptake by GMppNp could be induced dose-dependently by preloading the platelets with serotonin. Preincubation with 15 µM serotonin resulted in a maximum effect of 40% inhibition of uptake because of GMppNp. The GMppNp-induced inhibition slightly declined, when higher concentrations of serotonin were applied. The uptake in the presence of GMppNp is expressed as a percentage of control, representing the serotonin uptake in the absence of GMppNp for each preincubation condition. Unspecific uptake in the presence of tetrabenazine was subtracted before calculating values. The data represent the means of three determinations ± S.D. C, incubation of alpha -toxin permeabilized Tph1-/- platelets with 15 µM of either serotonin or noradrenaline before performing the uptake with [3H]serotonin reconstituted GMppNp-mediated inhibition. The ef- fects of GMppNp are expressed as a percentage of inhibition of the respective control condition. Unspecific uptake in the presence of tetrabenazine was subtracted before calculating the values. The data represent the means of three determinations ± S.D.

To determine whether the reconstitution of the GMppNp-mediated down-regulation of VMAT2 in Tph1-/- platelets is also mediated by other monoamines, such as noradrenaline, a second experimental approach was performed. Generally, platelets take up serotonin because of the activity of the plasma membrane transporter SERT, which less accepts other monoamines. To directly preload secretory vesicles with noradrenaline, pemeabilization with alpha -toxin instead of SLO was used. alpha -Toxin generates only small pores and therefore yields longer living permeabilized preparations. alpha -Toxin-permeabilized Tph1-/- platelets were preloaded with 15 µM noradrenaline or serotonin used for comparison or just received buffer. Following preloading, the usual uptake procedure using [3H]serotonin dissolved in uptake buffer and supplemented with or without GMppNp was performed. Whereas GMppNp inhibition was less than 15% in control platelets, it increased to 42-48% because of the preloading with either noradrenaline or serotonin, respectively (Fig. 3C). The data so far suggest that preloading with serotonin and noradrenaline can reconstitute GMppNp-mediated inhibition in Tph1-/- platelets.

Monoamine Content of Platelets from Wild Type and Tph1-/- Mice-- To prove that Tph1-/- platelets do not contain other monoamines, a HPLC analysis was performed (Fig. 4A). In wild type platelets the serotonin content was calculated to be 5.31 ng/µg protein, whereas in Tph1-/- platelets it was only 0.32 ng/µg protein, representing ~6% of the amount detected in wild type platelets (19). Additionally, the content of noradrenaline, adrenalin, and dopamine was examined. Both noradrenaline and adrenalin could only be detected in minute amounts, and dopamine was below detection level.


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Fig. 4.   HPLC analysis of monoamine content in wild type and Tph1-/- platelets. A, sedimented platelets were resuspended with 0.1 N perchloric acid, sonicated, and centrifuged. The resulting supernatant was subjected to HPLC analysis. The serotonin content in wild type platelets was 5.31 ng/µg protein, whereas in Tph1-/- platelets only 0.32 ng of serotonin/µg of protein were detected. Noradrenaline and adrenalin were detected at very low levels, and dopamine was below detection level (not shown). The values represent the means of three determinations ± S.D. B, the serotonin content in wt or Tph1-/- platelets before and after a 30-min preincubation with 15 µM serotonin was compared by HPLC analysis. In the wild type, the amount of serotonin slightly increased by 20% from 2.15 to 2.66 ng serotonin/µg protein after preincubation. In Tph1-/- platelets, an approximately 8-fold increase from 0.18 to 1.51 ng serotonin/µg protein could be detected. The values represent the means of three determinations ± S.D. wt, wild type.

To demonstrate that preincubation with serotonin does considerably alter the vesicular filling in platelets, we compared the amount of serotonin before and after a 30-min preincubation with 15 µM serotonin. In this set of experiments, the platelet serotonin content before preincubation was 2.15 and 0.18 ng/µg protein in wild type and Tph1-/- platelets, respectively (Fig. 4B). After preincubation with serotonin, the serotonin content was increased by about 20% (2.66 ng/µg) in wild type platelets. In Tph1-/- platelets the increase was much more pronounced, reaching 8-fold the amount of untreated platelets (1.51 ng/µg). The resulting serotonin content approximates serotonin levels found in wild type platelets (Fig. 4B).

VMAT2 and G Protein Subunits in Wild Type and Tph1-/- Mice-- The ultrastructure of the platelets was examined by electron microscopy to see whether the deficiency in serotonin synthesis in Tph1-/- mice causes morphological changes of the vesicular storage organelles. However, the comparison with platelets of wild type mice revealed no differences in the ultrastructural morphology (Fig. 5A). All platelets contained some few vesicular elements with an average diameter of ~120 nm and a sharp membraneous envelope, which identified them as dense bodies, although most of them had lost their dense core because of the histological preparation for electron microscopy. The dense bodies could be easily discriminated from profiles of the open canalicular system of the platelet plasma membrane that is characterized by a fuzzy glycocalyx coat on its luminal appearing membrane. In addition to the dense bodies, two other types of opaque vesicular elements could be observed in platelets of both, Tph1-/- and wild type mice. These comprise large alpha -granules with an approximate diameter of 100-250 nm. Some of them contained vesicular structures inside and may therefore represent an immature stage of alpha -granules, also called multivesicular bodies (31). Also, small dense granules with a diameter of 30 nm were observed throughout in the platelet cytoplasm.


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Fig. 5.   Detection of VMAT2 and G protein subunits in wt and Tph1-/- platelets. A, platelets isolated from wt and Tph1-/- mice show no ultrastructural differences. Three different types of vesicular organelles could be identified within the platelets of both preparations: dense bodies (black asterisk), alpha -granules (white asterisk), and small dense vesicles (arrowheads). Scale bars, 200 nm. B, brain postnuclear supernatants and platelets from wild type and Tph1-/- mice were subjected to SDS-PAGE and subsequent immunoreplica analysis. Note that Galpha o2 showed a strong immunoreaction in brain, but only minute amounts were detectable in platelets. Gbeta 5 exhibited a strong signal in brain, but no immunoreactivity was found in platelets. C, application of antisera against VMAT2, Galpha q/11 or synaptobrevin (syb) revealed a membrane-associated immunogold labeling of dense bodies as well as of small dense vesicles and alpha -granules from Tph1-/- platelets. Scale bars, 100 nm. wt, wild type.

To see whether a change in the occurrence of some proteins required for proper function of vesicular serotonin storage might account for the observed differences, we compared by immunoreplica analysis the amount of VMAT2 and G proteins in SSV and platelet preparations from both types of mice. An antiserum against VMAT2 detected major protein bands at 55 kDa as well as a few minor bands in the platelets and brain of either group, corresponding to the apparent molecular weight and possible protein modifications of VMAT2 (32, 33). An antiserum against Galpha o2 exhibited a strong immunosignal in brain, but virtually no signal could be detected in platelets of either type. Antisera against Galpha q/11 and Gbeta 2 showed strong signals in platelets and brain of either group, whereas Gbeta 5 was only detected in brain preparations. Protein bands in the wild type appeared to be a little stronger than in knockout animals, but no significant differences were seen in the general pattern (Fig. 5B).

The application of immunogold electron microscopy revealed that platelets from Tph1-/- and wild type mice also do not differ with respect to the subcellular distribution of the vesicular proteins synaptobrevin, VMAT2, as well as the G protein Galpha q subunit. Immunogold signals for all three proteins were found to be associated with membranes of the three vesicular organelles described above (Fig. 5C). Interestingly, the immunoreactivities for VMAT2 and synaptobrevin were more pronounced on alpha -granules and small dense vesicles compared with dense bodies. On the other hand dense bodies have been described to be the only serotonin storage sites in human platelets (18, 34). Probably, mouse platelets for which no ultrastructural data were found in the literature differ from human platelets in this respect.

Galpha q Regulates Vesicular Monoamine Transport in Platelets-- In contrast to the situation in neurons and neuroendocrine cells, the absence of Galpha o2 in platelets (Ref. 35 and this work) excludes its involvement in the inhibition of VMAT2 activity. In platelets Galpha q is one of the most important G proteins (35). In addition to its localization on the plasma membrane, it also sediments with the dense granule fraction in human platelets (36) and localized together with VMAT2 on secretory vesicles (Fig. 5C). Therefore, Galpha q might be well suited as a regulator of vesicular monoamine transport in platelets. Consequently, we performed uptake experiments on mice lacking the gene for the alpha -subunit of Gq. Galpha q-/- mice have increased bleeding times, because of a defective platelet activation mediated by Galpha q signaling (26). They also suffer from several deficits like motor discoordination, probably because of a reduced developmental regression of surplus cerebellar climbing fibers innervating Purkinje cells (37).

When we compared the GMppNp-induced inhibition of vesicular serotonin uptake into platelets from wild type and Galpha q-/- mice, no significant down-regulation of vesicular serotonin uptake could be detected in Galpha q-/- platelets (Fig. 6A). To rule out the possibility that the lack of GMppNp-mediated modulation of VMAT2 simply results from monoamine depleted granules in the Galpha q-/- platelets (comparable with the situation in Tph1-/- platelets), we analyzed the platelet serotonin content. The HPLC analysis (Fig. 6B) demonstrated that there was no significant difference in the amount of serotonin stored in wild type and Galpha q-/- platelets. In addition, no significant difference was detected in the duodenum where serotonin secreting enterochromaffin cells provide the serotonin taken up by the platelets during their passage through the capillary net of the duodenal mucosal villi. Thus, Galpha q is responsible for the regulation of VMAT2 in mouse platelets.


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Fig. 6.   Galpha q-deficient mice exhibit no GMppNp-mediated inhibition of vesicular serotonin uptake. A, platelets from wild type and Galpha q-/- mice were incubated for 15 min at 37 °C with 80 nM [3H]serotonin in the absence or presence of 100 µM GMppNp. In platelets obtained from wild type animals, the addition of GMppNp inhibited the serotonin uptake to about 40%. Conversely, only a very weak GMppNp effect was observed in Galpha q-/- mice. Data from five individual experiments, each performed with material obtained from eight mice, were summarized. The values given in percentages inhibition (controls in the absence of GMppNp set as 100%) are expressed as the means of the five experiments ± S.D. Unspecific uptake in the presence of tetrabenazine was subtracted prior to calculations. Statistical significance (p = 0.011) was verified using Student's t test. B, an HPLC analysis demonstrates that there is no significant difference in the amount of serotonin stored in platelets or EC cells of the duodenum between wild type and Galpha q-/- mice. The values represent the means of three determinations ± S.D. wt, wild type.

Down-regulation of VMAT2 by Galpha q Does Not Involve Plasma Membrane Receptor Activation-- The data so far indicate that the vesicular content and Galpha q are crucial for the regulation of platelet VMAT2 activity. However, preloading of Tph1-/- platelets with serotonin could cause activation of receptors that in turn may mediate the change in the modulation of VMAT2 by GMppNp. Even under permeabilization such an involvement of receptors may not be ruled out with certainty. To distinguish whether G protein regulation of VMAT2 is mediated by plasma membrane receptors or directly by vesicular properties, a variety of control experiments were performed using antagonists or agonists of receptors known to be expressed in platelets.

The Galpha q-coupled 5HT2A receptor is one of the key elements in Ca2+ mobilization during platelet activation and aggregation (38). Activation of the Galpha s-coupled alpha 2A adrenoreceptor is reported to inhibit platelet serotonin release (39). The expression of dopamine receptors (D3 type) in platelets was recently published (40). Preloading of intact Tph1-/- platelets was performed with either serotonin or noradrenaline in the absence or presence of spiperone, which blocks 5HT2A and D2-like receptors, BRL 44408, which interferes with alpha 2A receptors, and GR 103691, antagonizing D3 receptor-mediated effects at 100 nM each. Fig. 7A illustrates that neither spiperone alone nor a combination of all three antagonists significantly affected the GMppNp-mediated inhibition of vesicular serotonin uptake into cells preincubated with serotonin. GMppNp-mediated down-regulation of VMAT2 could also be reconstituted by noradrenaline using permeabilized platelets (Fig. 3C). For preincubation with noradrenaline, intact platelets had to be incubated with relatively high concentrations (100 µM) to circumvent substrate specificity of SERT. In platelets pretreated with noradrenaline (Fig. 7B), the addition of GMppNp inhibited the [3H]serotonin uptake by 20% comparable with the situation in permeabilized platelets. About the same effect was observed, when receptor antagonists were added during preincubation.


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Fig. 7.   Receptor activation is not required for G protein regulation of VMAT2. A and B, during preloading with serotonin (A) or noradrenaline (B), intact Tph1-/- platelets were incubated with or without the monoamine receptor antagonists spiperone (5HT2A, D2-like), BRL44408 (alpha A2), and GR 103601 (D3) at a concentration of 100 nM each. Neither spiperone nor a combination of all three ligands affected the GMppNp-mediated inhibition of [3H]serotonin uptake observed after preloading with 15 µM serotonin (A) or 100 µM noradrenaline (B). C, incubation of permeabilized wild type platelets with 1 µM of the TXA2 receptor agonist U46619 had no effect on vesicular [3H]serotonin uptake.

The impaired platelet function in Galpha q deletion mutants is due to a defective activation by several physiological activators that all signal via Galpha q (26), the most important in this respect being the thromboxane A2 (TXA2) receptor. To exclude the possibility that the Galpha q-mediated modulation of VMAT2 is mainly due to an activation of Galpha q-coupled receptors even under permeabilized conditions, we performed uptake experiments in the presence of the TXA2 receptor agonist U46619. The addition of 1 µM U46619 to permeabilized wild type platelets had no effect on vesicular serotonin uptake (Fig. 7C). These data suggest that Galpha q-mediated down-regulation of VMAT2 is independent from plasma membrane receptor activation and sustained by vesicular properties.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

G protein heterotrimers localize to secretory vesicles (11) and nonhydrolyzable GTP analogues known to activate G proteins down-regulate the activity of VMATs (12, 13). Here we present the first evidence that the vesicular content itself mediates the G protein regulation. In addition we show that although VMAT2 is a downstream target for Galpha o2 in brain (13), it is regulated by Galpha q in platelets.

Vesicular Content as a Regulator for Vesicular Filling-- From electrophysiological studies it is evident that quantal release of neurotransmitters from a given neuron, synapse, or neuroendocrine cell varies irrespective of the transmitter phenotype released. This leads to the idea that vesicular content may be regulated and adapt to environmental changes.

Overexpression of VACHT (41) or VMAT2 (42) indicates that the vesicular content estimated by quantal release correlates with the amount of transmitter transporter molecules per vesicle. The addition of precursors to increase the monoamine content (43) or gamma -vinyl-GABA to inhibit degradation of GABA by GABA transaminases (44) led to an increased amplitude of quantal transmitter release. These findings suggest that secretory vesicles can accept more neurotransmitter if available. Although these manipulations increased the transmitter content over a given amount, the opposite model characterized by empty vesicles is more difficult to find. In the case of monoaminergic vesicles reducing transmitter content by directly interfering with inhibitors of tyrosine or tryptophan hydroxylases for catecholamine or serotonin synthesis, respectively, proved to be difficult because the inhibitors may also interfere with VMAT itself. Direct inhibition of VMAT by reserpine expectedly reduced vesicular content but resulted in secretory granules of reduced volume at least in PC12 cells (8). Although these data suggest that vesicular volume is critical in regulating transmitter content, further analysis of putative regulatory mechanisms focusing on vesicular content is hindered by the irreversible inhibition of VMAT caused by reserpine treatment. Less filled or probably empty secretory vesicles have been generated at the frog neuromuscular junction by repetitive stimulation in combination with an impaired uptake because of hemicholinium, vesamicol, and NH<UP><SUB>4</SUB><SUP>+</SUP></UP> at the level of the cholin or acetycholin transporters, respectively (45). These less filled or empty vesicles were recruited for the releasable pool (45), suggesting that exocytotic release and vesicular filling may work independently from each other.

Using a genetic model characterized by the depletion of Tph1, a peripheral form of tryptophan hydroxylase (19) resulting in platelets that are fully equipped for serotonin storage and release but that contain only minute amounts of serotonin, we provide evidence that vesicular content is crucial for regulation of transmitter uptake. Preloading of Tph1-/- platelets with serotonin fully reconstitutes the G protein-mediated inhibition. In this respect it is noteworthy that reconstitution of the GMppNp inhibition is not serotonin-specific and works also with noradrenaline, which is transported by VMAT at a comparable Km. These data indicate that signal transduction starts from the luminal site of the vesicle. As required for a general regulation, other monoamines in addition to serotonin do the same job. So far we do not know the "receptor" working from the luminal site that senses the transmitter content. VMAT2 by one of its intravesicular loops may be a candidate.

A mechanism that enables secretory vesicles to modulate their transmitter content might prevent them from getting overloaded. A more attractive interpretation is a general regulation performed by the vesicles themselves that allows them to modulate their transmitter content in correlation with their environment. Generally in the central nervous system by such a kind of regulation fine-tuning of vesicular concentration can be achieved in different synapses by the respective vesicular transmitter transporters.

Regulation of vesicular content may function at the level of the vesicle itself depending on the filling stage and does not require direct receptor activation. Evidence came from the failure of inhibitors of plasma membrane receptors to directly interfere with the reconstitution of G protein-mediated regulation in Tph1-/- platelets. These include serotonergic (5HT2A), noradrenergic, or dopaminergic receptors relevant for platelet activation. This does not, however, exclude cross-talk between membrane receptors and vesicles, which would enable neurons, neuroendocrine cells, or even platelets to adapt their vesicular transmitter content to the requirements of the network or their respective environment. Reduction of vesicular content has been achieved by activating D2 receptors using quinpirol, which inhibited tyrosine hydroxylase and reduced quantal size in PC12 cells (43). The reduction in quantal size may be directly linked to the reduced catecholamine content. Alternatively, activation of D2 receptors may turn on Galpha o (46) present in chromaffin granules (11) and in PC12 cells by which VMAT activity can be down-regulated (12). Future investigations will define the signal cascade between plasma membrane receptors and vesicular content.

Galpha q as a Regulator of VMAT2 in Platelets-- Platelets resemble neurons with respect to transmitter storage and release and thus are sometimes regarded as a peripheral indicator of functional aspects of central serotonergic and other monoaminergic neurons. Platelets, however, do not contain Galpha o, which is mainly present in neurons and neuroendocrine cells where it is involved in VMAT regulation (12, 13). Using a Galpha q deletion mutant, we show here that VMAT2 of platelets is regulated by Galpha q. Galpha q-mediated regulation of VMAT2 appears to be restricted to platelets because VMAT2 activity in the brain-derived SSV was inhibited by Galpha o2 (13) and not affected in Galpha q-/- mice.2 Failure of Galpha q-/- platelets to respond to a GMppNp stimulus is not due to a reduced monoamine content as revealed by HPLC analysis. This indicates that VMAT2 may be regulated by different G proteins depending on the respective tissue. Whether such promiscuity also applies to VMAT1 or other vesicular transmitter transporters is not known so far.

Galpha q deletion causes a severe phenotype in platelets characterized by a complete failure of aggregation. Receptors including the one for TXA2, which is the most important receptor for platelet activation (35), are linked to this G protein. So regulation of VMAT2 might at least partially be obtained by activation of Galpha q-linked receptors. However, the addition of a TXA2 agonist to permeabilized wild type platelets has no effect, excluding the possibility that under permeabilized conditions an activation of TXA2 (or other Galpha q-linked receptors) may activate Galpha q, which in turn down-regulates VMAT2.

Thus, Galpha q appears to work at the vesicular membrane. This assumption is substantiated by electron microscopic studies revealing that VMAT2 and Galpha q occur on vesicular structures including dense bodies, alpha -granules, and a third vesicular population we called small dense vesicles. From human platelets it is well known that especially dense bodies are the main source of serotonin storage (18, 34). The small dense vesicles we observed in mouse platelets appeared to be absent in human platelets. The fact that these small dense vesicles also contain, in addition to VMAT2 and Galpha q, synaptobrevin immunoreactivity suggests that they are important storage organelles for serotonin at least in mouse platelets.

In summary, VMAT2 can be regulated by either Galpha q (this work) or Galpha o2 (13). The vesicular filling mediates the G protein effects, which by an as yet unknown signal cascade, probably depending on the G protein involved, modulates VMAT2.

    ACKNOWLEDGEMENTS

We are indebted to Stefan Offermanns for generously providing the Galpha q-deficient mice. We also thank Ursel Tofote for expert technical assistance.

    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

Dagger Dagger To whom correspondence should be addressed. E-mail: gudrun.ahnert@charite.de.

Published, JBC Papers in Press, February 25, 2003, DOI 10.1074/jbc.M212816200

2 S. Winter, D. Walther, M. Höltje, I. Pahner, and G. Ahnert-Hilger, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: VMAT, vesicular monoamine transporter; SSV, small synaptic vesicle; AS, antiserum; SLO, streptolysin O; TH, Tyrode-Hepes; HPLC, high pressure liquid chromatography; PBS, phosphate-buffered saline; TXA2, thromboxane A2; GABA, gamma -aminobutyric acid; GMppNp, guanosine 5'-(beta igamma -imido)triphosphate.

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