Impaired neurotransmitter uptake in PC12 cells overexpressing human Cu/Zn-superoxide dismutase--implication for gene dosage effects in Down syndrome

Rat PC12 cells expressing elevated levels of transfected human Cu/Zn-superoxide dismutase (CuZn-SOD) gene were generated. These transformants (designated PC12-hSOD) closely resembled the parental cells in their morphology, growth rate, and response to nerve growth factor, but showed impaired neurotransmitter uptake. The lesion was localized to the chromaffin granule transport mechanism. We found that the pH gradient (delta pH) across the membrane, which is the main driving force for amine transport, was diminished in PC12-hSOD granules. These results show that elevation of CuZnSOD activity interferes with the transport of biogenic amines into chromaffin granules. Since neurotransmitter uptake plays an important role in many processes of the central nervous system, CuZnSOD gene-dosage may contribute to the neurobiological abnormalities of Down's syndrome.     

Down's syndrome: abnormal neuromuscular junction in tongue of transgenic mice with elevated levels of human Cu/Zn-superoxide dismutase

To investigate the possible involvement of Cu/Zn-superoxide dismutase (CuZnSOD) gene dosage in the neuropathological symptoms of Down's syndrome, we analyzed the tongue muscle of transgenic mice that express elevated levels of human CuZnSOD. The tongue neuromuscular junctions (NMJ) in the transgenic animals exhibited significant pathological changes, namely, withdrawal and destruction of some terminal axons and the development of multiple small terminals. The ratio of terminal axon area to postsynaptic membrane decreased, and secondary folds were often complex and hyperplastic. The morphological changes in the transgenic NMJ were similar to those previously seen in muscles of aging mice and rats as well as in tongue muscle of patients with Down's syndrome. The findings suggest that CuZnSOD gene dosage is involved in the pathological abnormalities of tongue NMJ observed in Down's syndrome patients.     

Gene dosage of CuZnSOD and Down''s syndrome: diminished prostaglandin synthesis in human trisomy 21, transfected cells and transgenic mice.

Patients with Down's syndrome (DS) exhibit elevated activity of copper zinc superoxide dismutase (CuZnSOD) caused by the trisomy 21 state. To investigate the possible involvement of CuZnSOD gene dosage in perturbation of prostaglandin biosynthesis we analyzed transfected cells and transgenic mice that express elevated levels of human CuZnSOD. It was found that the synthesis of prostaglandin E2 (PGE2) was diminished in transfected PC12-CuZnSOD cells as well as in fibroblasts from DS patients. Primary cells derived from transgenic CuZnSOD mice showed similar reduction. Impaired biosynthesis of prostaglandins was not confined to cells grown in culture since secretion of PGE2 and PGD2 by kidney and cerebellum of transgenic CuZnSOD was significantly lower than in non-transgenic littermate mice. These findings strongly suggest that overexpression of the CuZnSOD gene induces a demotion in PGE2 and PGD2 formation and establish a connection between alteration of prostaglandin biosynthesis in trisomy 21 cells and gene dosage of CuZnSOD.     

Role of Ca(2+) and protein kinase C in the serotonin (5-HT) transport in human platelets

Accumulation of serotonin (5-HT) into human platelets was not affected by the presence of the extra-cellular calcium chelator EGTA, while decreased by platelet incubation with the membrane permeant chelator BAPTA-AM. Serotonin uptake also diminished upon platelet exposure to EGTA/thapsigargin or EGTA/ionomycin which increased the cytosolic [Ca(2+)] to levels lower than those inducing secretion of dense granules. The latter inhibition depended in part on changes of intra-granular pH, since the accumulation of acridine orange, which is driven into the dense granules by the intra-granular acid pH gradient, was slightly decreased in the presence of EGTA/thapsigargin. These compounds also inhibited the 5-HT uptake in platelets pre-incubated with reserpine and bafilomycin that prevent 5-HT from entering into the dense granules. Inhibitors of protease, protein phosphatase, Na(+)/H(+) exchanger or ciclo-oxygenase activities did not modify the serotonin accumulation. Addition of EGTA/thapsigargin to reserpine-treated, [(14)C]5-HT-loaded, platelets caused an imipramine-insensitive release of labelled serotonin. This release was reduced by both BAPTA-AM or protein kinase C inhibitor bisindoylmaleimide (GF). The latter compound, either alone or together with EGTA/thapsigargin, inhibited the 5-HT accumulation in reserpine-treated platelets. It is concluded that both cytosolic [Ca(2+)] and protein kinase C are involved in the regulation of the plasma membrane 5-HT transport.     

Munc13-4 is a GTP-Rab27-binding protein regulating dense core granule secretion in platelets

Platelets store self-agonists such as ADP and serotonin in dense core granules. Although exocytosis of these granules is crucial for hemostasis and thrombosis, the underlying mechanism is not fully understood. Here, we show that incubation of permeabilized platelets with unprenylated active mutant Rab27A-Q78L, wild type Rab27A, and Rab27B inhibited the secretion, whereas inactive mutant Rab27A-T23N and other GTPases had no effects. Furthermore, we affinity-purified a GTP-Rab27A-binding protein in platelets and identified it as Munc13-4, a homologue of Munc13-1 known as a priming factor for neurotransmitter release. Recombinant Munc13-4 directly bound to GTP-Rab27A and -Rab27B in vitro, but not other GTPases, and enhanced secretion in an in vitro assay. The inhibition of secretion by unprenylated Rab27A was rescued by the addition of Munc13-4, suggesting that Munc13-4 mediates the function of GTP-Rab27. Thus, Rab27 regulates the dense core granule secretion in platelets by employing its binding protein, Munc13-4.     

Zwitterionic and anionic forms of a serotonin analog as transport substrates

4,6-Difluoroserotonin, a serotonin analog with an acidic 5-hydroxyl proton (pK alpha = 7.97) relative to serotonin (pK alpha = 10.73), was tested as a substrate for the biogenic amine transporter of bovine chromaffin granules and the plasma membrane serotonin transporter of human blood platelets. The platelet serotonin transporter transports this analog with identical rates as those for serotonin, both at pH 6.7, where the hydroxyl group is predominantly protonated and at pH 9, where it is largely dissociated. In contrast, the chromaffin granule biogenic amine transporter prefers the form of 4,6-difluoroserotonin with a protonated 5-hydroxyl group. Thus, the KM for 4,6-difluoroserotonin increases, and Vmax decreases (relative to the values for serotonin) as the pH increases from 7 to 9. This effect may reflect a specific requirement for the protonated hydroxyl group in substrate translocation, as opposed to binding, since the KI for 4,6-difluoroserotonin inhibition of serotonin transport is the same as the KM for serotonin from pH 7 to 9.     

Transport and storage of serotonin by thrombin-treated platelets

Repeated thrombin treatment of washed platelets prepared from rabbits can decrease the serotonin content of the platelets by about 80%. When these platelets are deaggregated they reaccumulate serotonin but their storage capacity for serotonin is reduced by about 60%. If thrombin-pretreated platelets are allowed to equilibrate with a high concentration of serotonin (123 mu M), they release a smaller percentage of their total serotonin upon further thrombin treatment, in comparison with the percentage of serotonin released from control platelets equilibrated with the same concentration of serotonin calculations indicate that in thrombin-treated platelets reequilibrated with serotonin, two-thirds of the serotonin is in the granule compartment and one-third is in the extragranular compartment, presumably the cytoplasm. Analysis of the exchange of serotonin between the suspending fluid and the platelets showed that thrombin treatment does not alter the transport rate of serotonin across the platelet membrane and does not cause increased diffusion of serotonin from the platelets into the suspending fluid. The primary reason for the reduced serotonin accumulation by the thrombin-treated platelets appears to be loss of amine storage granules or of the storage capacity within the granules.     

Thymic abnormalities and enhanced apoptosis of thymocytes and bone marrow cells in transgenic mice overexpressing Cu/Zn-superoxide dismutase: implications for Down syndrome.

The copper-zinc superoxide dismutase (CuZnSOD) gene resides on chromosome 21 and is overexpressed in Down syndrome (DS) patients. Transgenic CuZnSOD mice with elevated levels of CuZnSOD were used to determine whether, as in DS, overexpression of CuZnSOD was also associated with thymus and bone marrow abnormalities. Three independently derived transgenic CuZnSOD strains had abnormal thymi showing diminution of the cortex and loss of corticomedullary demarcation, resembling thymic defects in children with DS. Transgenic CuZnSOD mice were also more sensitive than control mice to in vivo injection of lipopolysaccharide (LPS), reflected by an earlier onset and enhanced apoptotic cell death in the thymus. This higher susceptibility to LPS-induced apoptosis was associated with an increased production of hydrogen peroxide and a higher degree of lipid peroxidation. When cultured under suboptimal concentrations of interleukin 3 or in the presence of tumour necrosis factor, bone marrow cells from transgenic CuZnSOD mice produced 2- to 3-fold less granulocyte and macrophage colonies than control. The results indicate that transgenic CuZnSOD mice have certain thymus and bone marrow abnormalities which are similar to those found in DS patients, and that the defects are presumably due to an increased oxidative damage resulting in enhanced cell death by apoptosis.     

Sandy: a new mouse model for platelet storage pool deficiency

Sandy (sdy) is a mouse mutant with diluted pigmentation which recently arose in the DBA/2J strain. Genetic tests indicate it is caused by an autosomal recessive mutation on mouse Chromosome 13 near the cr and Xt genetic loci. This mutation is different genetically and hematologically from previously described mouse pigment mutations with storage pool deficiency (SPD). The sandy mutant has diluted pigmentation in both eyes and fur, is fully viable and has prolonged bleeding times. Platelet serotonin levels are extremely low although ATP dependent acidification activity of platelet organelles appears normal. Also, platelet dense granules are extremely reduced in number when analysed by electron microscopy of unfixed platelets. Platelets have abnormal uptake and flashing of the fluorescent dye mepacrine. Secretion of lysosomal enzymes from kidney and from thrombin-stimulated platelets is depressed 2- and 3-fold, and ceroid pigment is present in kidney. Sandy platelets have a reduced rate of aggregation induced by collagen. The sandy mutant has an unusually severe dense granule defect and thus may be an appropriate model for cases of human Hermansky-Pudlak syndrome with similarly extreme types of SPD. It represents the tenth example of a mouse mutant with simultaneous defects in melanosomes, lysosomes and/or platelet dense granules.     

Serotonin transport in isolated platelet granules. Coupling to the electrochemical proton gradient

The effect of the transmembrane proton gradient (delta pH) and potential gradient (delta psi) upon the rate and extent of amine accumulation was investigated in intact 5-hydroxytryptamine (serotonin) containing dense granules. The granules were isolated and purified from other subcellular organelles under isotonic conditions utilizing a newly developed continuous density gradient of Percoll. As measured by [14C]methylamine distribution, isolated granules suspended in a highly buffered medium at pH 7.0 had an intragranular pH of 5.40, independent of the pH of the external medium. This pH gradient could be collapsed by the addition of 60 mM ammonia. In the presence of Mg-ATP, a transmembrane potential (delta psi) of 30-40 mV, inside positive, was generated and sustained for over 30 min, as measured by [14C]thiocyanate distribution. The addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, a proton translocator, resulted in the reversal of the potential to negative values. The Mg-ATP-dependent generation of the delta psi was prevented by addition of dicyclohexylcarbodiimide and trimethyltin, inhibitors of proton-translocating ATPases in this and other subcellular organelles. Ammonia (1-50 mM) addition to highly buffered suspensions of serotonin granules caused a dose-dependent decrease in the delta pH, while thiocyanate added at varying concentrations produced a dose-related collapse of the delta psi and had no effect upon the delta pH. Both the delta pH and delta psi were found to independently drive accumulation of [14C]serotonin into the granules; stepwise collapse of each gradient resulted in a corresponding diminution of [14C]serotonin accumulation. The maximum rate and extent of [14C]serotonin uptake, however, were observed in the presence of both the delta pH and delta psi. The conclusions provide support for the existence of a proton-translocating ATPase in the serotonin granule membrane responsible for the generation of the delta pH and delta psi. Moreover, the results demonstrate a primary role for the electrochemical proton gradient (delta mu H+) in the carrier-mediated active transport of 5-hydroxytryptamine into the platelet granule.     

p-Chloroamphetamine induces serotonin release through serotonin transporters.

p-Chloroamphetamine (PCA) interacts with serotonin transporters in two membrane vesicle model systems by competing with serotonin for transport and stimulating efflux of accumulated serotonin. In plasma membrane vesicles isolated from human platelets, PCA competes with [3H]imipramine for binding to the serotonin transporter with a KD of 310 nM and competitively inhibits serotonin transport with a KI of 4.8 nM. [3H]Serotonin efflux from plasma membrane vesicles is stimulated by PCA in a Na(+)-dependent and imipramine-sensitive manner characteristic of transporter-mediated exchange. In membrane vesicles isolated from bovine adrenal chromaffin granules, PCA competitively inhibits ATP-dependent [3H]serotonin accumulation with a KI of 1.7 microM and, at higher concentrations, stimulates efflux of accumulated [3H]serotonin. Stimulation of vesicular [3H]serotonin efflux is due in part to dissipation of the transmembrane pH difference (delta pH) generated by ATP hydrolysis. Part of PCA's ability to stimulate efflux may be due to its transport by the vesicular amine transporter. Flow dialysis experiments demonstrated uptake of [3H]PCA into chromaffin granule membrane vesicles in response to the delta pH generated in the presence of Mg2+ and ATP. In plasma membrane vesicles, no accumulation was observed using an NaCl gradient as the driving force. We conclude that rapid nonmediated efflux of transported PCA prevents accumulation unless PCA is trapped inside by a low internal pH.     

Normal serotonin uptake by blood platelets and brain synaptosomes but selective impairment of platelet serotonin storage in mice with Chediack-Higashi syndrome

The Chediack-Higashi syndrome (CHS) is an autosomal recessive disorder reported in man and in several animal species including the "beige mice" (bg/bg). Among several manifestations of this genetic trait, deficiency of secretable substances - including serotonin - normally stored in platelet dense granules is a characteristic feature. The animal model of Chediak-Higashi syndrome used in the present study provides a unique opportunity to compare the kinetics of serotonin (5-hydroxytryptamine, 5-HT) uptake in platelets and brain synaptosomes in conditions of selective reduction of 5HT concentration in the platelets. The kinetics of 5HT uptake, as measured in the present study, was normal in synaptosomes and platelets from the same animals. The lower intraplatelet 5HT levels in bg/bg animals as compared to normal synaptosomes levels in the presence of normal uptake offer an indirect proof that the 5HT defect described in the CHS is due to an impaired 5HT storage mechanism. This is supported by the observation that spontaneous release of 5HT was markedly increased in platelets from CH5 mice but was normal in synaptosomes from the same animals. Thus platelets are a reliable model to study 5HT uptake, but not 5HT storage and release in brain synaptosomes.     

Energy dispersive X-ray microanalysis of neurosecretory granules of mouse pituitary on fresh air-dried tissue spreads

Electron microscopy of fresh air dried spreads of unstained posterior lobe tissue from mouse pituitary disclosed neurosecretory granules. Each granule showed a seemingly homogeneous dense core surrounded by a halo and a bounding membrane. The area between granules in the cytoplasm was relatively well preserved. The energy dispersive X-ray microanalysis revealed peaks for sulfur, chlorine and potassium in two granules. The third granule displayed peaks for phosphorus and chlorine. These elements probably contribute to the high electron density of the granules. There was no peak for calcium, in contrast to the dense bodies of human blood platelets.     

The internal pH of isolated serotonin containing granules of pig platelets.

The [14C]methylamine distribution method was utilized to measure the internal pH of isolated serotonin containing granules of pig blood platelets under varying conditions. The granules used were isolated by a new protocol which stressed platelet rupture under controlled conditions and preservation of isotonicity throughout the isolation procedure, In a well buffered external medium, pH 6.85, The deltapH was measured as 1.11 with the internal pH being found acidic (pH 5.74). Increasing the external pH produced a corresponding increase in the deltaH. The pH gradient could be collapsed by the addition of ionophores and uncouplers which are known to transport protons across biological membranes. In addition, the deltapH was constant for granules suspended in various ionic media, thus suggesting that the deltaH did not arise secondarily due to the establishment of a Donnan equilibrium. The existence of the acidic intragranular space is discussed with respect to previous ancillary findings. Also, an explication of the possible physiological significance of the deltaH is presented.     

Participation of a transmembrane proton gradient in 5-hydroxytryptamine transport by platelet dense granules and dense-granule ghosts.

Dense granules, the storage organelles for 5-hydroxytryptamine in blood platelets, have been isolated from porcine platelets and are shown to transport 5-hydroxytryptamine in response to a transmembrane proton gradient (delta pH). Transport in the absence of delta pH is minimal, and it is shown that a rapid increase in transport takes place as delta pH increases. Direct measurements with [14C]methylamine show a delta pH of 1.1 units (acid inside) for intact granules. Osmotically active ghosts of dense granules from which 95% of the endogenous 5-hydroxytryptamine content has been released have also been prepared. Ghosts swell in the presence of ATP and Mg2+, and this swelling is shown to be due to the entry of protons via a process linked to ATP hydrolysis. Proton entry is also apparently linked to anion penetration in ghosts. Steady-state 5-hydroxytryptamine transport in ghosts is stimulated approx. 3-fold on the addition of ATP to the incubation medium, and the stimulation of 5-hydroxytryptamine transport in ghosts correlates with the formation of a transmembrane delta pH. Ghosts generate a delta pH of 1.1-1.3 pH units (acid inside) in the presence of 5 mM-ATP/2.5 mM-MgSO4. delta pH is generated within 3 min at 37 degrees C and is dissipated by the ionophore nigericin and by NH4Cl. It is shown that an Mg2+-stimulated ATPase activity is present on the ghost membrane, and inhibition of the ATPase leads to a corresponding decrease in 5-hydroxytryptamine transport. The results presented support the idea that 5-hydroxytryptamine transport into platelet dense granules is dependent on the presence of a transmembrane delta pH and, together with previous findings by others, suggest a generalized mechanism for biogenic amine transport into subcellular storage organelles.     

Serotonin (5-HT) transport in human platelets is modulated by Src-catalysed Tyr-phosphorylation of the plasma membrane transporter SERT.

BACKGROUND/AIM: platelets possess tightly regulated systems for serotonin (5-HT) transport. This study analysed whether the 5-HT transport mediated by the plasma-membrane transporter SERT is regulated by its Tyr-phosphorylation. METHODS: 5-HT transport was determined by filtration techniques, while immunoblotting procedures were adopted for detecting the Tyr-phosphorylation of SERT in human platelet fractions. RESULTS: 5-HT accumulation in platelets pre-treated with reserpine, which prevents the neurotransmitter transport into the dense granules, decreased upon cellular exposure to PP2 and SU6656, two structurally unrelated inhibitors of Src-kinases. By contrast, the protein Tyr-phosphatase inhibitor pervanadate increased the 5-HT accumulation. Anti-SERT immunostaining of the platelet fractions showed a major band displaying an apparent molecular mass of 50 kappaDa, indicating that, during the analytical procedure, SERT underwent proteolysis, which was counteracted by addition of 4 M urea in the cellular disrupting medium. The Tyr-phosphorylation degree of SERT immunoprecipitated from membrane extracts decreased by platelet treatment with SU6656 or PP2, and enhanced upon pervanadate treatment. The anti-SERT immunoprecipitates displayed anti-Src immunostaining and in vitro kinase activity towards a Src-specific peptide-substrate. Platelet treatment with PP2 or SU6656 also caused a decrease in the imipramine binding to platelets. It was concluded that the Src-mediated SERT Tyr-phosphorylation regulates the 5-HT transport by affecting the neurotransmitter binding sites.     

Transport of 5-hydroxytryptamine by dense granules from porcine platelets

A method is described for the isolation of a homogeneous preparation of dense granules from procine platelets. The purified dense granule fraction contained approximately 400 nmol of 5-hydroxytryptamine/mg of protein and appeared to be homogeneous when examined by electron microscopy. Isolated dense granules transport exogenously added 5-hydroxytryptamine via two mechanisms: 1) a carrier-mediated process predominating at low substrate concentrations and 2) a diffusion-controlled process predominating at high substrate concentrations. Temperature studies revealed an apparent energy of activation of 14.9 kcal/mol for the carrier-mediated transport. Kinetic data yielded a Km of 3.3 micron and a Vmax of 0.79 nmol/min/mg of protein for the mediated transport process. Steady state uptake was sensitive to changes in medium osmotic pressure and a decline in uptake below 300 mosM was correlated with release of endogenous 5-hydroxytryptamine. The transport was inhibited by a number of structural analogs of 5-hydroxytryptamine. These results demonstrate the existence of a carrier-mediated transport system for 5-hydroxytryptamine in the membranes of the platelet dense granules.     

RhoG Protein Regulates Platelet Granule Secretion and Thrombus Formation in Mice

Rho GTPases such as Rac, RhoA, and Cdc42 are vital for normal platelet function, but the role of RhoG in platelets has not been studied. In other cells, RhoG orchestrates processes integral to platelet function, including actin cytoskeletal rearrangement and membrane trafficking. We therefore hypothesized that RhoG would play a critical role in platelets. Here, we show that RhoG is expressed in human and mouse platelets and is activated by both collagen-related peptide (CRP) and thrombin stimulation. We used RhoG^−/− mice to study the function of RhoG in platelets. Integrin activation and aggregation were reduced in RhoG^−/− platelets stimulated by CRP, but responses to thrombin were normal. The central defect in RhoG^−/− platelets was reduced secretion from α-granules, dense granules, and lysosomes following CRP stimulation. The integrin activation and aggregation defects could be rescued by ADP co-stimulation, indicating that they are a consequence of diminished dense granule secretion. Defective dense granule secretion in RhoG^−/− platelets limited recruitment of additional platelets to growing thrombi in flowing blood in vitro and translated into reduced thrombus formation in vivo. Interestingly, tail bleeding times were normal in RhoG^−/− mice, suggesting that the functions of RhoG in platelets are particularly relevant to thrombotic disorders.     

Organellar proteomics of human platelet dense granules reveals that 14-3-3zeta is a granule protein related to atherosclerosis

Dense granules, a type of platelet secretory organelle, are known to accumulate high concentrations of small molecules such as calcium, adenine nucleotides, serotonin, pyrophosphate, and polyphosphate. Protein composition of these granules has been obscure, however. In this paper, we use proteomics techniques to describe, for the first time, the soluble protein composition of platelet dense granules. We have isolated highly enriched human platelet dense granule fractions that have been analyzed using two proteomics methods. Using this approach, we have identified 40 proteins, and most of them, such as actin-associated proteins, glycolytic enzymes, and regulatory proteins, have not previously been related to the organelle. We have focused our efforts on studying 14-3-3zeta, a member of a conserved family of proteins that interact with hundreds of different proteins. We have demonstrated that 14-3-3zeta is localized mostly on dense granules and that it is secreted after platelet activation. As some proteins secreted from activated platelets could promote the development of atherosclerosis and thrombosis, we have studied the expression of 14-3-3zeta in sections of human abdominal aorta of patients with aneurysm, identifying it at the atherosclerotic plaques. Together, our results reveal new details of the composition of the platelet dense granule and suggest an extracellular function for 14-3-3zeta associated with atherosclerosis.