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.     

The thiol proteinase inhibitors improve the abnormal rapid down-regulation of protein kinase C and the impaired natural killer cell activity in (Chediak-Higashi syndrome) beige mouse

Protein kinase C (PKC) is essential in intracellular signal transduction for various cell functions including natural killer (NK) cell activity. This enzyme is hydrolysed by calpain, which is Ca2+-dependent thiol proteinase. We showed here that in NK activity-deficient beige (bg/bg) mouse, the model of Chediak-Higashi syndrome, the translocated membrane-bound PKC activity declined rapidly in NK cell-enriched lymphocytes after TPA stimulation. However, the rapid decline was abolished by the pretreatment of cells with leupeptin (a thiol and serine proteinase inhibitor) or E64 (a thiol proteinase inhibitor). Furthermore, these reagents improved the impaired NK cell activity in beige mouse whereas they did not affect NK cell activity in C57BL/6 (+/+) and the heterozygous (+/bg) mice. Meanwhile, TPA stimulation induced only low levels in NK cytotoxic factors (NKCF) release from beige NK cells, but these reagents augmented the lowered NKCF release. These results suggest that the improvement of impaired NK cell activity in beige mouse by the thiol proteinase inhibitors may be due to the elimination of abnormal rapid down-regulation of PKC, resulting in the augmentation of the lowered PKC activity.     

Primary dysfunction in aggregation and secretion of SHRSP platelets: not secondary to the circulation of "exhausted" platelets

The thrombin-induced secretion of [14C]-serotonin and adenine nucleotides from stroke-prone spontaneously hypertensive rats (SHRSP) platelets was markedly reduced with the development of hypertension accompanying hypo-aggregability compared with that from age-matched Wistar Kyoto rats (WKY) platelets. Calcium Ionophore A23187-induced secretion and aggregation were also attenuated in SHRSP platelets. Additionally, an enhancement of platelet secretion as well as aggregation by extracellular Ca2+ was less in SHRSP platelets than in WKY platelets. The platelet contents of adenine nucleotides and serotonin were not different between SHRSP and WKY at 5-16 weeks of age whereas they became significantly lower in SHRSP beginning at 22 weeks. The serotonin content in SHRSP platelets at 36 weeks of age was only 55% of that in WKY platelets. It is suggested that the reduced platelet aggregation and secretion observed in SHRSP platelets at ages lower than approximately 20 weeks are not secondary phenomena to the circulation of degranulated platelets, but the primary defect of SHRSP platelets appears to be an impaired function of Ca2+.     

Discrete but simultaneous release of adenine nucleotides and serotonin from mouse megakaryocytes as detected with patch- and carbon-fiber electrodes

Using patch- and carbon-fiber electrodes, we studied release phenomena of adenine nucleotides and serotonin from megakaryocytes isolated from the bone marrow of the mouse. Megakaryocytes express ionotropic purinergic receptors on their surfaces. Under the condition of whole cell recording, the cells showed spikelike spontaneous inward currents. The spontaneous currents were carried by cations and had amplitudes of 30–800 pA at –43 mV and durations of 0.1–0.3 s. Pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 100 µM) and suramin (100 µM), purinoceptor-blocking agents, depressed the currents reversibly. It is thought that the receptor involved was the P_2X1 subtype on the cell and that the currents were due to activation of the P_2X1 receptor by adenine nucleotides released from the cell. The currents showed a skewed amplitude distribution, suggesting variation of vesicular contents and/or distinct localization or varied density of receptors on the cell. Frequency of the spontaneous inward currents was enhanced by external application of platelet-activating substances, thrombin (0.4 U/ml), phorbol ester (100 nM), and ADP (2 µM), at low concentrations. With a carbon-fiber electrode, which can detect oxidizable substances including serotonin, spikelike oxidation currents from the external surface of the megakaryocyte were detected. The frequency of the oxidation currents increased remarkably after the application of thrombin (10 U/ml). The majority of the oxidation currents coincided with the rising phase of the whole cell currents, suggesting corelease of serotonin and adenine nucleotide from the same vesicle. We concluded that megakaryocytes store adenine nucleotides and serotonin in the same vesicle and release them simultaneously in a discrete manner. ADP; ATP; serotonin; patch clamp; P_2X1 receptor     

Platelet aggregation independent of adp release or prostaglandin synthesis in patients with Hermansky-Pudlak syndrome

Platelets from patients with the Hermansky-Pudlak (HPS) syndrome are deficient in the storage pool of adenine nucleotides and serotonin. As a result, the storage pool deficient (SPD) platelets develop only single waves of clumping when stimulated by threshold concentrations of aggregating agents which cause irreversible, biphasic aggregation of normal platelets. Yet, patients with HPS either have no bleeding problems or only mild symptoms.In the present study we have evaluated the importance of prostaglandin synthesis and secretion to the irreversible aggregation of HPS platelets. Results of the study demonstrate that aspirin-treated SPD platelets, which cannot form thromboxane or undergo the release reaction on stimulation by arachidonate, can still undergo irreversible aggregation in response to thrombin and ADP if treated first with epinephrine. A mechanism of membrane modulation mediated by a-adrenergic receptors cooperatively linked to the endoperoxide and thromboxane receptor can secure irreversible aggregation of normal or abnormal platelets despite absence of secretion and prostaglandin synthesis.     

Inducible beige adipocytes improve impaired glucose metabolism in interscapular BAT-removal mice

Inducible beige adipocytes are emerging as an interesting issue in obesity and metabolism research. There is a neglected possibility that brown adipocytes are equally activated when external stimuli induce the formation of beige adipocytes. Thus, the question is whether beige adipocytes have the same functions as brown adipocytes when brown adipose tissue (BAT) is lacking. This question has not been well studied. Therefore we determine the beneficial effects of beige adipocytes upon cold challenge or CL316243 treatments in animal models of interscapular BAT (iBAT) ablation by surgical denervation. We found that denervated iBAT were activated by cold exposure and CL316243 treatments. The data show that beige adipocytes partly contribute to the improvement of impaired glucose metabolism resulting from denervated iBAT. Thus, we further used iBAT-removal animal models to abolish iBAT functions completely. We found that beige adipocytes upon cold exposure or CL316243 treatments improved impaired glucose metabolism and enhanced glucose uptake in iBAT-removal mice. The insulin signaling was activated in iBAT-removal mice upon cold exposure. Both the activation of insulin signaling and up-regulation of glucose transporter expression were observed in iBAT-removal mice with CL316243 treatments. The data show that inducible beige adipocytes may have different mechanisms to improve impaired glucose metabolism. Inducible beige adipocytes can also enhance energy expenditure and lipolytic activity of white adipose tissues when iBAT is lacking. We provide direct evidences for the beneficial effect of inducible beige adipocytes in glucose metabolism and energy expenditure in the absence of iBAT in vivo.     

Presence of mast cell precursors in fetal liver of mice

When liver cells obtained from 13- to 18-day embryos of beige (Chediak-Higashi syndrome) mice were transplanted into irradiated normal adult mice, tissue mast cells with giant granules showing beige mouse origin developed in the normal recipient mice. Mast cell precursors seem to have developed earlier in the liver of embryos than mast cells themselves since no mast cells were detectable in any tissues of 13- and 14-day embryos. This result suggests that tissue mast cells originate from hematopoietic tissues not only in adult mice but also in mouse embryos.     

Metabolic aspects of the secretion of stored compounds from blood platelets. IV. Effects of ionophore X537A on washed platelets.

1. X537A at concentrations below 10 muM can liberate platelet serotonin from washed human platelets without inducing the platelet release reaction. Up to 100% of serotonin preabsorbed by the platelets can be liberated before initiation of the release reaction. 2. Concentrations of X537A above 10muM initiate the platelet release reaction, with a maximum release of adenine nucleotides and platelet factor 4 antigen comparable to that obtained with 1.25 units thrombin/ml. 3. The changes in ATP metabolism at the concentration necessary for X537A-induced release are more profound than those in platelets exposed to concentrations of thrombin or A23187 giving the same degree of release, and approach those seen with high concentrations of A23187. At concentrations where serotonin is liberated but no adenine nucleotide or platelet factor 4 antigen is released, short time incubation causes no change in the level of metabolic ATP.     

Impaired mitophagy in Sanfilippo a mice causes hypertriglyceridemia and brown adipose tissue activation

Lysosomal storage diseases result in various developmental and physiological complications, including cachexia. To study the causes for the negative energy balance associated with cachexia, we assessed the impact of sulfamidase deficiency and heparan sulfate storage on energy homeostasis and metabolism in a mouse model of type IIIa mucopolysaccharidosis (MPS IIIa, Sanfilippo A syndrome). At 12-weeks of age, MPS IIIa mice exhibited fasting and postprandial hypertriglyceridemia compared with wildtype mice, with a reduction of white and brown adipose tissues. Partitioning of dietary [³H]triolein showed a marked increase in intestinal uptake and secretion, whereas hepatic production and clearance of triglyceride-rich lipoproteins did not differ from wildtype controls. Uptake of dietary triolein was also elevated in brown adipose tissue (BAT), and notable increases in beige adipose tissue occurred, resulting in hyperthermia, hyperphagia, hyperdipsia, and increased energy expenditure. Furthermore, fasted MPS IIIa mice remained hyperthermic when subjected to low temperature but became cachexic and profoundly hypothermic when treated with a lipolytic inhibitor. We demonstrated that the reliance on increased lipid fueling of BAT was driven by a reduced ability to generate energy from stored lipids within the depot. These alterations arose from impaired autophagosome–lysosome fusion, resulting in increased mitochondria content in beige and BAT. Finally, we show that increased mitochondria content in BAT and postprandial dyslipidemia was partially reversed upon 5-week treatment with recombinant sulfamidase. We hypothesize that increased BAT activity and persistent increases in energy demand in MPS IIIa mice contribute to the negative energy balance observed in patients with MPS IIIa.     

Defective lysosomal enzyme secretion in kidneys of Chediak-Higashi (beige) mice

The beige mouse is an animal model for the human Chediak-Higashi syndrome, a disease characterized by giant lysosomes in most cell types. In mice, treatment with androgenic hormones causes a 20-50-fold elevation in at least one kidney lysosomal enzyme, beta-glucuronidase. Beige mice treated with androgen had significantly higher kidney beta-glucuronidase, beta-galactosidase, and N-acetyl-beta-D-glucosaminidase (hexosaminidase) levels than normal mice. Other androgen-inducible enzymes and enzyme markers for the cytosol, mitochondria, and peroxisomes were not increased in kidney of beige mice. No significant lysosomal enzyme elevation was observed in five other organs of beige mice with or without androgen treatment, nor in kidneys of beige females not treated with androgen. Histochemical staining for glucuronidase together with subcellular fractionation showed that the higher glucuronidase content of beige mouse kidney is caused by a striking accumulation of giant glucuronidase-containing lysosomes in tubule cells near the corticomedullary boundary. In normal mice lysosomal enzymes are coordinately released into the lumen of the kidney tubules and appreciable amounts of lysosomal enzymes are present in the urine. Levels of urinary lysosomal enzymes are much lower in beige mice than in normal mice. It appears that lysosomes may accumulate in beige mice because of defective exocytosis resulting either from decreased intracellular motility of lysosomes or from their improper fusion with the plasma membrane. A similar defect could account for characteristics of the Chediak-Higashi syndrome.     

Characteristics of mast cells in Chediak-Higashi mice: light and electron microscopic studies of connective tissue and mucosal mast cells

The beige mouse, a homologue of the Chediak-Higashi syndrome in man, possesses abnormally large granules in many tissue cells. The granules in the mucosal mast cells (MMC) of the small intestine of beige and littermate C57BL/6J mice were examined after infecting the mice with the intestinal parasite, Nippostrongylus brasiliensis. MMC in both beige and littermate mice had irregular granules which contained paracrystalline substructures embedded in an amorphous matrix. Granules were not observed in fusion with the cell membrane. Instead, in late-stage mast cells, the granule membrane broke down, the granule contents were spread throughout the cytoplasm, and the cell organelles disintegrated. Unlike connective tissue mast cells, MMC were poorly demonstrated with formalin fixation and toluidine blue staining.     

Participation of macrophages in atherosclerotic lesion morphology in LDLr-/- mice

Lystbeige (beige) mice crossed with LDL receptor-deficient (LDLr-/-) mice had a distinct atherosclerotic lesion morphology that was not observed in LDLr-/- mice. This morphology is often associated with a stable plaque phenotype. We hypothesized that macrophage expression of the beige mutation accounted for this distinct morphology. Cultured bone marrow-derived macrophages from LDLr-/- and beige,LDLr-/- mice were compared for their ability to accumulate cholesterol, efflux cholesterol, migrate in response to chemotactic stimuli through Matrigel-coated membranes, and express matrix metalloproteinase 9 (MMP9). No differences in cholesterol metabolism were identified. Beige,LDLr-/- macrophage invasion in vitro appeared to be less than LDLr-/- macrophage invasion but did not achieve significance. Nevertheless, tumor necrosis factor-alpha-induced MMP9 expression, secretion, and enzymatic activity of beige,LDLr-/- macrophages were all significantly decreased compared with those of LDLr-/- macrophages (P < 0.05). For in vivo analyses of macrophage function, bone marrow transplantation (BMT) studies were performed. LDLr-/- mice and beige,LDLr-/- mice were irradiated and reconstituted with wild-type or beige bone marrow from mice expressing green fluorescent protein (GFP). Identification of GFP cells provided for direct identification of donor-derived cells within lesions. Only expression of the beige mutation in the BMT recipients altered the macrophage location and collagen content of the lesions. These results suggested that impaired macrophage function by itself did not account for the stable lesion morphology of beige,LDLr-/- double-mutant mice.     

A simple fluorimetric microassay for adenine compounds in platelets and plasma and its application to studies on the platelet release reaction

1. The formation of a stable fluorescent product between chloroacetaldehyde and adenine or its derivatives provides the basis of a rapid simple assay for total adenine compounds in blood platelets and in plasma. The assay will measure down to 200pmol of adenine nucleotides. An evaluation of the method established the optimum conditions for the production of maximum fluorescence. 2. Values obtained for total adenine compounds in platelets were 12.9nmol/10(8) cells in man and 7.8nmol/10(8) cells in rat. These closely agree with previous values for total platelet adenine nucleotides found by using a firefly luciferase assay, or a recycled NAD-linked photometric assay. This supports the concept that the chloroacetaldehyde reaction measures total adenine nucleotides in platelets. 3. Platelet aggregation induced by collagen was studied photometrically in 0.1ml volumes of citrated platelet-rich plasma, and total adenine nucleotides were assayed in platelets and plasma before and after aggregation. During aggregation 58% of adenine nucleotides were released from human platelets, and 36% from rat platelets. 4. The chloroacetaldehyde assay is no substitute for more sophisticated procedures, but is a simple sensitive means of monitoring the release of adenine nucleotides from blood platelets and is particularly valuable when small plasma samples must be used.     

Aggregating platelets increase intracellular calcium in endothelial cells through release of adenine nucleotides

Aggregating platelets relax isolated coronary arteries through the release of endothelium-derived relaxing factor (EDRF). Since release of EDRF may be calcium dependent, we tested if and how aggregating platelets stimulated a calcium response in cultured endothelial cells. Aggregating platelets caused a transient increase in intracellular calcium in endothelial cells loaded with the fluorescent calcium indicator fura-2. The adenine nucleotides ADP and ATP, but not other platelet-derived mediators, mimicked the platelet-induced calcium response, and inhibition of adenine nucleotides impaired the response to aggregating platelets. Thus, aggregating platelets release adenine nucleotides and stimulate a rise in intracellular calcium in cultured endothelial cells. This calcium response may represent the intracellular transduction mechanism by which aggregating platelets induce endothelial release of EDRF and subsequent relaxation of coronary arteries.     

Nuclear magnetic resonance studies of blood platelets

Blood platelets contain membrane-enclosed granules which have inside them high concentrations of 5-hydroxytryptamine (serotonin) along with adenine nucleotides and divalent metal ions. 19F n.m.r. of fluorinated serotonin incorporated into the granules of both human and pig intact platelets has shown that the motional state of the serotonin is restricted. Comparison with 31P n.m.r. experiments indicates that this restriction of motion is a consequence of high molecular weight aggregates formed by the adenine nucleotides and metal ions, and that it varies with the species from which the platelets are obtained. In the case of human platelet granules, at least, these high molecular weight aggregates are present in the absence as well as in the presence of serotonin. The biological significance of these data is briefly discussed.     

The heat shock protein HSP70 promotes mouse NK cell activity against tumors that express inducible NKG2D ligands

The stress-inducible heat shock protein (HSP) 70 is known to function as an endogenous danger signal that can increase the immunogenicity of tumors and induce CTL responses. We show in this study that HSP70 also activates mouse NK cells that recognize stress-inducible NKG2D ligands on tumor cells. Tumor size and the rate of metastases derived from HSP70-overexpressing human melanoma cells were found to be reduced in T and B cell-deficient SCID mice, but not in SCID/beige mice that lack additionally functional NK cells. In the SCID mice with HSP70-overexpressing tumors, NK cells were activated so that they killed ex vivo tumor cells that expressed NKG2D ligands. In the tumors, the MHC class I chain-related (MIC) A and B molecules were found to be expressed. Interestingly, a counter selection was observed against the expression of MICA/B in HSP70-overexpressing tumors compared with control tumors in SCID, but not in SCID/beige mice, suggesting a functional relevance of MICA/B expression. The melanoma cells were found to release exosomes. HSP70-positive exosomes from the HSP70-overexpressing cells, in contrast to HSP70-negative exosomes from the control cells, were able to activate mouse NK cells in vitro to kill YAC-1 cells, which express NKG2D ligands constitutively, or the human melanoma cells, in which MICA/B expression was induced. Thus, HSP70 and inducible NKG2D ligands synergistically promote the activation of mouse NK cells resulting in a reduced tumor growth and suppression of metastatic disease.     

Signal transduction in normal and pathological thrombin-stimulated human platelets

Human blood platelets stimulated by thrombin undergo very rapid morphological changes, the most characteristic of which are pseudopod formation and granule centralization. These early changes in shape are accompanied by a transient decrease (30%) in phosphatidyl inositol 4,5-bisphosphate (PIP2) which occurs in the first 10 s after thrombin addition. Transient decreases in phosphatidyl inositol 4-phosphate (PIP) and phosphatidyl inositol (PI) occur later (20-30 s). These events lead to the formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG) and hence phosphatidate (PA). Two polypeptides are phosphorylated during the same time span: the myosin light chain (P20) and a 43 kDa protein (P43). Concomitant with these molecular changes, platelet 'release reaction' occurs, i.e., liberation of the different granule constituents into the external medium: the earliest concerns dense bodies which liberate adenine nucleotides, calcium and serotonin; alpha-granules then liberate adhesive and specific proteins and are followed by lysosomes which liberate hydrolases. Pathological platelets from patients with inherited disorders, presenting well-characterized and specific defects of either the platelet membrane (GT) or storage granules (GPS and HPS), have also been studied. The results obtained lead to the following conclusions: (1) the transducing system is normal in platelets unable to aggregate; (2) phosphorylation of P20 and P43 proteins can be complete with impaired release; and (3) when platelets lack alpha-granules the transducing system as well as the release of other granule populations are impaired. These results evidence the relationship between the absence of intraplatelet components and metabolic events.     

THE BIOSTIMULATORY EFFECT OF RED LASER IRRADIATION ON PIG BLOOD PLATELET FUNCTION

The molecular mechanisms of laser-induced changes in the cell structure and function are not well known. The authors examined the effects of low-power laser irradiation on unnucleated pig blood platelets. The obtained results showed that laser irradiation (1–5J) caused in blood platelets lipid peroxidation (measured as thiobarbituric acid reactive substances) and super-oxide anion generation, concomitant with the release of adenine nucleotides and proteins from platelets. The maximum platelet response to laser irradiation was observed when doses of 1.8–2J were used. Our results indicate that red laser irradiation induces: (1) platelet secretory process and the release of substances stored in the specific granules (adenine nucleotides, proteins); and (2) lipid peroxidation partly due to stimulation of endogenous arachidonate and production of its metabolites reacting with thiobarbituric acid.     

Adenine nucleotides in thrombocytes of birds

Analysis of free nucleotide composition of both avian thrombocytes and pig platelets showed quantitative differences in the level of adenine nucleotides. 3H-adenine taken up by turkey thrombocytes was metabolized mainly to adenine nucleotides was not released after thrombin action. Thrombin liberated non-radioactive adenine nucleotides (18.2 +/- 1.5%, 20.6 +/- 1.9%) of the total, probably localized in a storage pool. Malonyldialdehyhyde (MDA) production due to thrombin was observed in both platelets and thrombocytes.