Subcellular distribution of low molecular weight guanosine triphosphate-binding proteins in adipocytes: colocalization with the glucose transporter Glut 4

Insulin stimulation of glucose transport involves the translocation of vesicles containing the glucose transporter Glut 4 to the plasma membrane. Since low mol wt GTP-binding proteins (LMW-GTP-binding proteins) have been implicated in the regulation of vesicular trafficking, we have analyzed these proteins in adipocytes. Isolated adipocytes were incubated in the absence or presence of insulin before separation of plasma membranes (PM) and low density microsomes (LDM). [alpha-32P]GTP binding to proteins transferred to nitrocellulose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed specific and distinct subsets of proteins in the PM and LDM; those proteins were more abundant in PM than in LDM. [alpha-32P]GTP binding to these proteins was specific for the guanylnucleotides, since it was competed for by GTP and guanosine 5'-O-(3-thiotriphosphate), but not by ATP or adenosine 5'-O-(3-thiotriphosphate). The LMW-GTP-binding proteins were tightly associated with the membranes, as treatment with 1.5 M KCl did not modify this association. The distribution of the LMW-GTP-binding proteins in the fractions and their affinity for guanylnucleotides were the same in control and insulin-treated adipocytes. When the presence of Gi alpha subunits was looked for with a specific antibody, Gi alpha 1 and Gi alpha 2 were found almost exclusively in PM. By contrast, the same antibody revealed the presence of a 100 kDa band in the LDM. Insulin treatment of adipocytes did not modify the amounts of those G-proteins in PM or LDM fractions, although it promoted the translocation of Glut 4 proteins from LDM to PM. LDM fractions contain a specific subset of vesicles markedly enriched in Glut 4 molecules. When those vesicles were isolated from the total LDM fraction by immunoadsorption on highly specific antibodies to Glut 4 protein, LMW-GTP-binding proteins were found in the immune pellet. Those proteins were absent when immunoprecipitation was performed after solubilization of the vesicles with 1% Triton X-100. Our results strongly suggest that the vesicles containing the Glut 4 protein also contained LMW-GTP-binding proteins and indicate that these GTP-binding proteins could play a role in the exocytosis of the Glut 4-containing vesicles.     

Inherited thrombocytopenia caused by reduced platelet production in mice with the gunmetal pigment gene mutation

Hereditary macrothrombocytopenia and prolonged bleeding times are associated with the recessive mouse pigment dilution gene gunmetal (gm). Other platelet abnormalities include a mild storage pool deficiency and abnormal expression of two low-molecular-weight guanosine triphosphate binding proteins. These studies were designed to further elucidate the cause of the macrothrombocytopenia. The life span of gunmetal mouse platelets was not significantly different from normal. However, rates of platelet synthesis, measured by sulfate incorporation, were decreased to 25% of normal values. Bone marrow transplantation of normal marrow cells corrected the thrombocytopenia. Furthermore, direct morphologic analysis of mature mutant marrow megakaryocytes by transmission electron microscopy showed reductions in the normal cytoplasmic demarcation membrane system, areas of abnormal membrane complexes, and an increased incidence of emperipolesis. Mutant platelets were relatively more heterogeneous in size and contained unusual elongated and striated inclusions. Mutant megakaryocyte numbers were increased threefold to fivefold over normal numbers in marrow and spleen. Thus, the efficiency of platelet production from gunmetal megakaryocytes is reduced by an order of magnitude. Mutant marrow had a greater proportion of 32N and a smaller proportion of 8N megakaryocytes. Collectively, the results indicate that the gunmetal gene acts intrinsically in megakaryocytes and that an abnormality in this gene causes significant qualitative and quantitative effects on platelet production.     

Inherited platelet abnormalities associated with low factor VIII activity in the same family.

Six of eight examined members belonging to two generations of the same (NEG-TUR) family were shown to have functional changes in platelets and/or a moderate decrease of factor VIII activity (FVIII:C) in plasma, with normal values of factor VIII-related antigen (VIII R:AG). Platelet defects (mainly a reduced PF3 availability, present in five patients) and factor VIII decrease were combined differently in individual members. Only two male members with both the PF 3 and FVIII:C defects had moderate haemorrhagic symptoms following traumatic injuries. One of them had also an absent adhesiveness to glass, the other one an absent adhesiveness to collagen and a reduced platelet aggregation by ADP and by collagen. Bleeding time, platelet function tests (in the other members), and routine coagulation tests were within normal range; ristocetin aggregation was also normal in all members. We think that two inherited defects, a mild haemophilia A and a "sui generis" thrombocytopathy, co-exist in this family.     

Identification, partial purification, and characterization of two guanosine triphosphate-binding proteins associated with insulin receptors.

We have previously suggested that at least two different G-proteins are involved in mediating insulin receptor functions. Here we identify and partially purify two G-proteins with apparent molecular masses of 41 and 67 kilodaltons (kDa) that interact with insulin receptors in rat adipocytes and human placenta. Treatment of isolated rat adipocytes with insulin inhibited pertussis toxin-catalyzed ADP-ribosylation of a 41-kDa G-protein in subsequently isolated plasma membranes by 30.2 +/- 3.0% and in partially purified insulin receptor preparations by 35.6 +/- 5.7%. There was no associated decrease in the concentration of the 41-kDa G-protein in the plasma membranes, as determined by immunoblot with a common G alpha antibody. The common G alpha antibody also recognized a 67-kDa protein in the plasma membranes, the concentration of which was not affected by insulin. However, the 67-kDa protein was enriched in partially purified solubilized insulin receptor preparations. Two similar, 41- and 67-kDa G-proteins were identified in the wheat germ-purified insulin receptor preparations obtained from human placenta. Removal of these two G-proteins from insulin receptor preparations results in loss of the ability of insulin to stimulate receptor kinase activity. Addition of a fraction enriched with 41- and 67-kDa G-proteins to the G-protein-depleted insulin receptor restores the insulin sensitivity of the insulin receptor kinase activity. Furthermore, addition of G-protein-depleted insulin receptors to the fraction containing partially purified 41- and 67-kDa G-proteins enhances pertussis toxin-catalyzed ADP-ribosylation of the 41-kDa G-protein. These results indicate that either the 41- or 67-kDa G-protein, or both, interact with the insulin receptor mediating insulin receptor kinase activity. Such mutual interaction and regulation between the insulin receptor and G-proteins could be an important component of the signal transduction mechanism for insulin.     

Role of pertussis toxin-sensitive guanosine triphosphate-binding proteins in the response of erythroblasts to erythropoietin

Human progenitor-derived erythroblasts have been recently shown to respond to erythropoietin (Epo) with an increase in intracellular free calcium concentration [Cac]. To explore the role of guanosine triphosphate (GTP)-binding proteins in mediating the rise in [Cac], single day 10 erythroid burst forming unit (BFU-E)-derived erythroblasts loaded with Fura-2 were pretreated with pertussis toxin (PT), stimulated with Epo, and [Cac] measured over 18 minutes with fluorescence microscopy coupled to digital video imaging. The [Cac] increase in day 10 erythroblasts stimulated with Epo was blocked by pretreatment with PT in a dose-dependent manner but not by heat-inactivated PT. These observations provided strong evidence that a PT-sensitive GTP-binding protein is involved. To further characterize the GTP-binding protein, day 10 erythroblast membrane preparations were solubilized, electrophoresed, and immunoblotted with antibodies specific for the known PT-sensitive G-protein subunits: the three subtypes of Gia (1,2, and 3) and Goa, Gia1 or Gia3 and Gia2 were identified but no Goa was found. To examine the influence of Epo on adenylate cyclase activity, day 10 erythroblasts were initially treated with Epo, isolated membrane preparations made, and cyclic adenosine monophosphate (cAMP) production by adenylate cyclase in membrane preparations in the presence of theophylline measured. Epo did not inhibit but significantly stimulated adenylate cyclase activity. However, the mechanism of increase of [Cac] appears to be independent of adenylate cyclase stimulation because treatment of erythroblasts with the cell-permeant dibutyryl cAMP failed to increase [Cac]. In summary, pertussis toxin blocks the increase in [Cac] in erythroblasts after Epo stimulation suggesting that this response is mediated through a pertussis toxin-sensitive GTP-binding protein. Candidate PT-sensitive GTP-binding proteins identified on day 10 erythroblasts were Gia 1, 2, or 3, but not Goa.     

Cell-specific abnormal prenylation of Rab proteins in platelets and melanocytes of the gunmetal mouse

The mutant gunmetal mouse exhibits reduced rates of platelet synthesis, abnormalities of platelet alpha and dense granules and hypopigmentation. Several of these features resemble those of human alpha/delta platelet storage pool disease, grey platelet syndrome and Hermansky-Pudlak syndrome. Gunmetal mice have reduced levels of Rab geranylgeranyltransferase (RabGGTase), which adds lipophilic prenyl groups to the carboxyl terminus of Rab proteins. The degree of prenylation and the subcellular distribution of several Rab proteins were evaluated in mutant platelets, melanocytes and other tissues. Significant deficits in prenylation and membrane binding of most Rabs were observed in platelets and melanocytes. In contrast, minimal alterations in Rab prenylation were apparent in several other gunmetal tissues despite the fact that RabGGTase activity was equally diminished in these tissues. The mutant tissue-specific effects are probably due to increased concentrations of Rab proteins in platelets and melanocytes. These experiments show that Rab proteins are differentially sensitive to levels of RabGGTase activity and that normal platelet synthesis, platelet organelle function and normal pigmentation are highly sensitive to the degree of prenylation and membrane association of Rab proteins. Further, the tissue-specific effects of the gunmetal mutation suggest that RabGGTase is a potential target for therapy of thrombocytosis.     

Rab geranylgeranyl transferase alpha mutation in the gunmetal mouse reduces Rab prenylation and platelet synthesis

Few molecular events important to platelet biogenesis have been identified. Mice homozygous for the spontaneous, recessive mutation gunmetal (gm) have prolonged bleeding, thrombocytopenia, and reduced platelet alpha- and delta-granule contents. Here we show by positional cloning that gm results from a G-->A substitution mutation in a splice acceptor site within the alpha-subunit of Rab geranylgeranyl transferase (Rabggta), an enzyme that attaches geranylgeranyl groups to Rab proteins. Most Rabggta mRNAs from gm tissues skipped exon 1 and lacked a start codon. Rabggta protein and Rab geranylgeranyl transferase (GGTase) activity were reduced 4-fold in gm platelets. Geranylgeranylation and membrane association of Rab27, a Rab GGTase substrate, were significantly decreased in gm platelets. These findings indicate that geranylgeranylation of Rab GTPases is critical for hemostasis. Rab GGTase inhibition may represent a new treatment for thrombocytosis and clotting disorders.     

Characterization of a platelet membrane protein of low molecular weight associated with platelet activation following binding by monoclonal antibody AG-1

With the exception of the major platelet glycoproteins IIb/IIIa and Ib, which function as receptors for fibrinogen and von Willebrand factor, little is presently known regarding the possible role of other platelet surface proteins in mediating platelet aggregation. We report the production of a murine monoclonal antibody (AG-1) recognizing human platelet membrane surface protein of relatively low molecular weight (mol wt) that may be involved in this process. AG-1 added to human platelet-rich plasma induces dense granule secretion and aggregation, with lag phase and maximal extent of aggregation dependent on antibody concentration. Aggregation induced by AG-1 is inhibited by AG-1 Fab fragments, indicating that the response is not Fc receptor-mediated. Although AG-1 continues to produce platelet shape change in the presence of EDTA, aggregation is fully inhibited and appears to be mediated by fibrinogen binding to glycoproteins IIb/IIIa. AG-1 is a potent stimulus of thromboxane formation, but full inhibition of thromboxane production by 30 mumol/L indomethacin does not significantly inhibit platelet aggregation induced by 25 micrograms/mL AG-1, indicating that aggregation induced by AG-1 may proceed by way of an endoperoxide-independent pathway. Quantitation of AG-1 Fab binding to platelets reveals approximately 65,000 binding sites per platelet. When intact platelets are radioiodinated, immunoprecipitation of NP-40 lysates by AG-1 reveals an intensely labeled protein with an apparent mol wt of approximately 21,000 daltons, and several additional bands in the mol wt range of 22,000 to 28,000 daltons, all sharing the AG-1 epitope. These bands appear to be distinct from glycoprotein IX or from the beta-chains of glycoprotein Ib or IIb. Finally, studies with platelets labeled by the periodate-[3H]borohydride procedure suggest the possibility of complex formation between subpopulations of glycoprotein Ib and the low-mol-wt glycoproteins recognized by AG-1.     

Elucidation of the molecular mechanism of platelet activation: Dense granule secretion is regulated by small guanosine triphosphate-binding protein Rab27 and its effector Munc13-4

Cardiovascular diseases such as myocardial and cerebral infarction are common critical diseases occurring more frequently in the elderly. The trigger of the diseases is platelet activation following plaque rupture or erosion. Investigation of the molecular mechanism in platelet activation has been exclusively performed pharmacologically. We have succeeded in establishing the granule secretion and aggregation assays using permeabilized platelets. These systems enabled us to examine the molecular mechanism in platelet activation with molecular biological and biochemical methods. Using these assay systems, we have been investigating the molecular mechanism of platelet activation. With a support grant from the Novartis Foundation for Gerontological Research, we found several molecules involved in the regulation. In this report, I present the progress in the research of the granule secretion mechanism in activated platelets, which was reported in the Japanese Geriatric Society Meeting in 2005.     

Genetic variation responsible for mouse strain differences in integrin alpha 2 expression is associated with altered platelet responses to collagen

As mouse models have become commonplace for studying hemostasis and thrombosis, we considered whether the mouse system had utility for assessing genetic alterations in platelet receptors. Platelets from 5 mouse strains (C57BL/6 [C57], FVB/N [FVB], BALB/c, C3H/He, and 129Sv) showed only minor differences in the expression of integrin alpha(IIb), integrin beta(3), glycoprotein (GP) Ib alpha, or GPVI across strains. However, FVB platelets expressed approximately 50% the level of integrin alpha(2) as platelets from other strains (P <.0001). We bred FVB mice with C57 and assessed alpha(2) expression in FVB/C57xFVB/C57 (F2) offspring. Linkage analysis demonstrated the gene responsible for alpha(2) levels is tightly linked to the D13mit260 marker (log odds [lod] score 6.7) near the alpha(2) gene. FVB platelets showed reduced aggregation and a longer lag phase to collagen. FVB and C57 platelets aggregated similarly to collagen-related peptide, but FVB platelets showed a reduction in rhodocytin-induced Syk and PLC gamma 2 tyrosine phosphorylation. Thus, FVB platelets express half the level of alpha(2) as other mouse strains, a trait linked to the alpha(2) gene and seemingly responsible for reduced platelet aggregation to collagen. These strain differences serve as a useful model for the 2-fold difference in human platelet alpha(2)beta(1) expression and demonstrate that alpha(2)beta(1) participates in signaling during platelet activation.     

Effects of calcium-antagonists on dopamine release in the central nervous system--possible interactions with D2-autoreceptors and guanosine triphosphate-binding proteins.

The effects of calcium antagonists (verapamil and nicardipine) on central dopaminergic activity were investigated in vitro. Rat striatal slices prelabelled with (3H)dopamine and superfused with Krebs-solution were stimulated electrically at a frequency of 1 Hz. Exposure to verapamil (3.3 x 10(-7) - 1 x 10(-5) M) significantly increased both basal and stimulation-evoked (3H)dopamine release in a concentration-dependent manner. Nicardipine produced no changes in stimulation-evoked (3H)dopamine release, although a high concentration of nicardipine slightly increased basal release of (3H)dopamine. Exogenously applied unlabelled dopamine (1 x 10(-7) M) inhibited the stimulation-evoked (3H)dopamine release. Verapamil (1 x 10(-6) M) significantly antagonized the capacity of the unlabelled dopamine to inhibit stimulation-induced (3H)dopamine release. The blockade of D2-receptors by a preferential D2-antagonist, sulpiride, reduced the facilitatory effect of verapamil on stimulation-induced (3H)dopamine release. Pretreatment with pertussis toxin, which interferes with the coupling of the inhibitory guanosine triphosphate-binding proteins to adenylate cyclase, significantly diminished the effects of verapamil on stimulation-induced (3H)dopamine release. The results of the present study show that verapamil (but not nicardipine) increased dopamine release in rat striatum, at least partially via interactions with the D2-dopamine autoreceptors and the pertussis toxin-sensitive guanosine triphosphate-binding proteins. Furthermore, a close interaction between verapamil and the dopamine receptors might partially explain the central effects of verapamil.     

Induction of signal transduction in human neutrophils by Candida albicans hyphae: the role of pertussis toxin-sensitive guanosine triphosphate-binding proteins

Nonopsonized Candida hyphae elicit from human neutrophils a transient rise in cytosolic calcium concentrations and an oxidative burst without a detectable change in membrane potential. To determine if the signal-transduction pathway used by these organisms is mediated by guanine nucleotide-binding proteins (GNPs), we examined the functional responsiveness of neutrophils pretreated with pertussis toxin (PT). In response to serum-opsonized hyphae or zymosan, the rise in cytosolic calcium, membrane depolarization, and the respiratory burst were only partially abrogated. The transient rise in calcium induced by unopsonized hyphae was, however, completely eliminated in PT-treated neutrophils. Despite total abrogation of the calcium response, PT-treated cells could still mount a respiratory burst in response to these nonopsonized hyphae. Thus, neutrophil signaling by both serum-opsonized particles and nonopsonized hyphae is only partially mediated by PT-sensitive GNPs. Furthermore, the ability of unopsonized hyphae to elicit a respiratory burst without a calcium response suggests these events are separable and confirms the versatility of these organisms as probes for investigating neutrophil activation.     

低分子肝素对急性坏死性胰腺炎并肝损伤P-选择素和E-选择素表达的影响

目的研究低分子肝素(LWMH)对大鼠急性坏死性胰腺炎(ANP)并肝损伤中P-选择素和E-选择素表达的影响及其对急性坏死性胰腺炎的保护作用。方法将96只雄性SD大鼠随机分为三个组:对照组(C组,n=32)、急性坏死性胰腺炎组(A组,n=32)、低分子肝素干预组(G组,n=32)。A组以1.5%脱氧胆酸钠溶液逆行注入胰胆管建立急性坏死性胰腺炎模型(1 ml/kg),G组于造模后给予皮下注射低分子肝素(10 U/100 g),C组仅翻动胰腺后关腹。各组分别于造模后6、12、24、48 h四个时点分批处死大鼠,测定血清淀粉酶水平;E LISA检测血清TNF-α、IL-6水平,取胰腺及肝脏组织光镜下进行病理学评分;RT-PCR检测肝脏P-选择素和E-选择素mRNA表达。结果三组间和不同时点之间各项观察指标比较均有统计学意义(P0.01),A组6、12、24、48 h各时点胰腺及肝脏病理评分、血清淀粉酶及TNF-oα、IL-6水平,P-选择素和E-选择素mRNA表达均较C组相应时点显著升高(P0.05);G组各时点胰腺及肝脏病理评分、血清淀粉酶及TNF-oα、IL-6水平,P-选择素和E-选择素mRNA表达均较A组对应时点显著降低(P0.05)。结论 P-选择素和E-选择素在急性坏死性胰腺炎并肝损伤时的表达可能与病变严重程度有关;LWMH可能是通过下调P-选择素和E-选择素表达减轻急性坏死性胰腺炎的胰腺和肝脏病变。     

Platelet storage pool deficiency associated with inherited abnormalities of the inner ear in the mouse pigment mutants muted and mocha

Several inherited human syndromes have combined platelet, auditory, and/or pigment abnormalities. In the mouse the pallid pigment mutant has abnormalities of the otoliths of the inner ear together with a bleeding abnormality caused by platelet storage pool deficiency (SPD). To determine if this association is common, two other mouse pigment mutants, muted and mocha, which are known to have inner ear abnormalities, were examined for hematologic abnormalities. Both mutants had prolonged bleeding times accompanied by abnormalities of dense granules as determined by whole mount electron microscopy of platelets and by labeling platelets with mepacrine. When mutant platelets were treated with collagen, there was minimal secretion of adenosine triphosphate and aggregation was reduced. Lysosomal enzyme secretion in response to thrombin treatment was partially reduced in muted platelets and markedly reduced in mocha platelets. Similar reductions in constitutive lysosomal enzyme secretion from kidney proximal tubule cells were noted in the two mutants. These studies show that several mutations that cause pigment dilution and platelet SPD are associated with abnormalities of the inner ear. Also, these mutants, like previously described mouse pigment mutants, are models for human Hermansky-Pudlak syndrome and provide additional examples of single genes that simultaneously affect melanosomes, lysosomes, and platelet dense granules.     

Pancreatitis and alcoholism disorder the renal tubule and impair reclamation of some low molecular weight proteins

We sought to determine whether the clinical setting in which pancreatitis occurs affects the incidence and distribution of increased values of renal clearance of amylase relative to creatinine, CAm/CCr, and whether the increased values reflect a tubular disorder that impairs renal reclamation of certain low molecular weight proteins. We measured the renal clearance of three low molecular weight proteins (amylase, beta 2-microglobulin, and lysozyme) and urinary excretion of three lysosomal enzymes that originate from the renal tubule in three groups of patients (alcoholic pancreatitis, pancreatitis without alcoholism, and alcoholism without pancreatitis). When compared to normal controls, the mean CAm/CCr was significantly elevated in alcoholic pancreatitis (p less than 0.05) but not in equally severe pancreatitis without alcoholism nor in alcoholism without pancreatitis. The clearance ratio of beta 2-microglobulin was significantly increased in each of the three patient groups; mean clearance ratio of lysozyme was not significantly increased in any of the patient groups. Excretion of each of the three lysosomal enzymes was significantly increased in each of the patient groups. We conclude that the etiology of pancreatitis affects the distribution of values for CAm/CCr, impaired tubular reclamation of amylase is the mechanism of the increase in CAm/CCr, and a factor or factors associated with both pancreatitis and with alcoholism per se appear to disorder the renal tubule and to impair tubular reclamation of some but not all low molecular weight proteins-a novel finding of considerable potential significance.     

Effects of low molecular weight heparin on platelet surface P-selectin expression and serum interleukin-8 production in rats with trinitrobenzene sulphonic acid-induced colitis

AIM:To observe the effects of low molecular weight heparin(LMWH) on platelet surface P-selectin expression and serum interleukin-8 production in rats with trinitrobenzene sulphonic acid (TNBS) induced colitis.METHODS:Colitis was induced in female Sprauge-Dawley rats by colonic administration of 2, 4, 6-TNBS. LMWH, a dalteparin (150U/kg, 300U/kg) was subcutaneously administrated one hour before induction of colitis and went on once a day for 6 days. Then a half dose was given for the next 7 days. Control animals received the same volume of normal saline once a day for 14 days after treated by TNBS.Animals were sacrificed at 24h, days 7 and 14 after induction of colitis. The colon was excised for the evaluation of macroscopic and histological findings and TNF-α immunohistochemical assay. Platelet surface P-selectin expression was determined by radioimmunoassay and serum IL-8 production was assayed by ELISA method.RESULTS:LMWH treatment in a dose of 300U/kg for 14 days significantly improved colonic inflammation by histological examination. Serum IL-8 production in the 300U/kg treatment group was more significantly decreased at day 14 than that at 24h (P&lt;0.05). However, platelet surface P-selectin expression and TNF-α staining in colonic tissue were not significantly different among the three groups.CONCLUSION:LMWH has an anti-infiammatory effect on TNBS induced colitis in rats. The effect is possibly related to inhibition of proinflammatory cytokine IL-8, but not involved platelet surface P-selectin expression.     

Isolation and partial characterization of human low molecular weight protein associated with pulmonary surfactant

A low molecular weight (MW) protein was isolated from the bronchoalveolar lavage fluid of a patient with alveolar proteinosis. The protein was isolated on DEAE-cellulose and CM-cellulose columns by a cross-reaction with the monoclonal antibody against pig low MW protein (15 kDa) used as a marker. Acidic ethanol-soluble proteins obtained from the fractions eluted by 0.09 to 0.16 M NaCl concentration from the CM-cellulose column migrated mainly as a 15-kDa band in the SDS-PAGE system without urea but mainly as a 5-kDa band in a system with 8 M urea. The isoelectric point of the protein was pH 10 to 11, and it contained a large proportion of hydrophobic amino acids (72%), especially leucine (17%). The arginine content was also high (9%). Two monoclonal antibodies were raised against this low MW protein, and immunohistochemical studies revealed that the antigen was located in the inclusions of alveolar wall cells in normal lungs and in lungs from a patient with alveolar proteinosis. These results indicate that the low MW protein originates from lamellar inclusions of alveolar wall cells (possibly type II epithelial cells) and is secreted into alveolar spaces.