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PubMed1987

Fluid trapping of erythrocytes under hypoosmolar conditions.

125I albumin was used to assess the amount of trapped fluid after microhematocrit centrifugation of erythrocytes suspended in buffers of different osmolality. Surprisingly the total amount of trapped fluid per volume unit of packed erythrocytes decreased with decreasing osmolality of the suspending buffer despite erythrocyte swelling. However, if the contribution of the individual erythrocyte to the trapped fluid was calculated, the trapped fluid per erythrocyte did not change between 311 mosm/kg and 256 mosm/kg. For osmolalities below 256 mosm/kg a significant increase of trapped fluid was obtained. It is concluded that the packing ability of erythrocytes is not impaired in suspending fluid of moderate to severe infraphysiological tonicity. The daily clinical experience that considerable degrees of plasma hypoosmolality are tolerated in vivo without hemolysis or impairment of oxygen transport by erythrocytes may be explained by the excellent ability of shape adaptation of erythrocytes to each other and to other surfaces such as vascular endothelia. The method of trapped fluid determination might be of potential value as a complementary method in the evaluation of erythrocyte rheology if the amount of trapped fluid is related to the individual erythrocyte.

Adult↗
PubMed1997

Galectin-4 and small intestinal brush border enzymes form clusters.

Detergent-insoluble complexes prepared from pig small intestine are highly enriched in several transmembrane brush border enzymes including aminopeptidase N and sucrase-isomaltase, indicating that they reside in a glycolipid-rich environment in vivo. In the present work galectin-4, an animal lectin lacking a N-terminal signal peptide for membrane translocation, was discovered in these complexes as well, and in gradient centrifugation brush border enzymes and galectin-4 formed distinct soluble high molecular weight clusters. Immunoperoxidase cytochemistry and immunogold electron microscopy showed that galectin-4 is indeed an intestinal brush border protein; we also localized galectin-4 throughout the cell, mainly associated with membraneous structures, including small vesicles, and to the rootlets of microvillar actin filaments. This was confirmed by subcellular fractionation, showing about half the amount of galectin-4 to be in the microvillar fraction, the rest being associated with insoluble intracellular structures. A direct association between the lectin and aminopeptidase N was evidenced by a colocalization along microvilli in double immunogold labeling and by the ability of an antibody to galectin-4 to coimmunoprecipitate aminopeptidase N and sucrase-isomaltase. Furthermore, galectin-4 was released from microvillar, right-side-out vesicles as well as from mucosal explants by a brief wash with 100 mM lactose, confirming its extracellular localization. Galectin-4 is therefore secreted by a nonclassical pathway, and the brush border enzymes represent a novel class of natural ligands for a member of the galectin family. Newly synthesized galectin-4 is rapidly "trapped" by association with intracellular structures prior to its apical secretion, but once externalized, association with brush border enzymes prevents it from being released from the enterocyte into the intestinal lumen.

Amino Acid Sequence↗
PubMed1990

Transport of thyroid hormones by human erythrocytes: kinetic characterization in adults and newborns.

The uptake of [125I]T3 and [125I]T4 by human erythrocytes was studied. The erythrocytes were obtained from adult subjects (28-41 yr old) and suspended in a protein-free medium. The half-times of equilibration for both T3 and T4 were 6 min. At equilibrium, T3 was concentrated 55-fold inside the cells, while T4 was concentrated 40 times, but these accumulations were not dependent on either cellular ATP or the transmembrane Na+ gradient. The amounts of cell-associated thyroid hormones were 20 times (T3) and 17 times (T4) higher than the amounts of free extracellular hormones at 5 X 10(9) erythrocytes/mL (the blood concentration). Oligomycin and phloretin inhibited T3-saturable transport (but not T4 transport) independently of cellular energy. We suggest that thyroid hormones are concentrated by intracellular trapping. The rates of T3 and T4 efflux from preloaded erythrocytes were similar to the influx rates. The initial velocities of T3 (but not T4) uptake and efflux were 70% saturable. The uptake was specific because the unlabeled analogs T4, triiodothyroacetic acid, rT3, D-T3, and D,L-thyronine inhibited [125I]T3 uptake 60, 125, 160, 190, and 1600 times less, respectively, than did unlabeled T3. The kinetic parameters of T3-saturable uptake, Km, and maximum velocity were determined for three groups of subjects: newborns, 28 to 41-yr-old adults, and 76 to 90-yr-old adults. The Km (67 nmol/L in 28 to 41-yr-old adults) was not age dependent, BUT the maximum velocity was significantly higher in newborns than in adults. We conclude that T3 transport across the human erythrocyte membrane is mediated mainly by facilitated diffusion, whereas T4 transport results from free diffusion. Human erythrocytes might act as a circulating pool of thyroid hormones, especially T3 in newborns.

Adenosine Triphosphate↗
PubMed1993

Influence of extracellular matrix on the proliferation and differentiation of adrenocortical cells in culture.

Extracellular matrix is indispensable for cell differentiation in vivo. The aim of this study is to examine effects of extracellular matrix on adrenocortical cells embedded in collagen gel in terms of cellular proliferation and differentiation. Dissociated human or bovine adrenocortical cells were embedded in collagen gel; the gel was then immediately thin-coated on the bottom of culture dishes. The cortical cells were thus trapped in collagen and cultured in monolayer, a suitable means for observation of culture cells. To the authors' best knowledge, this "collagen gel-embedded" monolayer culture is described here for the first time. Cortical cells were spherical in shape and had the activities of proliferation and steroid production in collagen gel matrix of this culture method. Aldosterone production in particular tended to be maintained much longer than in conventional monolayer culture. This method seems to provide a physiological environment in which to study the differentiation of cortical cells.

Adrenal Cortex↗
PubMed1992

Sequential changes in histologic pattern and extracellular matrix deposition during the healing of chronic venous ulcers.

As part of a major clinical trial, sequential biopsies were taken from the margins of venous leg ulcers during their healing. The changing patterns of tissue architecture and extracellular matrix synthesis during healing were documented histologically and immunocytochemically. Initial biopsies were similar in appearance: prominent fibrin cuffs, variable inflammation, hemosiderin, and red blood cell extravasation. So called "fibrin cuffs" were highly organized structures composed of laminin, fibronectin, tenascin, and collagen as well as trapped leukocytes and fibrin. Fibronectin was absent from the ulcer tissue although collagen was abundant. Major histologic changes were observed after 2 weeks' pressure bandage therapy; hemosiderin, acute inflammation, and granulation tissue with the deposition of fibronectin had all increased and epithelial migration had commenced. Complete epithelialization was frequent by the fourth week of treatment, but the basement membrane was incomplete. At this time, hemosiderin and red blood cell extravasation had decreased and "fibrin cuffs" were virtually absent although chronic inflammation remained. The complex organization of the so-called "fibrin cuffs" may inhibit angiogenesis (but offer protection against increased venous pressure) in addition to their previously ascribed role in causing tissue ischemia.

Biopsy↗
PubMed1996

Glucose uptake in Plasmodium falciparum-infected erythrocytes is an equilibrative not an active process.

The uptake of glucose into human erythrocytes infected with Plasmodium falciparum was investigated using a number of different glucose analogues. In short time-courses with cells suspended in media containing 5 mM glucose, 2-deoxy-D-glucose equilibrated rapidly between the intracellular and extracellular compartments. Its transport into the infected cell was primarily via the host cell (cytochalasin B-sensitive) transporter. 2-Deoxy-D-glucose did permeate the broad-specificity pathway that is induced in infected cells by the intracellular parasite. However, this pathway made little contribution to the total uptake of 2-deoxy-D-glucose under physiological conditions. In parasitised cells incubated with [14C]2-deoxy-D-glucose for prolonged periods the intracellular concentration of radiolabel increased to values higher than that in the external medium; it reached a maximum value three to six times higher than the extracellular concentration before falling back to a concentration similar to that outside the cells. This transient intracellular accumulation of radiolabel was due entirely to the phosphorylation of the [14C]2-deoxy-D-glucose and its consequent trapping within the cell. The specific characteristics of the 2-deoxy-D-glucose uptake time courses measured under different conditions were accounted for by the kinetics of the phosphorylation process and the energy status of the cell. The data indicate that 2-deoxy-D-glucose (as well as the non-phosphorylated compounds 3-O-methyl-D-glucose and L-glucose) enter the intracellular parasite via a passive (i.e. equilibrative) rather than an active (i.e. concentrative) transport process.

3-O-Methylglucose↗
PubMed2005

Nitrogen-containing bisphosphonate, YM529/ONO-5920 (a novel minodronic acid), inhibits RANKL expression in a cultured bone marrow stromal cell line ST2.

Increase in bone resorption by osteoclasts can cause metabolic bone diseases, such as osteoporosis. Recent attention has been paid to the receptor activator of the NF-kappaB ligand (RANKL), an accelerator of osteoclast differentiation. RANKL is expressed on the bone marrow-derived stromal cell membrane and induces the differentiation of osteoclasts by binding to RANK expressed on the osteoclast precursor cell membrane. Since the inhibition of RANKL expression can lead to the inhibition of osteoclastic bone resorption, the clinical application of RANKL inhibition could be expected to have a major effect on metabolic bone disease therapy. In this study, we investigated whether or not YM529/ONO-5920, a nitrogen-containing bisphosphonate (a novel minodronic acid), inhibits RANKL expression in a bone marrow-derived stromal cell line (ST2 cells). Reverse transcription-polymerase chain reaction revealed that the administration of YM529/ONO-5920 to ST2 cells inhibited RANKL mRNA expression and reduced RANKL proteins as assessed by Western blot analysis. The inhibition of RANKL mRNA expression was reversed when geranylgeranyl pyrophosphate (GGPP), an intermediate in the mevalonate pathway, was used in combination. Furthermore, YM529/ONO-5920 reduced phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), and similarly, U0126, a mitogen-activated protein kinase kinase 1/2 inhibitor, inhibited RANKL expression. Pretreatment with GGPP reversed the YM529/ONO-5920-induced decrease in phosphorylation of ERK. Furthermore, YM529/ONO-5920 decreased TRAP-positive cells in co-culture of ST2 cells and an osteoclast cell line, C7 cells, and this decrease was inhibited by pretreatment with GGPP. This indicates that YM529/ONO-5920 inhibits GGPP biosynthesis in the mevalonate pathway and then signal transduction in the Ras-mitogen-activated protein kinase pathway, thereby inhibiting RANKL expression on ST2 cells. These results suggest a newly elucidated action of bisphosphonates in the inhibition of bone resorption.

Animals↗
PubMed2005

Protons trap NR1/NR2B NMDA receptors in a nonconducting state.

NMDA receptors are highly expressed in the CNS and are involved in excitatory synaptic transmission, as well as synaptic plasticity. Given that overstimulation of NMDA receptors can cause cell death, it is not surprising that these channels are under tight control by a series of inhibitory extracellular ions, including zinc, magnesium, and H+. We studied the inhibition by extracellular protons of recombinant NMDA receptor NR1/NR2B single-channel and macroscopic responses in transiently transfected human embryonic kidney HEK 293 cells using patch-clamp techniques. We report that proton inhibition proceeds identically in the absence or presence of agonist, which rules out the possibility that protonation inhibits receptors by altering coagonist binding. The response of macroscopic currents in excised patches to rapid jumps in pH was used to estimate the microscopic association and dissociation rates for protons, which were 1.4 x 10(9) m(-1) sec(-1) and 110-196 sec(-1), respectively (K(d) corresponds to pH 7.2). Protons reduce the open probability without altering the time course of desensitization or deactivation. Protons appear to slow at least one time constant describing the intra-activation shut-time histogram and modestly reduce channel open time, which we interpret to reflect a reduction in the overall channel activation rate and possible proton-induced termination of openings. This is consistent with a modest proton-dependent slowing of the macroscopic response rise time. From these data, we propose a physical model of proton inhibition that can describe macroscopic and single-channel properties of NMDA receptor function over a range of pH values.

Animals↗
PubMed2007

Degradation of extracellular matrix and its components by hypobromous acid.

EPO (eosinophil peroxidase) and MPO (myeloperoxidase) are highly basic haem enzymes that can catalyse the production of HOBr (hypobromous acid). They are released extracellularly by activated leucocytes and their binding to the polyanionic glycosa-minoglycan components of extracellular matrix (proteoglycans and hyaluronan) may localize the production of HOBr to these materials. It is shown in the present paper that the reaction of HOBr with glycosaminoglycans (heparan sulfate, heparin, chondroitin sulfate and hyaluronan) generates polymer-derived N-bromo derivatives (bromamines, dibromamines, N-bromosulfon-amides and bromamides). Decomposition of these species, which can occur spontaneously and/or via one-electron reduction by low-valent transition metal ions (Cu+ and Fe2+), results in polymer fragmentation and modification. One-electron reduction of the N-bromo derivatives generates radicals that have been detected by EPR spin trapping. The species detected are consistent with metal ion-dependent polymer fragmentation and modification being initiated by the formation of nitrogen-centred (aminyl, N-bromoaminyl, sulfonamidyl and amidyl) radicals. Previous studies have shown that the reaction of HOBr with proteins generates N-bromo derivatives and results in fragmentation of the polypeptide backbone. The reaction of HOBr with extracellular matrix synthesized by smooth muscle cells in vitro induces the release of carbohydrate and protein components in a time-dependent manner, which is consistent with fragmentation of these materials via the formation of N-bromo derivatives. The degradation of extracellular matrix glycosaminoglycans and proteins by HOBr may contribute to tissue damage associated with inflammatory diseases such as asthma.

Animals↗
PubMed2001

In-vitro differentiation of pancreatic beta-cells.

Stem cell biology is a new field that holds promise for in-vitro mass production of pancreatic beta-cells, which are responsible for insulin synthesis, storage, and release. Lack or defect of insulin produces diabetes mellitus, a devastating disease suffered by 150 million people in the world. Transplantation of insulin-producing cells could be a cure for type 1 and some cases of type 2 diabetes, however this procedure is limited by the scarcity of material. Obtaining pancreatic beta-cells from embryonic stem cells would overcome this problem. We have derived insulin-producing cells from mouse embryonic stem cells by a 3-step in-vitro differentiation method consisting of directed differentiation, cell-lineage selection, and maturation. These insulin-producing cells normalize blood glucose when transplanted into streptozotocin-diabetic mice. Strategies to increase islet precursor cells from embryonic stem cells include the expression of relevant transcription factors (Pdx1, Ngn3, Isl-1, etc), together with the use of extracellular factors. Once a high enough proportion of islet precursors has been obtained there is a need for cell-lineage selection in order to purify the desired cell population. For this purpose, we designed a cell-trapping method based on a chimeric gene that fuses the human insulin gene regulatory region with the structural gene that confers resistance to neomycin. When incorporated into embryonic stem cells, this fusion gene will generate neomycin resistance in those cells that initiate the synthesis of insulin. Not only embryonic, but also adult stem cells are potential sources for insulin-containing cells. Duct cells from the adult pancreas are committed to differentiate into the four islet cell types; other possibilities may include nestin-positive cells from islets and adult pluripotent stem cells from other origins. Whilst the former are committed to be islet cells but have a reduced capacity to expand, the latter are more pluripotent and more expandable, but a longer pathway separates them from the insulin-producing stage. The aim of this review is to discuss the different strategies that may be followed to in-vitro differentiate pancreatic beta-cells from stem cells.

Animals↗
PubMed1987

Basolateral membrane Cl/HCO3 exchange in the rat proximal convoluted tubule. Na-dependent and -independent modes.

To examine whether Cl-coupled HCO3 transport mechanisms were present on the basolateral membrane of the mammalian proximal tubule, cell pH was measured in the microperfused rat proximal convoluted tubule using the pH-sensitive, intracellularly trapped fluorescent dye (2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein. Increasing the peritubular Cl concentration from 0 to 128.6 meq/liter caused cell pH to decrease from 7.34 +/- 0.04 to 7.21 +/- 0.04 (p less than 0.001). With more acid extracellular fluid (pH 6.62), a similar increase in the peritubular Cl concentration caused cell pH to decrease by a similar amount from 6.97 +/- 0.04 to 6.84 +/- 0.05 (p less than 0.001). This effect was blocked by 1 mM SITS. To examine the Na dependence of Cl/HCO3 exchange, the above studies were repeated in the absence of luminal and peritubular Na. In alkaline Na-free solutions, peritubular Cl addition caused cell pH to decrease from 7.57 +/- 0.06 to 7.53 +/- 0.06 (p less than 0.025); in acid Na-free solutions, peritubular Cl addition caused cell pH to decrease from 7.21 +/- 0.04 to 7.19 +/- 0.04 (p less than 0.05). The effect of Cl on cell pH was smaller in the absence of luminal and peritubular Na than in its presence. To examine whether the previously described Na/(HCO3)n greater than 1 cotransporter was coupled to or dependent on Cl, the effect of lowering the peritubular Na concentration from 147 to 25 meq/liter was examined in the absence of ambient Cl. Cell pH decreased from 7.28 +/- 0.03 to 7.08 +/- 0.03, a response similar to that observed previously in the presence of Cl. The results demonstrate that Cl/HCO3 (or Cl/OH) exchange is present on the basolateral membrane. Most of Cl/HCO3 exchange is dependent on the presence of Na and may be coupled to it. The previously described Na/(HCO3)n greater than 1 cotransporter is the major basolateral membrane pathway for the coupling of Na and HCO3 and is not coupled to Cl.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1988

Proton release associated with respiratory burst of polymorphonuclear leukocytes.

The stimulation of polymorphonuclear leukocytes (PMNs) by phorbol-12-myristate-13-acetate in the presence of sodium fluoride caused the release of protons into the reaction medium concomitant with the generation of superoxide anions. The rates of oxygen consumption and proton release due to the metabolic burst were 16.3 +/- 3.5 and 10.2 +/- 1.1 nmol/min/10(7) cells respectively. When the superoxide anions were trapped with cytochrome c, the proton release was increased (35.8 +/- 0.5 nmol/min/10(7) cells) until the cytochrome c was reduced. Since the protons released from the activated cells would be consumed by the generated superoxide anions in the extracellular medium, the net amount of the protons released was 3-4-fold greater than that observed in the absence of extracellular cytochrome c. The increased proton release may be coupled to increased cellular respiration, since the inhibition of the respiratory burst with deoxyglucose, p-chloromercuribenzoic acid or chlorpromazine decreased the proton release. Amiloride (2 mM) inhibited the proton release by up to 40%. These observations suggest that some mechanisms other than a Na+/H+ antiport and carbon dioxide diffusion could be transporting the H+ generated in the cytosol of the activated PMNs.

Animals↗
PubMed2000

Bicarbonate exacerbates oxidative injury induced by antitumor antibiotic doxorubicin in cardiomyocytes.

Doxorubicin, a broad-spectrum antitumor antibiotic, causes dose-dependent cardiomyopathy and heart failure. Although the exact molecular mechanisms of cardiotoxicity are not well established, oxidative mechanisms involving doxorubicin-induced superoxide anion production have been proposed. In this study, we show that bicarbonate, a physiologically relevant tissue component, greatly amplified doxorubicin-induced cardiomyocyte injury. Bicarbonate also enhanced inactivation of aconitase, a crucial tricarboxylic acid cycle enzyme, in cardiomyocytes exposed to doxorubicin. The cell-permeable superoxide dismutase mimetic, Mn(III)tetrakis (4-benzoic acid) porphyrin, reversed doxorubicin-induced cardiomyocyte injury. Bicarbonate enhanced the inactivation of purified mitochondrial aconitase in the xanthine/xanthine oxidase system, generating superoxide. The results suggest that bicarbonate amplifies the prooxidant effect of superoxide. Bicarbonate also caused an increased loading of cardiomyocytes with doxorubicin. We conclude that the bicarbonate-mediated increase in doxorubicin toxicity is due to increased intracellular loading of doxorubicin in cardiomyocytes and subsequent exacerbation of superoxide-mediated cardiomyocyte injury.

Aconitate Hydratase↗
PubMed1989

Effect of acetylcholine on membrane potential of cultured human nonpigmented ciliary epithelial cells.

Human nonpigmented ciliary epithelial cells (NPE) were grown in tissue culture after transformation with an origin-defective mutant of SV-40 DNA. In these cells membrane potentials (V) were measured using the microelectrode technique. Addition of 10(-4) M acetylcholine led to a bisphasic voltage response. An immediate, transient hyperpolarization was followed by a sustained depolarization below the steady state level. These responses were irreversibly blocked by 10(-5) M atropine. In Ca2+-free media the initial addition of acetylcholine resulted in an unchanged voltage response. A second application of acetylcholine in Ca2+-free solution evoked only an abortive response of V, and further addition had no effect on V. In the presence of Ca2+ channel blockers (10(-5) M verapamil, 1 mM Co2+) the acetylcholine-induced response of the membrane potential was not changed. The initial hyperpolarization induced by acetylcholine was reduced by 33 +/- 3% (n = 6) in the presence of 2 mM Ba2+ and by 79 +/- 6% (n = 6) in the presence of 1 mM quinidine. Moreover, the amplitude of the hyperpolarization was dependent on the extracellular K+ concentration. With increasing extracellular K+ concentration (and decreasing transmembrane K+ gradient) the acetylcholine-induced hyperpolarization was reduced. To further elucidate the role of Ca2+ in the acetylcholine-induced responses, we measured cytoplasmic Ca2+ activity using the fluorescence of intracellularly trapped Fura-2. Cytoplasmic Ca2+ activity increased immediately and transiently upon addition of acetylcholine. We conclude that acetylcholine transiently hyperpolarizes V in cultured human NPE by activation of K+ channels mediated by mobilization of Ca2+ from intracellular stores.

Acetylcholine↗
PubMed1993

Contact signalling and cell motility.

There is now clear evidence that signals can be generated when cells make adhesions. This happens when contacts are made with the extracellular matrix, with other cells or with experimental substrata. It is shown that intracellular signals are triggered in a variety of situations by the aggregation of transmembrane glycoproteins. Such aggregation occurs at adhesion sites, and it is suggested that adhesion molecules which are capable of lateral diffusion become trapped at developing adhesion sites because their headgroups bind to arrays of external ligands. Signals generated by molecular clustering can influence cell spreading and motility via familiar transduction pathways. Although it is not yet possible to reconstruct with confidence the entire sequence of events between initial glycoprotein clustering and motility, there is a lot of information which points to the identities of the molecular ingredients. I shall focus the discussion on the thin peripheral lamellar extensions produced by cell types ranging from amoebae to vertebrate nerve growth cones, and discuss the experimental evidence relating to their cytoskeletal architecture and dynamics. Finally I shall try to construct models of motility consistent with the experimental data, and suggest how lamellar motility may be modulated by signals generated by clustered adhesion molecules.

Animals↗
PubMed2000

TROY, a newly identified member of the tumor necrosis factor receptor superfamily, exhibits a homology with Edar and is expressed in embryonic skin and hair follicles.

In a signal sequence trap screening of the murine brain, we identified a new member of the tumor necrosis factor receptor superfamily designated TROY. TROY is a type I membrane protein of 416 amino acids with characteristic cysteine-rich motifs in the extracellular domain and a tumor necrosis factor receptor-associated factor (TRAF) 2 binding sequence in the cytoplasmic domain of 223 amino acids. In fact, activation of nuclear factor kappaB was induced by the overexpression of TROY and inhibited by dominant negative forms of TRAF2, TRAF5, and TRAF6, indicating that TRAFs and nuclear factor kappaB are involved in the signal transduction of TROY. We also cloned a cDNA for a human counterpart, which showed a 75% homology with mouse TROY at the amino acid level. The extracellular domain of TROY exhibits an extensive homology with that of Edar, a receptor that specifies hair follicle fate. TROY mRNA is strongly expressed in brain and embryo and moderately expressed in the heart, lung, and liver but not the spleen. In the embryo, the expression level is particularly strong in the skin. Interestingly, in situ hybridization analysis of the embryo showed that TROY mRNA was exclusively expressed in the epithelium of many tissues. On the other hand, in neonatal mice, TROY is expressed in hair follicles like Edar as well as in the cerebrum, suggesting pleiotropic functions of TROY in development as well as in the adult mice. The Troy gene is located near the waved coat (Wc) locus, a mutant related to abnormalities in skin and hair.

Amino Acid Sequence↗
PubMed1994

Role of hydroxyl radicals in the activation of human platelets.

Platelets primed by exposure to subthreshold concentrations of arachidonic acid or collagen are known to be activated by nanomolar levels of hydrogen peroxide. We here demonstrate that this effect is mediated by hydroxyl radicals (OHzero) formed in an extracellular Fenton-like reaction. H2O2-induced platelet aggregation, serotonin release and thromboxane A2 productions were inhibited by OHzero scavengers and by the iron chelator desferrioxamine; hydroxyl radicals were detected directly by ESR measurements of the spin-trapped OHzero adduct. The role of OHzero was confirmed in experiments with exogenously added iron; free or EDTA-bound ferrous iron activated platelets in a process blocked by deoxyribose, mannitol or catalase, whereas ferric iron was without effect unless reductants were included. The activation by OHzero depended on concomitant release of arachidonic acid and was blocked by the phospholipase A2 inhibitors mepacrine and aristolochic acid, and by the Na+/K+ antiporter inhibitor ethylisopropylamiloride. In contrast, neomycin and staurosporin were without effects, indicating that phospholipase C and protein kinase C were not involved in the initial phase of activation. Neither radical formation nor arachidonic acid release was blocked by aspirin. In whole blood aggregation of platelets could be induced by H2O2 generated upon specific stimulation of neutrophils by N-formyl-methionyl-leucyl-phenylalanine; platelet activation and radical formation were blocked by the NADPH oxidase inhibitor diphenyliodonium as well as by catalase and mannitol. These results suggest that reactive oxygen species act as 'second messengers' during the initial phase of the platelet activation process.

Arachidonic Acid↗