scholarly journals The involvement of Na+/K(+)-ATPase in the development of platelet procoagulant response.

2007 ◽  
Vol 54 (3) ◽  
pp. 625-639 ◽  
Author(s):  
Marian Tomasiak ◽  
Halina Stelmach ◽  
Tomasz Rusak ◽  
Michał Ciborowski ◽  
Piotr Radziwon
Keyword(s):  
Morphological Changes ◽  
Membrane Curvature ◽  
Procoagulant Activity ◽  
Calcium Signal ◽  
Left Shift ◽  
Cell Sizing ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
In Vitro Treatment

In circulation, platelets may come into contact with both exogenous (cardiac glycoside treatment) and endogenously produced inhibitors of Na+/K(+)-ATPase. We examined whether blocking of platelet Na+/K(+)-ATPase by ouabain results in generation of procoagulant activity. It was shown that an in vitro treatment of platelets with ouabain (20-200 microM for 20 to 60 min) is associated with an intracellular accumulation of sodium ([Na+](i)), generation of a weak calcium signal, and expression of procoagulant activity. The ouabain-induced procoagulant response was dose- and time-related, less pronounced than that evoked by collagen and similar to that produced by gramicidin, not affected by EDTA or aspirin, and strongly reduced in the absence of extracellular Na+ or by hyperosmolality. Flow cytometry studies revealed that ouabain treatment results in a unimodal left shift in the forward and side scatter of the entire platelet population indicating morphological changes of the plasma membrane. The shift was dose related, weaker than that evoked by collagen and similar to that produced by gramicidin. Ouabain-treated platelets express phosphatidylserine (PS). The ouabain-evoked PS expression was dose- and time-dependent, weaker than that produced by collagen and similar to that evoked by gramicidin. Electronic cell sizing measurements showed a dose-dependent increase in mean platelet volume upon treatment with ouabain. Hypoosmotically-evoked platelet swelling resulted in the appearance of procoagulant activity. Thromboelastography measurements indicate that, in whole blood, nanomolar (50-1000 nM, 15 min) concentrations of ouabain significantly accelerate the rate of clot formation initiated by contact and high extracellular concentration of calcium. We conclude that inefficiently operating platelet Na+/K(+)-ATPase results in a rise in [Na+](i). An increase in [Na+](i) and the swelling associated with it may produce PS exposure and a rise in membrane curvature leading to the generation of a procoagulant activity.

355 DEVELOPMENTAL COMPETENCE OF IMMATURE BUBALINE CUMULUS - OOCYTE COMPLEXES VITRIFIED BY THE OPEN PULLED STRAW AND CONVENTIONAL STRAW METHODS

2007 ◽  
Vol 19 (1) ◽  
pp. 293
Author(s):  
A. Sharma ◽  
G. N. Purohit
Keyword(s):  
Morphological Changes ◽  
Developmental Competence ◽  
Control Group ◽  
Equal Concentration ◽  
Nuclear Maturation ◽  
Developmental Capacity ◽  
Phosphate Buffered Saline ◽  
Dose Dependent Increase ◽  
Dose Dependent

The in vitro maturation (IVM), fertilization (IVF), and morphological changes in buffalo cumulus–oocyte complexes (COCs) cryopreserved by ultrarapid freezing using conventional (CON) and open pulled staw (OPS) methods were tested. COCs were cryopreserved using a vitrification solution comprised of Dulbecco's phosphate-buffered saline+0.5 M sucrose+0.4% BSA and two concentrations (4.5 or 5.5 M) of each cryoprotectant ethylene glycol (EG) and dimethylsulfoxide (DMSO) by either the CON or the OPS method. Vitrified COCs were stored in LN for 7 days and then thawed; morphologically normal COCs were used for IVM (n = 1070) and IVF (n = 933) in 2 separate experiments to record morphological damage of COCs due to vitrification, nuclear maturation 24 h after culture (9 replicates), and fertilization 24 h after insemination (10 replicates). The COCs were matured in vitro in TCM-199 media with hormone supplements and fertilized using TALP-BSA as described previously (Purohit et al. 2005 Anim. Reprod. Sci. 87, 229–239). Freshly collected COCs were separately used for IVM (n = 110) and IVF (n = 130) and kept as controls. The arcsin transformed data of the proportions of oocytes matured or fertilized was compared by Duncan's new multiple range test. The highest proportion of morphologically normal oocytes was seen in 5.5 M EG with the CON method (94.5%) and the lowest was seen in 4.5 M DMSO with the OPS method (82.4%). At the end of experiment 1, it was apparent that IVM in all vitrification groups was significantly lower (P < 0.05) compared to the control group (66.4%). Among the various vitrification treatments, the highest IVM occurred in 5.5 M EG with the OPS method (39.2%) and the lowest in 4.5 DMSO with the CON method (19.3%). Comparison of both concentrations of EG and DMSO showed that the proportion of COCs attaining Metaphase-II (M-II) increased with increasing concentration of both of the cryoprotectants. However, at equal concentration of EG and DMSO, the proportion of COCs attaining M-II was significantly higher in the OPS method compared to the CON method. In experiment 2, a significantly higher (P < 0.05) IVF was seen for fresh COCs (45.4%) compared to vitrified COCs. Among the vitrification treatments, the highest fertilization was seen in 5.5 M EG with the OPS method (33.6%) and the lowest in 4.5 M DMSO with the CON method (15.17%). A dose-dependent increase in the proportion of oocytes fertilized was seen with increasing concentration of both EG and DMSO [CON: 4.5 M (15.2%), 5.5 M (25.6%); OPS: 4.5 M (21.3%), 5.5 M (27.5%)] in both CON and OPS methods. Comparison of the 2 cryoprotectants revealed that EG was better compared to DMSO.At equal concentrations of EG or DMSO, a significantly higher (P < 0.05) proportion of fertilized oocytes was seen in the OPS method compared to the CON method. It was concluded that vitrification results in some damage to oocytes, with decrease in their subsequent IVM and IVF. Developmental capacity of vitrified buffalo oocytes can be improved by using OPS instead of conventional straws.

scholarly journals Involvement of Na+/H+ exchanger in desmopressin-induced platelet procoagulant response.

2004 ◽  
Vol 51 (3) ◽  
pp. 773-788 ◽  
Author(s):  
Maria M Tomasiak ◽  
Halina Stelmach ◽  
Anna Bodzenta-Łukaszyk ◽  
Marian Tomasiak
Keyword(s):  
Flow Cytometry ◽  
Thrombin Generation ◽  
Annexin V ◽  
Underlying Mechanism ◽  
Dependent Manner ◽  
Platelet Volume ◽  
Cell Sizing ◽  
Dose Dependent ◽  
In Vitro Treatment

Desmopressin (DDAVP) action on platelets is associated with the development of procoagulant response but the underlying mechanism of this phenomenon is not known. We investigated whether this effect of DDAVP might be due to activation of plasma membrane Na+/H+ exchanger. The DDAVP-induced platelet procoagulant response, measured as phospholipid-dependent thrombin generation, was dose dependent and significantly weaker than that produced by collagen or monensin (mimics Na+/H+ antiport). Both the DDAVP- and collagen-produced procoagulant responses were less pronounced in the presence of EIPA, an Na+/H+ exchanger inhibitor. Flow cytometry studies revealed that in vitro treatment of platelets with DDAVP or collagen was associated with the appearance of both degranulated (and fragmented) and swollen cells. The DDAVP-evoked rise in size and granularity heterogeneity was similar to that produced by collagen or monensin and was not observed in the presence of EIPA. Using flow cytometry and annexin V-FITC as a probe for phosphatidylserine (PS) we demonstrated increased and uniform binding of this marker to all subsets of DDAVP-treated platelet population. The DDAVP-evoked PS expression was dose dependent, strongly reduced by EIPA and weaker than that caused by monensin or collagen. As judged by optical swelling assay, DDAVP in a dose dependent manner produced a rise in platelet volume. The swelling was inhibited by EIPA and its kinetics was similar to that observed in the presence of monensin. Electronic cell-sizing measurements showed an increase in mean platelet volume and a decrease in platelet count and platelet crit upon treatment with DDAVP. DDAVP elicited a slow (much slower than collagen) alkalinization of platelet cytosol. Altogether the data indicate an involvement of Na+/H+ exchanger in the generation of procoagulant activity in DDAVP-treated platelets.

Discrepancy between tissue factor activity and tissue factor expression in endotoxin-induced monocytes is associated with apoptosis and necrosis

2005 ◽  
Vol 94 (12) ◽  
pp. 1236-1244 ◽  
Author(s):  
Olav Klingenberg ◽  
Reidun Øvstebø ◽  
Gun-Britt Joø ◽  
Åse-Brit Westvik ◽  
Peter Kierulf ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tissue Factor ◽  
Procoagulant Activity ◽  
Clinical Samples ◽  
Meningococcal Infection ◽  
High Concentrations ◽  
Apoptosis And Necrosis ◽  
Dose Dependent Increase ◽  
Dose Dependent

SummaryTissue factor (TF), the main initiator of blood coagulation, contributes to the manifestation of disseminated intravascular coagulation following septic shock in meningococcal infection. Since a direct relationship between disease severity and lipopolysaccharide (LPS) concentration in the circulation has been shown, we hypothesized that the procoagulant and cytotoxic effects of endotoxin also in vitro were related to its concentration. In vitro studies, however, have frequently used much higher LPS concentrations than those observed in clinical samples. Using elutriation-purified human monocytes, we observed that LPS up to 1000 ng/ml exerted a concentration-dependent increase in TF activity (tenase activity, fibrin formation in plasma). Although there was a dose-dependent increase in TF activity, there was not a concomitant increase in TF expression at LPS concentrations above 1 ng/ml (flow cytometry, Western blotting, TF mRNA). Flow cytometry revealed that this discrepancy between TF activity and TF expression at endotoxin concentrations above 1 ng/ml, coincided with an LPS dose-dependent increase in cell surface phosphatidylserine (PS), considered to promote coagulation. The increased PS expression was associated with an increased number of 7-AAD-positive cells indicating cell death. We conclude that enhancement of monocyte procoagulant activity in vitro by high concentrations of LPS may result from increased PS exposure due to apoptosis and necrosis. Therefore, the LPS concentrations used to examine monocyte procoagulant activity in vitro, should be carefully chosen.

Stimulation of HCO3- transport in isolated proximal bullfrog duodenum by prostaglandins

1980 ◽  
Vol 239 (3) ◽  
pp. G198-G203 ◽  
Author(s):  
G. Flemstrom
Keyword(s):  
Electrical Potential ◽  
Potential Difference ◽  
Electrical Potential Difference ◽  
Morphological Examination ◽  
Minimal Concentration ◽  
Dependent Transport ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Stimulation Of

An in vitro preparation of proximal duodenum from the bullfrog transported alkali into the luminal solution (approximately 1 mueq x h-1 x cm-2) and generated a transepithelial electrical potential difference (5-10 mV, lumen negative). Transport was inhibited by 2,4-dinitrophenol (10(-5) M), CN- (5 X 10(-3) M), indomethacin (5 X 10(-5) M), and acetazolamide (5 X 10(-3) M) indicating that metabolism is required. Both alkali transport and the electrical potential difference showed a dose-dependent increase on administration of the prostaglandins E2, 16,16-dimethyl E2, and F2 alpha. The minimal concentration stimulating transport was lower with the E-type prostaglandins (10(-8) M than with F2 alpha (10(-6) M), and the former also produced greater maximal responses. In addition to metabolic-dependent transport of alkali, there was passive transmucosal migration of HCO3-, amounting to approximately 40% of basal (unstimulated) transport and sensitive to variation of the transmucosal hydrostatic pressure. Morphological examination showed that the preparation is devoid of Brunner glands. Stimulation of duodenal epithelial HCO3- transport by prostaglandins may contribute to their previously demonstrated ability to prevent duodenal ulceration.

Metabolic and Pharmacologic Interaction of Ethanol and Metronidazole in the Rat

1972 ◽  
Vol 50 (6) ◽  
pp. 476-484 ◽  
Author(s):  
H. Kalant ◽  
A. E. LeBlanc ◽  
M. Guttman ◽  
J. M. Khanna
Keyword(s):  
Incidental Finding ◽  
Repeated Administration ◽  
Volatile Material ◽  
Alcohol Dehydrogenase Activity ◽  
Liver Homogenates ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Concentration Curves ◽  
Central Depressant

Metronidazole, added in vitro, did not act either as an inhibitor or as a substrate for the alcohol dehydrogenase activity of rat liver homogenates. Concentration curves of ethanol and acetaldehyde in the blood after an oral dose of ethanol were not altered by pretreatment with metronidazole; in contrast, disulfiram caused marked elevation of acetaldehyde levels. When given once only, metronidazole (or possibly a metabolite of it) exerted a mild central depressant effect of its own and produced a dose-dependent increase in the intoxicant effect of ethanol. After repeated administration of metronidazole, synergism with ethanol was not seen. An incidental finding was the production of a volatile material during incubation of solutions containing NAD, which gives an acetone-like peak in gas-liquid chromatograms.

scholarly journals Effect of glucose on fermentation heat in sheep rumen fluid in vitro

1986 ◽  
Vol 56 (1) ◽  
pp. 305-311 ◽  
Author(s):  
A. Arieli
Keyword(s):  
Heat Production ◽  
High Glucose ◽  
Infinite Dilution ◽  
Rumen Fluid ◽  
Heat Production Rate ◽  
Sheep Rumen ◽  
Effect Of Glucose ◽  
Dose Dependent Increase ◽  
Dose Dependent

1. Heat production rate (H) of rumen fluid was measured in a direct calorimeter, Basal H of samples of 15 ml rumen fluid mixed with 45 ml buffer was 0.4 mW/ml rumen fluid.2. Addition of glucose (0.4–6.4 mg/sample) was followed by a dose-dependent increase in H. Maximal H was 1.1 rnW/ml and lasted up to 5 min, returning thereafter to the basal level.3. Expression of fermentation heat (Hf; kJ/mol substrate added) against glucose dose indicated an asymptotic dose response.4. Maximal Hf(at infinite dilution) agreed with stoichiometric calculations whereas minimal Hfsuggested a partial fermentation of the substrate at a high-glucose dose in the rumen environment.

scholarly journals Establishment and characterization of human B-cell lymphoma cell lines using B-cell growth factor

Blood ◽  
1990 ◽  
Vol 75 (6) ◽  
pp. 1311-1318 ◽  
Author(s):  
RJ Ford ◽  
A Goodacre ◽  
I Ramirez ◽  
SR Mehta ◽  
F Cabanillas
Keyword(s):  
Growth Factor ◽  
Cell Growth ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoma Cells ◽  
B Cell Growth Factor ◽  
Hodgkin's Lymphomas ◽  
Dose Dependent Increase ◽  
Dose Dependent

B-cell non-Hodgkin's lymphomas (NHL-B) have been difficult to establish in long-term cell culture using standard techniques. We report the establishment of five representative cell lines from high grade NHL-B using B-cell growth factor (BCGF). The five NHL-B cell lines display the morphologic, immunophenotypic, genotypic, and biologic characteristics of the lymphoma cells present in the original diagnostic specimen. The cell lines showed at least a sevenfold dose- dependent increase in proliferation in vitro over background in the presence of BCGF. Other putative B-cell growth-stimulating cytokines showed no significant proliferative activity or were inhibitory in some cases. NHL-B cell lines secreted growth factor(s) into culture supernatants that mediated at least a fivefold dose-dependent increase in cell proliferation in autochthonous lymphoma cells and a 10-fold or greater stimulation in growth factor-dependent normal B cell lines in vitro. The cell lines show monoclonal rearrangements of IgH genes and nonrandom chromosomal abnormalities characteristic of NHL-B, while the expression of Epstein-Barr virus associated antigen (EBNA-I) is present in two of the five cell lines. The studies show that lineage-specific growth factors may be used to establish neoplastic B cell lines in vitro, which are important experimental systems for cellular and molecular studies in the NHL-B.

scholarly journals The Model of D-Galactosamine-Induced Injury of Rat Hepatocytes in Primary Culture

2006 ◽  
Vol 49 (1) ◽  
pp. 59-65 ◽  
Author(s):  
Otto Kučera ◽  
Halka Lotková ◽  
Roman Kanďár ◽  
Renata Héžová ◽  
Vladimíra Mužáková ◽  
...  
Keyword(s):  
Morphological Changes ◽  
Animal Experiments ◽  
Rat Hepatocytes ◽  
Short Term ◽  
Hepatocyte Injury ◽  
Albumin Production ◽  
Potential Activity ◽  
Dose Dependent ◽  
Uridine Nucleotides

D-galactosamine (GalN) is a highly selective hepatotoxin that causes liver damage similar to human viral hepatitis via depletion of uridine nucleotides, which subsequently diminishes synthesis of RNA and proteins. Model of galactosamine hepatotoxicity is frequently used in animal experiments in vitro. The purpose of our study was to establish the model of GalN-induced hepatocyte injury in in vitro conditions using primocultures of rat hepatocytes as an important prerequisite for further experiments in which we would like to study potential hepatoprotective effect of various substances. Rate of hepatocyte injury was evaluated by morphological changes, changes in cell viability, albumin production, mitochondrial membrane potential, activity of mitochondrial dehydrogenases and glutathione content. Marked dose dependent hepatocyte injury was found after 24-hour incubation with GalN. Based on the results we suggest as an optimal model for short-term toxicity test exposure to GalN for 24 hours in dose of 40 mM.

Immunolocalization, binding, and biological activity of endothelin in rabbit uterus: effect of ovarian steroids

1991 ◽  
Vol 260 (2) ◽  
pp. E292-E305
Author(s):  
M. Maggi ◽  
G. B. Vannelli ◽  
A. Peri ◽  
M. L. Brandi ◽  
G. Fantoni ◽  
...  
Keyword(s):  
Endothelin Receptors ◽  
Binding Studies ◽  
Stromal Compartment ◽  
Ovarian Steroids ◽  
Specific Receptors ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Two Populations ◽  
Dissociation Constant Kd

Specific immunostaining for endothelin 1 (ET-1) was observed in the endometrium but not myometrium of rabbits. The staining was dramatically affected by subacute treatment with ovarian steroids: epithelial cells were predominantly positive in immature rabbits, whereas, in sex steroid-primed rabbits, ET-1 was mainly localized in the stromal compartment. Binding studies were performed in myometrium of estrogen-treated rabbits using labeled ET-1 and ET-3, the corresponding unlabeled peptides, and sarafotoxin b (SRTX). Mathematical modeling of experimental results indicates that two populations of sites are present in myometrium. One site (R1 = 1 pmol/mg protein) shows approximately the same affinity for ET-1, ET-3, and SRTX [dissociation constant (Kd) 100 pM], whereas the second site (R2 = 10 pmol/mg protein) selectively binds ET-1 (Kd 400 pM). According to binding studies, ET-1 was more potent than SRTX in stimulating uterine contraction "in vitro." The subacute administration of increasing concentrations of 17 beta-estradiol (0.2-200 micrograms/kg for 4 days), but not 17 beta-estradiol (200 micrograms/kg for 4 days) plus progesterone (5 mg/kg for 4 days), stimulates a dose-dependent increase in endothelin receptors in myometrium (half-maximal effective dose = 0.7 micrograms/kg for 4 days). However, estrogen treatment does not affect the concentration of endothelin receptors in myometrial cells in primary culture. Conversely, divalent ions like calcium and magnesium enhance the binding of ET-1 to both uterine membranes and cells. Our results indicate that in rabbit uterus endothelin is present in the endometrium, whereas specific receptors are located in myometrium.

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