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PubMed1995

Regulation of vascular smooth muscle soluble guanylate cyclase activity, mRNA, and protein levels by cAMP-elevating agents.

Although the biochemical properties of soluble guanylate cyclase (sGC) have been extensively studied, little is known about the regulation of gene expression of sGC subunits by second messengers. cAMP analogues and elevating agents have been previously shown to alter gene expression in vascular cells. The aim of the present study was to investigate the effects of cAMP-elevating agents on sodium nitroprusside-stimulated sGC activity and to correlate activity changes with mRNA and protein levels in cultured rat aortic smooth muscle cells. Pretreatment of cells with 50 to 1000 mumol/L isobutylmethyl-xanthine or 0.01 to 10 mumol/L forskolin led to a time- and concentration-dependent decrease in sodium nitroprusside-induced cGMP accumulation, first evident after 3 hours of pretreatment with forskolin and 6 hours of pretreatment with isobutylmethylxanthine. Incubation of cells with a protein kinase A-selective inhibitor (H89 or KT 5720) partially or fully prevented the downregulation in sodium nitroprusside-induced cGMP accumulation caused by cAMP-elevating agents. Quantification of reverse transcriptase-polymerase chain reaction products by high-performance liquid chromatography revealed that mRNA for both alpha1- and beta1-subunits of sGC were decreased in cells pretreated with isobutylmethylxanthine and forskolin but not with dideoxyforskolin (inactive analogue). Moreover, protein levels for the sGC alpha1 subunit of cells pretreated with isobutylmethylxanthine and forskolin but not with dideoxyforskolin were decreased as indicated by Western blot analysis. These data indicate that cAMP-elevating agents decrease sGC activity, possibly by decreasing mRNA or protein levels or both.

Animals↗

PubMed2002

Analysis of immunohistochemical label of Fos protein in the suprachiasmatic nucleus: comparison of different methods of quantification.

The induction of the proto-oncogene c-fos, and its phosphoprotein product Fos, has been extensively used to study the effects of light on the circadian pacemaker in the suprachiasmatic nucleus (SCN). Experimental approaches to the quantification of Fos induction have mainly been based on immunohistochemistry and subsequent measure of Fos immunoreactivity (IR) in sections of the SCN. In this study, the authors compare several methods of quantification using optical density image analysis or counts of Fos-IR labeled cells. To assess whether optical density measures using image analysis reflect the amount of Fos in brain tissue, the authors developed standards of known concentrations of Fos protein in an agar matrix. The agar standards were sectioned and treated simultaneously with sections of the SCN from animals exposed to different levels of irradiance. Optical density was found to be proportional to the quantity of Fos in the sections, indicating that this measure accurately reflects relative levels of Fos protein induction. Quantification by optical density analysis allows an objective measure in which the various parameters, conditions of illumination, and threshold can be maintained constant throughout the analysis. Counting cells by visual observation is more subjective because threshold values cannot be precisely defined and can vary according to the observer, illumination, degree of label, and other factors. In addition, cell counts involving direct visual observation, automated cell counts, or stereological methods do not take into account the difference in the density of label between cells, thus giving equal weight to lightly or densely stained cells. These measures are more or less weakly correlated with measures of optical density and thus do not accurately reflect the amount of bound Fos protein in the tissue sections. In contrast, labeled surface area as measured by image analysis shows a linear relationship with optical density. The main outcome of this study is that computer-assisted image analysis provides an accurate and rapid method to determine the relative amount of Fos protein in the SCN and the effects of light on intracellular signaling mechanisms involved in the circadian clock.

3,3'-Diaminobenzidine↗

PubMed1992

Indomethacin promotes germinal matrix microvessel maturation in the newborn beagle pup.

BACKGROUND AND PURPOSE: Although indomethacin has been demonstrated to prevent germinal matrix and intraventricular hemorrhage in clinical and animal studies, the mechanism of action of this agent to prevent hemorrhage remains unclear. Previous studies have demonstrated both that the microvessels in the germinal matrix of newborn beagle pups undergo basement membrane maturation during the first 4 postnatal days and that indomethacin may promote laminin deposition in tumor cell culture systems. METHODS: We employed the newborn beagle pup model to test the hypothesis that indomethacin may stimulate laminin deposition in germinal matrix microvessels. Newborn pups were randomized to receive either 0.1 mg/kg/dose i.p. indomethacin or an equal volume of saline diluent. Pups received doses of study medication once a day for 1, 2, or 3 days and were studied on postnatal days 1, 2, 3, or 4. Pups were anesthetized and systemically perfused with buffered formalin; the brains were removed and prepared for immunohistochemical study. RESULTS: Sections stained with Bandeiraea lectin demonstrated that there was no difference in germinal matrix vessel density among the postnatal ages studied; similarly, there were no differences in vessel density between saline- and indomethacin-treated animals at any postnatal age. Quantification of germinal matrix stained intensity by confocal microscopy demonstrated significant increases in indomethacin-treated pups for both laminin staining at postnatal days 2 (p = 0.05) and 3 (p = 0.0009) and type V collagen staining at postnatal day 2 (p = 0.011). Although staining for beta 1 integrins increased across postnatal ages, there were no differences between saline- and indomethacin-treated animals. CONCLUSIONS: These data suggest that indomethacin may stimulate basement membrane deposition in the germinal matrix microvessels of newborn beagle pups to prevent germinal matrix and/or intraventricular hemorrhage.

Animals↗

PubMed2004

Role of apoptosis in reperfusion injury.

Many changes occur during reperfusion of the myocardium after ischemic damage. Necrosis and apoptosis appear to be ongoing during ischemia, while apoptosis is boosted by the reperfusion event. In the past 10 years, distinct intracellular pathways important for hypertrophy, apoptosis, cardiac failure, ischemic preconditioning and reperfusion damage have been recognized. The eventual response of the cardiomyocyte will depend on energy and time available as well as changes in pH and ion handling and the delicate balance of activation of signaling molecules and transcription factors. There is agreement on the central role of mitochondria and nitric oxide (NO) in programmed cell death. However, although many groups analyzed the contribution of NO to cell death, still the circumstances and levels required for cardioprotection or death are unclear. Growth factors, cytokines, and downstream signaling molecules have been shown to influence programmed cell death through mechanisms reminiscent of preconditioning. Here, the role of apoptosis in ischemia reperfusion-related cell death is reviewed. Important data have been obtained in isolated cells, intact hearts and intact animals. Both pharmacological as well as genetic interventions are discussed. Proof for apoptosis in man post-myocardial infarction (MI) treated through primary Percutaneous Trans-luminal Coronary Angioplasty or other reperfusion therapy is reviewed. Finally, the currently available quantification methods for apoptosis post-MI are mentioned.

Animals↗

PubMed1998

Tyrosine phosphorylation of proteins in primary human myeloid leukemia cells stimulated by cytokines: analysis of the frequency of phosphorylation, and partial identification and semi-quantification of signaling molecules.

We investigated tyrosine phosphorylation of proteins in primary human leukemia cells stimulated by granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), interleukin-3 (IL-3), tumor necrosis factor (TNF), thrombopoietin (TPO) and phorbol myristate acetate (PMA) in 61 patients with acute myeloid leukemia (AML), nine patients with chronic myeloid leukemia (CML) in blastic crisis and four patients in chronic phase, and compared these data of leukemia with those of normal human immature hematopoietic cells. These cytokines and PMA induced tyrosine phosphorylation of proteins in a manner characteristic for each cytokine or PMA in AML cells. G-CSF, GM-CSF and IL-3 frequently phosphorylated p92, p80, p70, p44 and p42. p95 was frequently phosphorylated by G-CSF, and was phosphorylated in one third of the cases by TPO. On the other hand, TNF selectively induced tyrosine phosphorylation of p42, and PMA selectively induced that of p44 and p42. In marked contrast to AML cells, CML cells responded poorly to cytokines with protein tyrosine phosphorylation, and normal human bone marrow mononuclear cells and CD34-positive cells also showed poor response to cytokines. The results of the immunoprecipitation studies showed tyrosine phosphorylation of signal transducers and activators of transcription (Stat) 5 induced by G-CSF, GM-CSF, IL-3 and/or TPO in six cases, that of extracellular signal-regulated kinase (ERK) by GM-CSF in two cases and that of p38 by TNF in three cases. Intracellular amount of Stat5 was markedly increased in AML cells compared with that in CML cells and normal human bone marrow cells. whereas intracellular amount of ERK and p38 was uniformly abundant in both leukemic and normal cells. These results show cytokine-specific and amplified tyrosine phosphorylation of proteins in AML cells and suggest that amplified response might, at least in part, result from the increased amount of signaling molecules such as Stat5.

Acute Disease↗

PubMed2002

Quantification and distribution of equine oocyte cortical granules during meiotic maturation and after activation.

In vitro fertilization (IVF) is being routinely used in humans and several domestic species, however, limited success has been achieved in the horse. Although immature equine oocytes are capable of completing meiosis in vitro, subsequent fertilization, and embryonic development of those oocytes are questionable. The lack of development of these oocytes could be attributed to an impaired cytoplasmic maturation. In the horse, the study of oocyte cytoplasmic maturation and post-fertilization development has been hindered by the lack of progress in IVF. In mammalian oocytes, migration of cortical granules (CG) has been used as an important criterion to evaluate cytoplasmic maturation. The aim of this study was to describe and quantify the CG distribution of equine oocytes during in vitro meiotic maturation and to assess activation of oocytes with calcium ionophore based upon fluorescein isothiocyanate (FITC)-labeled Lens culinaris agglutinin (LCA) and laser confocal microscopy. The results of this study indicate that CG are distributed throughout the cytoplasm of oocytes at the germinal vesicle (GV) stage (immature). As maturation proceeds, a progressive centripetal migration of CG to the oocyte cortex occurs with the formation of a monolayer adjacent to the plasma membrane starting by the end of a 30 hr incubation period and increasing significantly after 36 hr. After activation, significant reduction in the number of CG was observed (P < 0.001) suggesting that oocytes cultured under the present conditions possess the ability to release CG in response to the elevation of intracellular free calcium.

Animals↗

PubMed2006

The expression of aromatase in gonadotropes is regulated by estradiol and gonadotropin-releasing hormone in a manner that differs from the regulation of luteinizing hormone.

The role of estrogens is dual: they suppress basal expression of gonadotropins and enhance GnRH responsiveness at the time of the LH surge. Estrogens are synthesized by cytochrome P450 aromatase (P450arom), encoded by the cyp19 gene. We focused on the cyp19 gene in rat and showed that it is expressed in gonadotropes through promoters PII and PI.f, using RT-PCR and dual fluorescence labeling with anti-P450arom and -LH antibodies. Real-time PCR quantification revealed that aromatase mRNA levels varied during the estrous cycle and were significantly increased after ovariectomy. This effect is prevented by estradiol (E2) as well as GnRH antagonist administration, suggesting that GnRH may mediate the steroid effect. Interestingly, the long-acting GnRH agonist that induces LH desensitization does not modify aromatase expression in ovariectomized rats. Administration of E2 in ovariectomized rats receiving either GnRH agonist or GnRH antagonist clearly demonstrated that E2 also reduces cyp19 expression at the pituitary level. The selective estrogen receptor-alpha ligand propyl pyrazole triol and the selective estrogen receptor-beta ligand diarylpropionitrile both mimic the E2 effects. By contrast, propyl pyrazole triol reduces LH beta expression whereas diarylpropionitrile does not. In addition, using transient transfection assays in an L beta T2 gonadotrope cell line, we provided evidence that GnRH agonist stimulated, in a dose-dependant manner, cyp19 promoters PII and PI.f and that E2 decreased the GnRH stimulation. In conclusion, our data demonstrate that GnRH is an important signal in the regulation of cyp19 in gonadotrope cells. Both common and specific intracellular factors were responsible for dissociated variations of LH beta and cyp19 expression.

Animals↗

PubMed1995

Mechanism of inhibition of glycolysis in Streptococcus mutans NCIB 11723 by chlorhexidine.

Inhibition of the rate of acid production from glucose by the cells of Streptococcus mutans NCIB 11723 was directly related to the concentrations of 0.075 to 0.20 mM chlorhexidine. Lactate production was inhibited to a greater extent than acetate and formate. Quantification of glycolytic intermediates revealed that the steps in glycolysis inhibited by chlorhexidine were the reactions catalyzed by phosphofructokinase and glyceraldehyde 3-phosphate dehydrogenase and/or phosphoglycerate kinase. However, the activities of these enzymes were not decreased in cells treated with the inhibitor. It was demonstrated that chlorhexidine caused leakage of metabolites from the cells. Our results indicate that the decreased rate of glycolysis caused by chlorhexidine is due to the leakage of metabolic intermediates and not to direct effects on enzymes involved in glycolysis by S. mutans NCIB 11723.

Acetates↗

PubMed2003

Quantification and glucocorticoid regulation of glucocorticoid receptor transcripts in two human leukemic cell lines.

We have quantified the basal and glucocorticoid-regulated levels of different transcripts from the human glucocorticoid receptor (GR) gene in the T-cell acute lymphoblastic leukemia cell line, CEM-C7, and in the B lymphoblastoid cell line, IM-9. Highly specific quantitative, reverse transcription-polymerase chain reaction assays measured total GR transcripts, transcripts encoding the isoforms glucocorticoid receptor alpha (GRalpha) and glucocorticoid receptor beta (GRbeta), and transcripts containing different forms of exon 1: 1A1, 1A2, 1A3, 1B, and 1C. GRalpha and GRbeta transcripts are coordinately upregulated in CEM-C7 cells and coordinately downregulated in IM-9 cells by dexamethasone. The concentration of GRalpha mRNA is more than a 1000-fold higher than that for GRbeta mRNA. Transcripts with different exon 1 forms are all upregulated in CEM-C7 cells and all downregulated in IM-9 cells by dexamethasone, but transcripts containing exons 1A1, 1A2, or 1A3 are regulated to a higher degree than transcripts containing exon 1B or exon 1C. However, exon 1B- and exon 1C-containing transcripts are substantially more abundant than exon 1A-containing transcripts, with exon 1A3-containing transcripts more abundant than exon 1A1- or exon 1A2-containing transcripts. Analysis using models for glucocorticoid receptor autoregulation kinetics suggests that the minor 1A3-containing transcript component could be important for GR protein upregulation, and hence apoptosis, in CEM-C7 cells. These studies suggest that GRalpha transcripts containing exons 1A3, 1B, and 1C contribute most to the intracellular level of GR mRNA and may be the most relevant for steroid-mediated apoptosis in T-lymphoblasts.

Blotting, Western↗

PubMed2001

Unique in vivo applications of spin traps.

The ultimate goal of in vivo electron spin resonance (ESR) spin trapping is to provide a window to the characterization and quantification of free radicals with time within living organisms. However, the practical application of in vivo ESR to systems involving reactive oxygen radicals has proven challenging. Some of these limitations relate to instrument sensitivity and particularly to the relative stability of these radicals and their nitrone adducts, as well as toxicity limitations with dosing. Our aim here is to review the strengths and weaknesses of both traditional and in vivo ESR spin trapping and to describe new approaches that couple the strengths of spin trapping with methodologies that promise to overcome some of the problems, in particular that of radical adduct decomposition. The new, complementary techniques include: (i) NMR spin trapping, which monitors new NMR lines resulting from diamagnetic products of radical spin adduct degradation and reduction, (ii) detection of *NO by ESR with dithiocarbamate: Fe(II) "spin trap-like" complexes, (iii) MRI spin trapping, which images the dithiocarbamate: Fe(II)-NO complexes by proton relaxation contrast enhancement, and (iv) the use of ESR to follow the reactions of sulfhydryl groups with dithiol biradical spin labels to form "thiol spin label adducts," for monitoring intracellular redox states of glutathione and other thiols. Although some of these approaches are in their infancy, they show promise of adding to the arsenal of techniques to measure and possibly "image" oxidative stress in living organisms in real time.

Animals↗

PubMed1996

Fluorescence lifetime imaging: an emerging technique in fluorescence microscopy.

Fluorescence microscopy is an important tool for biological research, in part because of the extremely high detection sensitivity that can be achieved, but also because fluorescent molecules can be used as probes on account of their environmental responsiveness, for example to measure intracellular pH or metal ion concentration. Unfortunately, the environmental sensitivity can sometimes be a source of problems because of enhancement or 'quenching', which can make it very difficult to relate emission intensity to the amount of fluorophore present. The measured intensity is essentially proportional to the product of the amount of fluorophore present in the sample and the local quantum yield of the fluorophore (the quantum yield can be thought of as the probability that an excited molecule decays by fluorescence emission rather than by other non-radiative processes). This is a particular difficulty in an environment such as a cell or tissue slice in which quantum yield and flurophore concentration can both vary within the sample. Ideally we would wish to be able to measure the quantum yield of fluorescence as well as the fluorescence intensity, as this would allow environmental effects to be compensated for. Unfortunately, this is not at all easy, and indirect means to achieve the same goal are more appropriate. A recently introduced technique, fluorescence lifetime imaging (Morgan et al. 1992, Wang et al. 1992), offers one such means to improve quantification of fluorescence microscopy. In addition, as will be explained, the technique offers the prospect of significantly improving detection sensitivity in appropriate circumstances.

Energy Transfer↗

PubMed1984

Sequential determination of calcium distribution in B cells at the various phases of glucose-induced insulin secretion.

Localization and quantification of calcium pyroantimonate precipitates within the B cells, and determination of insulin secretion were performed in rat pancreas perfused with 3.3 and 16.6 mmol/l glucose. Observations were carried out during the peak, the refractory period, and at 10 and 20 min in the second phase of glucose secretion after the start of a glucose challenge. Specific calcium pyroantimonate precipitates, assessed by EGTA cross-incubation, appeared attached to plasma membrane, Golgi complex, mitochondria, cytoplasmic matrix and secretory granules. The total number of cellular calcium pyroantimonate precipitates increased with perfusion time, being significantly higher at every time-point with the higher concentration of glucose (16.6 mmol/l) than with the 3.3 mmol/l glucose concentration. Calcium pyroantimonate precipitates showed a progressive increment both in plasma membranes and mitochondria. In the cytoplasmic matrix, B granules and Golgi complex, a sharp increase in the number of precipitates was detected at the refractory period, followed by a continuous decrease until the end of the experiment. These results show that the number of calcium pyroantimonate precipitates, localized in different organelles, changes according to the functional state of B cells. They stress the importance of intracellular readily exchangeable pools as regulators of calcium availability for insulin stimulus-secretion coupling.

Animals↗

PubMed2006

High-performance anion-exchange chromatography using on-line electrolytic eluent generation for the determination of more than 25 intermediates from energy metabolism of mammalian cells in culture.

In this work, we present an improved method for the determination of a wide range of intracellular metabolites from mammalian cells by anion-exchange chromatography. The analysis includes the measurement of intermediates from glycolysis and tricarboxylic acid cycle as well as several additional nucleotides and sugar nucleotides. The use of an electrolytic on-line eluent generation device made the method highly convenient, reliable and prone to errors. Due to short delay times of the eluent generator, rapid KOH gradient changes could be applied to improve separation and to speed up elution. Suppressed conductivity and UV in series was used for detection. The detection wavelength of the UV detector was switched from 220 to 260 nm during the elution for a more selective signal depending on the absorption of analytes. Standards from more than 50 metabolites of major cellular pathways were chromatographically tested and compared to chromatograms from extraction samples of Madin-Darby canine kidney (MDCK) and BHK21 cells. A validation for most substances was performed. Detection limits were below the micromolar range and the coefficient of correlation (R(2)) was above 0.99 for most analytes. Working ranges were between 0.125-3.875 and 4.5-139.5 microM. Sample pH had a major impact on the quantification of several metabolites, but measurements were robust within a pH range of 6.5-9.0.

Analytic Sample Preparation Methods↗

PubMed2000

Comparison of proteolytic enzymes and glutathione S-transferase levels in non-heart-beating donors' (NHBD) kidney perfusates.

UNLABELLED: In order to identify biochemical markers of kidney damage prior to transplantation, we determined the levels of activity of a range of proteolytic enzymes in kidney perfusate samples from non-heart-beating donor (NHBD) cases. Urinary protease activities have been described as indices of kidney damage in renal disease; their potential as markers of tissue damage in kidneys before transplantation has not been assessed. In an attempt to identify additional/improved biochemical markers, the present study compared the levels of total glutathione S-transferase (GST) with corresponding levels of several proteolytic enzymes in perfusate fluid from machine perfused NHBD kidneys. Proteases were selected to represent factors that may influence enzyme efflux, such as intracellular localization or molecular size. METHODS: Perfusate samples were obtained over an 8-hour period from machine-preserved NHBD kidneys. Protease activities in these samples were determined by fluorometric assays and comparison made with total GST activity. Individual proteases were analysed in the transplanted and non-transplanted kidney groups (discarded on the basis of other viability parameters). RESULTS: A correlation between protease activity and total GST was obtained for only leucyl- and pyroglutamyl aminopeptidase. Furthermore, in the transplanted group, it was possible to set nominal upper limits of activity for alanyl-arginyl- and dipeptidyl IV-aminopeptidase (AP). In the non-transplanted kidney group protease levels were increased above "normal" upper limits for the same enzyme types. By the use of alanyl AP it was possible to discriminate 75% of unsuitable kidneys discarded by the use of other criteria. CONCLUSION: The lack of correlation between total GST and protease activity for most of the enzymes investigated and alanyl AP levels in perfusate samples could be related to differences in cellular localisation, suggesting that assays of alanyl AP may give complimentary biochemical information relating to kidney tissue damage. Quantification of alanyl AP in machine perfusate samples may be a valuable additional independent biomarker of tissue damage.

Endopeptidases↗

PubMed1999

Distribution and quantification of immunoreactive orexin A in rat tissues.

A sensitive and specific radioimmunoassay for orexin A was developed. Orexin A immunoreactivity was found to be confined to the central nervous system (CNS) with the highest concentrations in the hypothalamus, inferior and superior colliculi and brainstem. Within the hypothalamus, the highest levels were found in the lateral and posterior hypothalamus. These regions had a greater orexin A content in females compared to males. The orexin A content of hypothalamic regions did not change with fasting and no difference was noted in hypothalami of rats fed a high fat diet. The hypothalamic orexin A content was not different in obese Zucker rats compared to lean controls. Thus, orexin A has a wide distribution in the CNS, but appetite regulation may not be its main function.

Animals↗

PubMed1995

Contact sensitizers modulate mechanisms of receptor-mediated endocytosis but not fluid-phase endocytosis in murine epidermal Langerhans cells.

In order to define the influence of contact allergens on the fluid-phase endocytosis (FPE) of soluble molecules of murine epidermal Langerhans cells (LC), we studied the internalization of FITC-labeled bovine serum albumin (FITC-BSA), TRITC-labeled dextrane (TRITC-DEX) as well as horseradish peroxidase by LC. A 3-parameter flow-cytometric technique was performed for quantification of internalized FITC-BSA in LC using quantum red-labeled reagents for detection of Ia-antigen expression by LC and propidium iodide for exclusion of dead cells from analysis. A temperature-dependent rapid accumulation of FITC-BSA was noticed in time-course studies reaching a plateau between 1 and 2 h of in vitro culture at 37 degrees C. The quantity of FPE under stimulation with phorbol 12-myristate 13-acetate (PMA), concanavalin A (Con A), staphylococcal enterotoxin B (SEB) and contact sensitizers (DNFB, Kathon CG, K2Cr2O7) as well as the irritant SLS was determined. Treatment of LC with PMA and Con A resulted in a significant increase of total FITC-BSA uptake. The contact sensitizers as well as SEB and SLS failed to mediate augmented fluid-phase endocytosis. By use of the pH-insensitive soluble marker, TRITC-DEX and a microscope photometer for evaluation these findings could be confirmed. This excluded any artificial influence of differences in pH values in endocytotic compartments which might have influenced the fluorescence intensity of the pH-sensitive fluorochrome FITC. For qualitative analysis of FPE, the intracellular distribution of internalized horseradish peroxidase in LC was studied. An aggregated pattern became apparent in untreated LC and did not change under stimulation with any of the substances used.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Primary cultures of rat islet capillary endothelial cells. Constitutive and cytokine-inducible macrophagelike nitric oxide synthases are expressed and activities regulated by glucose concentration.

We have succeeded in obtaining cultures of pure rat islet capillary endothelial cells. These multiply in vitro and exhibit the same antigenic phenotype as expressed in situ: von Willebrand factorhigh, Ox43 (rat endothelial marker)weak, and Ox2 (thymocyte and brain endothelium marker)high. This phenotype differs from both exocrine endothelium stained in situ and rat aorta endothelial cells cultured in vitro under identical conditions. Islet and aorta endothelial cells were cultured in the presence of various glucose concentrations. Nitrite and citrulline concentrations in culture supernatants were measured as an indirect quantification of nitric oxide formation. In islet endothelia, both nitrite and citrulline levels were found to be strongly glucose-dependent, with high levels at high glucose concentrations and vice versa, in contrast to aorta endothelial cells, where no glucose effect was found. Shifting islet endothelial cultures from high to low glucose levels or the reverse led to a slow decrease or increase in nitrite and citrulline formation with several cell generations needed to reach steady levels. Adding a combination of the cytokines interleukin-1 beta, tumor necrosis factor-alpha, and interferon-gamma to both endothelial cell cultures led to a dramatic increase of nitric oxide formation. Again with islet but not with aorta endothelial cells a modulating effect by glucose concentrations was found. Reverse-transcription-polymerase chain reaction with specific primers demonstrated the presence of constitutively expressed nitric oxide synthase-RNA in the islet capillary endothelial cells and confirmed the glucose effect. In addition, we found that cytokines indeed induce the expression of inducible synthase messenger RNA in both endothelial cells, which was not found in the absence of cytokines. Electron paramagnetic resonance spectroscopy of islet endothelial cells confirmed intracellular synthesis of nitric oxide in the presence of cytokines. In conclusion, we here for the first time provide evidence that constitutive nitric oxide synthase is also expressed in capillary endothelium and that cytokine challenge leads to the expression of the inducible isoform in these cells.

Amino Acid Oxidoreductases↗

PubMed1994

Immunofluorescent quantification of tyrosine phosphorylation of cellular proteins in whole cells by flow cytometry.

Tyrosine phosphorylation of proteins, a major event in the transduction of mitogenic signals, was analysed by flow cytometry with a fluorescent antiphosphotyrosine monoclonal antibody, on formaldehyde-fixed, permeabilized cells. We have used this method (PY-Facs) to study activation of normal human T lymphocytes and cells of a leukemic T-cell line: Jurkat. In contrast to normal T cells, Jurkat cells as well as three other leukemic cell lines display a higher constitutive level of tyrosine phosphorylation. This level of tyrosine phosphorylation results from an equilibrium that can be up-regulated by the tyrosine phosphatase inhibitor, vanadate peroxide, and down-regulated by the tyrosine kinase inhibitors, genistein and staurosporine. We have also observed an increased tyrosine phosphorylation of proteins after mitogenic stimulation of Jurkat cells via T-cell receptor triggering. In addition, the entry of normal purified T cells from G0 phase into the cell cycle after co-stimulation with a phorbol ester and an anti-receptor antibody is correlated with a pronounced increase in tyrosine phosphorylation. We thus confirmed that this biochemical event was tightly associated with the activation status of the cells. The rapidity and sensitivity of the method we describe here make it particularly convenient for routine use and processing of a large number of samples, e.g., during analysis of human tumors. Moreover, because it retains sufficiently the integrity of treated cells and does not alter expression of membrane antigens, this method is suitable for multiparametric analysis, particularly for simultaneous studies associating the measure of tyrosine phosphorylation levels with possible modifications of membrane or intracellular structures as well as with cell cycle status.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗