Biomedical subjects

A M Vollmar

Publications and source records attributed to A M Vollmar.

At least 37 records · Page 2Linked to original sources

PubMed1998

Effect of allicin and ajoene, two compounds of garlic, on inducible nitric oxide synthase.

Inducible nitric oxide synthase (iNOS) has recently been shown to be present in human atherosclerotic lesions and to promote the formation of deleterious peroxynitrite. Allicin and ajoene are discussed as active compounds with regard to the beneficial effects of garlic in atherosclerosis. The aim of this study was to investigate the effect of allicin and ajoene on the iNOS system in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Ajoene (IC50 2.5-5 microM) and allicin (IC50 15-20 microM) dose dependently reduced nitrite accumulation, a parameter for NO synthesis, in supernatants of LPS-stimulated (1 microg/ml, 20 h) macrophages. Accordingly, reduced iNOS enzyme activities were measured by conversion of L-[3H]arginine to L-[3H]citrulline in homogenates of LPS-activated cells treated with ajoene or allicin. None of these compounds, however, showed a direct effect on the catalytic-activity of iNOS. Consequently, iNOS protein and mRNA expression in ajoene (10 microM) or allicin (50 microM) treated cells were evaluated by Western blot and Northern blot analysis, respectively. Markedly reduced iNOS protein as well as mRNA levels were demonstrated. These observations indicate that allicin and ajoene inhibit the expression of iNOS in activated macrophages. The possible link of this effect to the beneficial features attributed to garlic is discussed.

Cell Line↗

PubMed1998

The Griess assay: suitable for a bio-guided fractionation of anti-inflammatory plant extracts?

In the field of inflammation research the inducible nitric oxide synthase (iNOS) became an important pharmacological target, since overproduction of nitric oxide (NO) after induction of this enzyme seems to be associated with numerous pathological conditions. NO released from cells can be detected and quantified photometrically as its stable product nitrite by a simple colorimetric reaction (Griess reaction). The aim of our study was to investigate whether this method might be suitable for the bio-guided fractionation of anti-inflammatory plant extracts. For this purpose we assayed extracts as well as fractions of the roots of Curcuma zanthorrhiza Roxb, which contain the known iNOS inhibitor curcumin, and compared the obtained activity with their curcumin content. Furthermore, leaf extracts of Betula pendula Roth, to which defined amounts of curcumin were added, were examined to clarify the question whether chlorophyll might interfere with the test system. The presented results suggest that the Griess assay is indeed suitable to guide fractionation of plant extracts in order to isolate highly active compounds. Factors, however, which might restrict the broad application of this assay are the limited selection of solvents which do not interfere with the system and high contents of chlorophyll in plant extracts.

Animals↗

PubMed1998

Ajoene, a compound of garlic, induces apoptosis in human promyeloleukemic cells, accompanied by generation of reactive oxygen species and activation of nuclear factor kappaB.

The pharmacological role of garlic in prevention and treatment of cancer has received increasing attention, but thorough investigations into the molecular mechanisms of action of garlic compounds are rare. The present study demonstrates that ajoene, a major compound of garlic induces apoptosis in human leukemic cells, but not in peripheral mononuclear blood cells of healthy donors. The effect was dose and time dependent. Apoptosis was judged by three criteria, morphology of cells, quantification of subdiploid DNA content by flow cytometry, and detection of DNA fragmentation by gel electrophoresis. Ajoene increased the production of intracellular peroxide in a dose- and time-dependent fashion, which could be partially blocked by preincubation of the human leukemic cells with the antioxidant N-acetylcysteine. Interestingly, N-acetylcysteine-treated cells showed a 50% loss of ajoene-induced apoptosis. Moreover, ajoene was demonstrated to activate nuclear translocation of the transcription factor nuclear factor kappaB, an effect that was abrogated in N-acetylcysteine-loaded cells. These results suggested that ajoene might induce apoptosis in human leukemic cells via stimulation of peroxide production and activation of nuclear factor kappaB. This is a novel aspect in the biological profile of this garlic compound and an important step in elucidating the underlying molecular mechanisms of its antitumor action.

Antineoplastic Agents↗

PubMed1997

The triterpenoid quinonemethide pristimerin inhibits induction of inducible nitric oxide synthase in murine macrophages.

Inducible nitric oxide synthase dependent production of nitric oxide (NO) plays an important role in inflammation. We investigated whether pristimerin ((20alpha)-3-hydroxy-2-oxo-24-nor-friedela-1(10),3,5,7-te traen-carboxylic acid-(29)-methylester), an antitumoral, antimicrobial as well as anti-inflammatory plant compound, has an effect on the inducible NO synthase system in lipopolysaccharide-activated RAW 264.7 macrophages. Pristimerin dose dependently (IC50: 0.2-0.3 microM) reduces nitrite accumulation, a parameter for NO synthesis, in supernatants of lipopolysaccharide-stimulated (1 microg/ml, 20 h) macrophages. This effect correlates with a reduced inducible NO synthase enzyme activity measured by conversion of [3H]L-arginine to [3H]L-citrulline and significantly lower levels of enzyme protein (Western blotting) in homogenates of cells cotreated with lipopolysaccharide and pristimerin (12 h). Northern blot analysis and polymerase chain reaction (PCR) showed decreased inducible NO synthase mRNA levels in activated macrophages exposed to pristimerin (4 h). Electrophoretic mobility shift assay (EMSA) demonstrated a markedly reduced binding activity of nuclear factor-kappa B (NFkappaB) in nuclear extracts of pristimerin-treated cells. These results suggest that pristimerin inhibits the induction of inducible NO synthase by a mechanism which involves inhibition of NFkappaB activation. This feature of pristimerin is likely to contribute to its anti-inflammatory activity.

Cell Line↗

PubMed1997

Effects of atrial natriuretic peptide on phagocytosis and respiratory burst in murine macrophages.

Atrial natriuretic peptide (ANP) is known to affect cardiovascular physiology displaying both hormone- and neurotransmitter characteristics. However, there is increasing evidence that ANP possesses additional biological activities referring to the immune system. To further strengthen this hypothesis the effect of ANP on two major functions of macrophages, i.e., phagocytosis and respiratory burst was tested. Both parameters were analyzed by flow cytometry employing bone marrow derived macrophages and the murine macrophage cell line J774. In both cell types preincubation with ANP dose dependently (10(-10)-10(-7) M) increases ingestion of opsonized fluorescent latex particles. The respiratory burst activity was monitored by oxidation of dihydrorhodamine-123 in cells stimulated either with phorbol-myristate (PMA, 10 ng/ml) or formyl-Met-Leu-Phe (fMLP, 1 microM). In both cases preexposure of cells to ANP (10(-8)-10(-6) M) for 2 h enhances reactive oxygen production. The data demonstrate an influence of ANP on important defense mechanisms of macrophages and thus extend the knowledge regarding the pharmacological profile of this natriuretic peptide.

Animals↗

PubMed1997

Influence of atrial natriuretic peptide on thymocyte development in fetal thymic organ culture.

The aim of the present study was to examine the effect of atrial natriuretic peptide (ANP) on T-cell development. The addition of ANP (10(-6)-10(-10) M, 7 days) to organ cultures of 14 day old fetal mouse thymuses (FTOC) resulted in a decreased cell yield (up to 65% of the amount in control cultures). Flow cytometric analysis revealed a decrease (up to 35%) in the percentage of the CD4+ CD8+ thymocytes accompanied by an increase (100%) of the CD4- CD8- cell population in FTOC incubated with ANP. Exposure of FTOC to ANP furthermore decreases the percentage of CD5 expressing cells and increases the mean size of thymocytes estimated by monitoring their forward scatter intensity (flow cytometry). Thus, ANP seems to affect maturation and differentiation of thymocytes.

Animals↗

PubMed1997

Differential gene expression of the three natriuretic peptides and natriuretic peptide receptor subtypes in human liver.

BACKGROUND: Various effects of atrial natriuretic peptide (ANP) on the liver have been observed. However, there is limited information about the types of receptors for natriuretic peptides expressed by the human liver. AIM: To investigate gene expression of the three NP receptor types (NPR) as well as of the NP in human liver. METHODS: Presence of mRNA coding for all three NPR and for ANP, brain and C-type natriuretic peptide (BNP, CNP) was investigated by reverse transcription-polymerase chain reaction (RT-PCR). Human liver tissues and hepatocellular carcinoma tissues were examined. RESULTS: Specific PCR products for all three NPR, namely NPR-A, B, and C, could be detected. Moreover, ANP and CNP, but not BNP mRNA was detectable. The concentration of ANP transcripts was up to fivefold higher in hepatocellular carcinoma compared with non-tumorous liver tissue of the same subjects. No difference in the expression of NP receptors relative to GAPDH mRNA of tumorous and non-tumorous tissue was observed except of slightly increased NPR-A transcripts. CONCLUSION: These data show that NPR transcripts are coexpressed with ANP and CNP mRNA in the human liver. This provides evidence for a local NP system in the human liver.

Aged↗

PubMed1997

Effects of different natriuretic peptides on nitric oxide synthesis in macrophages.

Atrial natriuretic peptide (ANP) has previously been suggested to inhibit the production of NO in LPS-activated primary macrophages. The aim of the present study was 1) to examine whether ANP elicits this effect also on macrophage cell lines (RAW 264.7, J774), 2) to elucidate whether ANP is the only natriuretic peptide (NP) inhibiting NO synthesis, 3) to look for the expression of natriuretic peptide receptors (NPR) on macrophages, 4) to consequently determine the type of receptor mediating the ANP effect and 5) to obtain first information on the underlying mechanism. Whereas ANP dose dependently (10(-6)-10(-8) M) inhibited NO synthesis (measured as nitrite accumulation, 20h) in all four types of macrophages (bone marrow derived and peritoneal macrophages; RAW 264.7 and J 774), urodilatin and atriopeptin I displayed only a weak effect restricted to the highest concentration (10(-6) M) employed. Importantly, C-type natriuretic peptide (CNP) showed no NO-inhibitory effect. The lack of effect of CNP was shown not to be due to its lower stability or its missing receptor. Macrophages were shown to express all three natriuretic peptide receptors (NPR-A, NPR-B, NPR-C) using RT-PCR technique. Furthermore, two types of NPR-B seem to be present in macrophages. The effect of ANP was mediated via the guanylate cyclase coupled NPR-A as shown by experiments employing stable cGMP analogs, the NPR-A antagonist HS-142-1, LY-83583, a cGMP inhibitor as well as C-ANF, a specific ligand of the NPR-C. Reduction of nitrite accumulation by ANP was highest when added simultaneously with LPS and abolished when added 12 h after LPS stimulation. In summary, ANP was shown to inhibit NO production of LPS-activated macrophages via cGMP.

Aminoquinolines↗

PubMed1996

Natriuretic peptide receptors on rat thymocytes: inhibition of proliferation by atrial natriuretic peptide.

Because the thymus expresses the natriuretic peptides (NP) as well as their respective receptors, an involvement of NP in the physiology of this organ has been suggested. To evaluate functional aspects of NP in the thymus, we looked for thymic cells bearing NP receptors (Npr). Furthermore, the regulation of Npr expression by activation of cells and the influence of NP on the proliferation of thymocytes was studied. Expression of receptor messenger RNAs (mRNAs) was examined by PCR and Northern blot. Existence of functional Npr was confirmed by measurement of cGMP, the second messenger of NP. Proliferation of thymocytes upon concanavalin A (Con A) stimulation was analyzed by incorporation of [3H]thymidine. We report her that thymocytes express mRNAs for the three Npr, namely Npra, Nprb, and Nprc and that activation of Npra and Nprb increases cGMP levels. Stimulation of thymocytes with Con A (1 microgram/ml, 48 h) resulted in an increase of mRNA coding for Npra, the receptor specific for atrial natriuretic peptide (ANP) and brain natriuretic peptide. Nprb and Nprc receptor expression was not altered under these conditions. In agreement with these data only ANP, but not the C-type natriuretic peptide, elicited increased cGMP response in Con A-stimulated cells. ANP inhibited also the proliferation of Con A stimulated thymocytes, whereas C-type natriuretic peptide did not show this effect. These results suggest that ANP affects the complex mechanisms of thymocyte proliferation and differentiation.

Animals↗

PubMed1995

Atrial natriuretic peptide inhibits nitric oxide synthesis in mouse macrophages.

The atrial natriuretic peptide (ANP) affects cardiovascular physiology, and, as has been suggested more recently, exerts immunomodulatory activities. In this context, we examined the effect of ANP on nitric oxide (NO) synthesis in murine bone marrow derived macrophages as well as in peritoneal macrophages. Cultured macrophages were stimulated with lipopolysaccharides (LPS 0.1-10 micrograms/ml) and NO synthesis was monitored by measuring increased concentrations of NO2 in the medium. In initial experiments employment of NG-monomethyl-L-arginine (L-NMMA) and dexamethasone, two specific inhibitors of nitric oxide synthase (NOS), confirmed the presence of inducible NOS activity in the cells. Exposure of cells to rat ANP99-126 in the range of 10(-8) to 10(-6) M significantly decreased LPS induced NO synthesis over 24 hours of incubation. Thus, ANP may alter macrophage function by affecting their nitric oxide synthesizing pathway.

Animals↗

PubMed1995

Co-expression of the natriuretic peptides (ANP, BNP, CNP) and their receptors in normal and acutely involuted rat thymus.

The natriuretic peptides (NP) ANP, BNP and CNP constitute circulating hormones as well as neuropeptides. The present investigation reveals that these peptides are also constituents of the immune system. BNP and CNP, in addition to ANP as previously communicated, are synthesized in the rat thymus. The NP seem to be produced by different types of thymic cells. Moreover, expression of NP is differentially affected by acute involution of the organ caused by dexamethasone (DEX). The concentration of BNP and CNP mRNA was not changed in gradually involuted thymi (i.e. at day 2, 3 and 4 after DEX administration). ANP mRNA was increased in parallel with the degree of involution (2-, 5- and 40-fold, respectively). The concentration of immunoreactive CNP (CNP-IR) was not affected by acute involution of the thymus (4th day after DEX); BNP-IR, however, was increased 2-fold and for comparison, ANP-IR as previously shown, was found 10-fold elevated. Our studies also revealed the presence of the specific mRNAs coding for the A-, B-, and C-type NP receptor in the thymus. The thymic NP system may be involved in the communication between the immune and neuroendocrine system.

Animals↗

PubMed1995

Expression and differential regulation of natriuretic peptides in mouse macrophages.

The coexpression of the natriuretic peptides ANP, BNP and CNP as well as their differential regulation in mouse macrophages was demonstrated by quantitative PCR, HPLC analysis, and specific radioimmunoassays. Exposure of peritoneal- and bone marrow-derived macrophages to various immunomodulators revealed that bacterial LPS strikingly increases (up to 300-fold) the mRNA coding for CNP as does zymosan (up to 15-fold). In this respect, neither the phorbol ester PMA nor the glucocorticoid dexamethasone had any effect. Examination of macrophages for ANP mRNA showed a similar response to LPS and zymosan, though only a three- to sixfold increase, confirming previous data. In contrast, the concentration of mRNA coding for brain natriuretic peptide in these cells was reduced by dexamethasone (up to twofold) as well as LPS (two- to fivefold). No change was observed upon challenge with zymosan or PMA. The findings at the mRNA level are complemented by their corresponding peptide products. Incubation of macrophages with LPS resulted in a two- and fivefold elevation of intracellular ANP and CNP immunoreactivity, respectively. The amount of peptides released from cells under these conditions was found increased for ANP (threefold) and CNP (10-fold). No changes were observed for both intra- and extracellular brain natriuretic peptide. The coexpression of natriuretic peptides in macrophages as well as their different regulations by immunomodulators suggest discrete functions of these peptides within the immune system.

Animals↗

PubMed1994

Atrial natriuretic peptide and plasma volume of dogs suffering from heart failure or dehydration.

Studies in humans and experimental animal models suggest that volume overload increases and volume underload decreases release of atrial natriuretic peptide (ANP), a hormone intimately linked to water and electrolyte homeostasis. This relationship was examined in dogs suffering from heart failure or dehydration and the data presented here are in support of this general concept. Plasma ANP concentration in dogs with congestive heart failure (CHF) was elevated with the severity of the disease (NYHA classification, class II: 21.4 +/- 9.2 fmol ANP/ml; class III: 65.5 +/- 72.6; class IV: 119.7 +/- 87.1; healthy dogs: 13.9 +/- 7.5). The increment in plasma ANP concentration in cardiac patients was also positively correlated with the plasma volume. The blood volume of dogs with moderate and severe CHF was significantly (P < 0.05) elevated and a normotonic blood pressure prevailed. In contrast, dehydrated dogs tend to display reduced ANP plasma concentration (7.7 +/- 5.6 fmol/ml) as well as significantly lower plasma volume and reduced blood pressure (P < 0.05). In dogs with severe CHF, ANP precursor material is present in the blood, which is normally undetectable. These data further support the concept of a regulatory function of ANP in volume homeostasis of dogs.

Animals↗

PubMed1994

Gene expression and secretion of atrial natriuretic peptide by murine macrophages.

An increased expression of atrial natriuretic peptide (ANP) has been reported in activated macrophages of the acutely involuted rat thymus. We communicate here that ANP may reflect a common constituent of macrophages, as mRNA coding for ANP is present in peritoneal- as well as in bone marrow-derived macrophages (PM, BMM). Furthermore, both types of macrophages synthesize and release ANP which was found to mainly represent the biologically active fragment ANP99-126. ANP expression in macrophages is regulated by compounds affecting the activity of these immune cells. For example, incubation of PM or BMM in vitro with LPS and zymosan, respectively, increased ANP-mRNA up to sixfold as determined by competitive PCR quantification. Exposure of macrophages to dexamethasone (Dex, 10(-7) M) elicits moderate effects (1.4-fold), while PMA (10(-7) M) failed to affect its abundance. These findings are complemented by data regarding ANP synthesis and secretion. Incubation of macrophages with LPS, Dex or a combination of both results in an up to 3.5-fold increase of intracellular ANP99-126 (basal 10 fmol/mg protein), and an up to 6.6-fold increase of its secretion (basal 40 fmol/mg protein, 24 h). Since macrophages synthesize and release ANP, the peptide may be involved in the complex mechanisms of host defense, a major function of these immune cells.

Animals↗

PubMed1993

Detection of C-type natriuretic peptide (CNP) transcript in the rat heart and immune organs.

Previous studies suggested the expression of mRNA, coding for CNP, exclusively in the central nervous system. In the present study, using the polymerase chain reaction (PCR) technique instead of the less sensitive Northern blot hybridization, CNP-specific sequences have also been detected in rat atria and ventricles of the heart as well as in organs of the immune system (thymus, spleen and lymph nodes). Parallel PCR-assays documented ANP-mRNA in these tissues. To verify specificity of the PCR-products, Southern blots have been hybridized with a third internal oligonucleotide and amplification products have been sequenced. The relative level of CNP-mRNA in these tissues was estimated to be in the range of 1-9% of total brain CNP transcripts. The results suggest that the peptide may have a peripheral as well as a central site of action. In light of its pronounced effect on cell proliferation, particular interest should focus on a possible role of CNP in the immune system.

Animals↗

PubMed1993

[Endothelin-1 and endothelin-3 in the plasma of dogs].

The endothelins represent a family of peptides with potent vasoconstrictory effects indicating a role in the regulation of vascular tone. An involvement of endothelins in the pathogenesis as well as pathophysiology of vascular diseases has been proposed. Therefore, the aim of the presented study was to develop the methodology to measure and characterize endothelin-1 (ET-1) and endothelin-3 (ET-3) in the plasma of dogs. Extraction of acidified plasma samples was performed by use of Sep-Pak C18 cartridges and plasma levels of ET-1- and ET-3-immunoreactivity (ET-1-IR; ET-3-IR) were determined by specific radioimmunoassays. The measured ET-immunoreactivities were verified by separation of plasma using high-pressure liquid chromatography (HPLC).

Amino Acid Sequence↗

PubMed1993

[Clinico-toxicologic aspects of non-steroidal anti-inflammatory agents in the dog and cat].

The use of nonsteroidal anti-inflammatory drugs (NSAID) is increasing in small animal practice. This has resulted in a greater incidence of acute as well as chronic toxicosis. NSAID toxicoses are usually manifested by gastrointestinal upset, such as nausea, vomiting, gastrointestinal hemorrhage and ulceration. The mechanisms by which these drugs cause their toxic effects are mainly associated with the impaired prostaglandin synthesis and therefore the reduced physiological cytoprotection of the mucosa. In addition, local cellular damage due to the acid effects of NSAID contribute to the GI-toxicity. Treatment of acute intoxication involves maintenance of vital function as well as early decontamination of the gastrointestinal tract and symptomatic treatment (correction of bleeding tendency and local acidosis). Symptomatic care of gastrointestinal damage is the most important aspect of dealing with chronic intoxication. A reduction of acid production as well as a stimulation of the repair mechanism of the mucosa is the aim of the therapy.

Animals↗

PubMed1992

Expression of atrial natriuretic peptide in thymic macrophages after dexamethasone treatment of rats.

The rat thymus represents a site of synthesis of atrial natriuretic peptide (ANP); the immunosuppressor dexamethasone strikingly increases ANP-expression in this immune organ. The presented data suggest that this increase can be attributed to macrophages. By means of immunohistochemistry and Northern blot analysis these immune cells were found to express ANP-immunoreactivity as well as mRNA coding for ANP. In contrast, macrophages of control thymi displayed only weak ANP-immunoreactivity. Thus, ANP appears to be a constituent of rat thymic macrophages, and its synthesis in the thymus is strongly elevated by acute exposure of the animals to glucocorticoids.

Animals↗