Biomedical subjects

A M Vollmar

Publications and source records attributed to A M Vollmar.

At least 19 recordsLinked to original sources

PubMed2002

Ajoene, an experimental anti-leukemic drug: mechanism of cell death.

The organosulfur compound ajoene, a constitutent of garlic, has been shown to induce apoptosis in a leukemic cell line as well as in blood cells of a leukemic patient. The mechanisms of action of ajoene, however, are unknown. The present study aims to characterize the molecular events leading to ajoene-triggered apoptosis. We show here that ajoene (20 microM) leads to a time-dependent activation of caspase-3-like activity as well as to the proteolytic processing of procaspase-3 and -8. Activation of caspases was necessary for ajoene-induced apoptosis since the broad-range caspase inhibitor zVAD-fmk completely abrogated ajoene-mediated DNA fragmentation. Although the initiator caspase-8 was activated, the CD95 death receptor was not involved in death signaling since the HL-60 clone used was shown to express a functionally inactive CD95 receptor. Furthermore, ajoene induced the release of cytochrome c, which was not inhibited by zVAD-fmk indicating that cytochrome c release precedes caspase activation. Ajoene also led to a dissipation of the mitochondrial transmembrane potential. Overexpression of Bcl-x(L) clearly diminished ajoene-induced caspase activation as well as apoptosis. These results indicate that apoptosis in leukemia cells triggered by ajoene is based on the activation of a mitochondria-dependent caspase cascade which includes also the activation of the initiator caspase-8.

Amino Acid Chloromethyl Ketones↗

PubMed2001

Reliable in vitro measurement of nitric oxide released from endothelial cells using low concentrations of the fluorescent probe 4,5-diaminofluorescein.

4,5-Diaminofluorescein (DAF-2) and its membrane-permeable derivate DAF-2 diacetate are fluorescent probes that have been developed to perform real-time biological detection of nitric oxide (NO). Their use for intracellular imaging, however, has recently been seriously questioned and data using DAF-2 for extracellular NO detection at low levels, as for example released from endothelial cells, are rare. Here we show that a reliable detection of low levels of NO in biological systems by DAF-2 is possible (a) by using low DAF-2 concentrations (0.1 microM) and (b) by subtracting the DAF-2 auto-fluorescence from the measured total fluorescence. The described method allows easy real-time detection of endothelial NO formation.

Cell Line↗

PubMed2001

Helenalin triggers a CD95 death receptor-independent apoptosis that is not affected by overexpression of Bcl-x(L) or Bcl-2.

Apoptosis is required for proper tissue homeostasis. Defects in apoptosis signaling pathways, thus, contribute to carcinogenesis and chemoresistance. A major goal in chemotherapy is, therefore, to find cytotoxic agents that restore the ability of tumor cells to undergo apoptosis. We show here that the sesquiterpene lactone helenalin (10-50 microM) induces apoptosis in leukemia Jurkat T cells even if they lack the CD95 death receptor or overexpress the antiapoptotic proteins Bcl-x(L) or Bcl-2. Activated peripheral blood mononuclear cells, however, are not affected (10-50 microM helenalin). Helenalin led to a time-dependent (0-24 h) cleavage of the specific caspase-3-like substrate Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin as well as to the proteolytic processing of procaspase-3 and -8. Caspase activation was a necessary requirement for apoptosis because the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk, 50 microM) completely abrogated helenalin-induced DNA fragmentation as well as phosphatidylserin translocation. Although the initiator caspase-8 was activated, the helenalin-induced signaling pathway did not require the CD95 death receptor as shown using cells without or with an antibody (ZB4)-blocked CD95 receptor. Helenalin also did not induce CD95 or CD95-ligand expression. On the other hand, helenalin was found to induce the release of cytochrome c from mitochondria that was not inhibited by the caspase inhibitor zVAD-fmk, which indicated that cytochrome c release precedes caspase activation. Cytochrome c release was accompanied by dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), which was partly inhibited by zVAD-fmk, which suggests that caspases are involved in loss of DeltaPsi(m). Most importantly, overexpression of the mitochondria protecting proteins Bcl-x(L) or Bcl-2 failed to confer resistance to helenalin-induced apoptosis, although the data presented here suggest that helenalin induces a mitochondria-dependent pathway. Thus, helenalin is a promising experimental cytotoxic agent that possibly points to new strategies to overcome apoptosis resistance attributable to overexpression of antiapoptotic Bcl-2 proteins.

Antineoplastic Agents, Phytogenic↗

PubMed2001

Ajoene, a natural product with non-steroidal anti-inflammatory drug (NSAID)-like properties?

The inducible isoform of cyclooxygenase (COX-2) is implicated in the pathogenesis of various inflammatory diseases as well as in carcinogenesis, especially of gastrointestinal tumors. Epidemiological as well as experimental data support a role for constituents of allium vegetables, such as garlic and onions, in the prevention of gastrointestinal cancer. Therefore, the aim of the present study was to examine whether the garlic-derived natural product ajoene interferes with the COX-2 pathway by using lipopolysaccharide (LPS)-activated RAW 264.7 cells as in vitro model. Ajoene was shown to dose-dependently inhibit the release of LPS (1 microg/mL)-induced prostaglandin E(2) in RAW 264.7 macrophages (IC(50) value: 2.4 microM). This effect was found to be due to an inhibition of COX-2 enzyme activity by ajoene (IC(50) value: 3.4 microM). Ajoene did not reduce COX-2 expression, but rather increased LPS-induced COX-2 protein and mRNA expression compared to LPS-stimulated cells only. In the absence of LPS, however, ajoene was unable to induce COX-2. The non-steroidal anti-inflammatory drug indomethacin was shown to act similarly in LPS-activated RAW 264.7 cells. These data suggest that ajoene works by a mechanism of action similar to that attributed to non-steroidal anti-inflammatory drugs. This finding may add a novel aspect to the biological profile of the garlic-derived natural product ajoene which might be important for understanding the usefulness of garlic for chemoprevention of gastrointestinal carcinomas.

Animals↗

PubMed2001

Elevation of intracellular calcium levels contributes to the inhibition of nitric oxide production by atrial natriuretic peptide.

Atrial natriuretic peptide (ANP) attenuates LPS-induced inducible nitric oxide synthase (iNOS) expression in murine macrophages by destabilizing iNOS mRNA. Because elevated intracellular free Ca2+ levels [Ca2+]i reduce iNOS mRNA stability, the aim of the present study was to determine whether inhibition of iNOS by ANP is due to alterations in intracellular calcium. As determined by fluorescence photometry, ANP (10(-7) and 10(-6) mol/L) was shown to elevate intracellular calcium levels in bone marrow-derived macrophages. This effect seemed to be mediated via the guanylate cyclase-coupled A receptor, because dibutyryl-cGMP mimicked and the A-receptor antagonist HS-142-1 partially abrogated the effect of ANP. Because the Ca2+ increase was also observed in Ca2+-free buffer, it is suggested that the liberation of intracellular calcium pools contributes to the elevation of [Ca2+]i by ANP. The B-receptor ligand C-type natriuretic peptide (CNP), which does not alter iNOS expression, had no effect on [Ca2+]i. The Ca2+-ionophore 4-Br-A23187 and thapsigargin, a compound known to liberate Ca2+ from intracellular stores, were further demonstrated to reduce LPS-induced NO production in macrophages (Griess assay), confirming a functional link for elevated [Ca2+]i and iNOS inhibition. These effects were abrogated by coincubation with extra- as well as intracellular Ca2+ chelators (EGTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)). The inhibitory effect of ANP on NO production was also abrogated by Ca2+ chelation. These findings support a causal relationship between reduced iNOS induction and elevation of [Ca2+]i. Taken together, the data indicate that intracellular Ca2+ elevation by ANP is involved in the inhibition of LPS-induced nitric oxide production in macrophages.

Aniline Compounds↗

PubMed2001

Low-molecular-weight hyaluronic acid induces nuclear factor-kappaB-dependent resistance against tumor necrosis factor alpha-mediated liver injury in mice.

Liver resident NK1.1+ T cells are supposed to play a pivotal role in the onset of inflammatory liver injury in experimental mouse models such as concanavalin A (Con A)-induced hepatitis. These cells, expressing the adhesion receptor, CD44, are largely depleted from the liver by a single intravenous injection of low-molecular-weight fragments of hyaluronic acid (LMW-HA). Here, we report that LMW-HA pretreatment protected mice from liver injury in several models of T-cell- and macrophage-dependent, tumor necrosis factor alpha (TNF-alpha)-mediated inflammatory liver injury, i.e., from liver injury induced by either Con A or Pseudomonas exotoxin A (PEA) or PEA/lipopolysaccharide (LPS). Interestingly, apart from inhibition of cellular adhesion, pretreatment of mice with LMW-HA was also capable of preventing hepatocellular apoptosis and activation of caspase-3 induced by direct administration of recombinant murine (rmu) TNF-alpha to D-galactosamine (GalN)-sensitized mice. LMW-HA-induced hepatoprotection could be neutralized by pretreatment with the nuclear factor-kappaB (NF-kappaB) inhibitor, pyrrolidine dithiocarbamate (PDTC), demonstrating the involvement of NF-kappaB in the observed protective mechanism. Indeed, injection of LMW-HA rapidly induced the production of TNF-alpha by Kupffer cells and the translocation of NF-kappaB into hepatocellular nuclei. Both LMW-HA-induced TNF-alpha production and NF-kappaB translocation were blocked by pretreatment with PDTC. Our findings provide evidence for an unknown mechanism of LMW-HA-dependent protection from inflammatory liver disease, i.e., induction of TNF-alpha- and NF-kappaB-dependent cytoprotective proteins within the target parenchymal liver cells.

ADP Ribose Transferases↗

PubMed2001

Cytotoxic sesquiterpene lactones mediate their death-inducing effect in leukemia T cells by triggering apoptosis.

Cytotoxicity is a well characterized property of sesquiterpene lactones. In the present study, the question was addressed whether sesquiterpene lactones mediate their cytotoxic effect by triggering apoptosis. Four compounds, ambrosin, alantolactone, hymenin and helenalin were shown to induce apoptosis in Jurkat leukemia T cells as judged by cell morphology, the appearance of apoptotic nuclei as well as the translocation of phosphatidylserine to the outer surface of the cell membrane.

Antineoplastic Agents, Phytogenic↗

PubMed2001

The atrial natriuretic peptide regulates the production of inflammatory mediators in macrophages.

The atrial natriuretic peptide (ANP), a member of the natriuretic peptide family, is a cardiovascular hormone which possesses well defined natriuretic, diuretic, and vasodilating properties. Most of the biological effects of ANP aremediated through its guanylyl cyclase coupled A receptor. Because ANP and its receptors have been shown to be expressed and differentially regulated in the immune system, it has been suggested that ANP has an immunomodulatory potency. Much investigation of the effects of ANP on the activation of macrophages has been carried out. ANP was shown to inhibit the lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS) in macrophages in an autocrine fashion. ANP in this context was shown to reduce significantly the activation of NF-kappaB and to destabilise iNOS mRNA. ANP, furthermore, can significantly reduce the LPS-induced secretion of tumour necrosis factor alpha (TNFalpha) in macrophages. The relevance of these findings on a regulatory role for ANP on TNFalpha in humans was shown by the fact that ANP significantly reduces the release of TNFalpha in whole human blood. It was furthermore shown to attenuate the release of interleukin 1beta (IL1beta). Interestingly, ANP did not affect the secretion of the anti-inflammatory cytokines IL10 and IL1 receptor antagonist (IL1ra). In summary, ANP was shown to reduce the secretion of inflammatory mediators in macrophages. Therefore, this cardiovascular hormone may possess anti-inflammatory potential.

Atrial Natriuretic Factor↗

PubMed2001

Induction of L-arginine transport is inhibited by atrial natriuretic peptide: a peptide hormone as a novel regulator of inducible nitric-oxide synthase substrate availability.

BACKGROUND: The inducible nitric-oxide synthase (iNOS) synthesizes NO from L-arginine. Availability of L-arginine is maintained by a lipopolysaccharide (LPS)-induced induction of the CAT-2B amino acids transporter. Recently, we could show that the cardiovascular hormone atrial natriuretic peptide (ANP) inhibits the induction of iNOS in LPS-stimulated macrophages via its guanylate cyclase-coupled A-receptor. PURPOSE: To investigate whether ANP exerts an effect on LPS-induced L-arginine uptake. METHODS: Murine bone marrow derived macrophages were activated with LPS (1 microg/ml, 20 h) in the presence or absence of ANP or C-type natriuretic peptide (CNP). L-Arginine transport was determined by measuring the uptake of L-[(3)H]arginine. L-[(3)H]Arginine influx was also determined in LPS-activated cells in the presence of N(G)-monomethyl-L-arginine (L-NMMA), competitor amino acids, or ANP. Nitrite accumulation was determined in supernatants of LPS-activated cells cultured in the presence or absence of L-ornithine. RESULTS: ANP dose dependently (10(-8)-10(-6)M) inhibited LPS-induced L-[(3)H]arginine uptake when added simultaneously with LPS, whereas it showed no effect when added simultaneously with L-[(3)H]arginine. The effect was abrogated by the A-receptor antagonist HS-142-1 (10 microg/ml). CNP (10(-6) M) did not influence L-arginine transport. Competitor amino acids (10(-2) M) inhibited L-[(3)H]arginine uptake. An excess of unlabeled L-arginine (10(-2) M) as well as its analog L-NMMA (10(-3) M) also reduced L-[(3)H]arginine influx. L-Arginine uptake was critical for production of NO because L-ornithine significantly decreased LPS-induced nitrite accumulation. CONCLUSION: This work demonstrates that ANP inhibits LPS-induced L-arginine uptake via its guanylate cyclase-coupled A-receptor. Besides its influence on the induction of iNOS, this effect may represent an important and unique mechanism by which ANP regulates NO production in macrophages.

Animals↗

PubMed2001

Immunomodulatory and cytoprotective function of atrial natriuretic peptide.

The atrial natriuretic peptide (ANP) was described as a peptide hormone synthesized and secreted by heart atria. It plays an important role in the regulation of volume homeostasis; however, the functions of ANP are not restricted to cardiovascular effects. The biological profile of ANP is much broader than originally thought. This article focuses on the immunomodulatory and anti-inflammatory functions of ANP addressing; for example, the influence of ANP on macrophage functions. Another important aspect of ANP reviewed here is its cytoprotective potential. The beneficial effect of ANP in preventing cell damage caused by ischemia and reperfusion warrants special attention. The therapeutic potential of ANP in organ preservation could be important for transplantation medicine.

Adjuvants, Immunologic↗

PubMed2000

cGMP-mediated inhibition of TNF-alpha production by the atrial natriuretic peptide in murine macrophages.

The atrial natriuretic peptide (ANP) is suggested to regulate inflammatory response by alteration of macrophage functions. The aim of this study was to investigate whether ANP influences production of TNF-alpha. TNF-alpha production in murine bone marrow-derived macrophages was induced by LPS, and TNF-alpha secretion (+/-ANP) was determined by L929 bioassay. ANP dose dependently (10-8-10-6 M) inhibited TNF-alpha release by up to 95%. The effect was mediated via the guanylate cyclase-coupled A receptor, as was shown by employing dibutyryl-cGMP, the cGMP-inhibitory compound Ly-83583, and the A receptor antagonist HS-142-1. A specific ligand of the natriuretic peptide "clearance" receptor inhibited TNF-alpha production only at 10-7 and 10-8 M, but not at 10-6 M. The B receptor ligand C-type natriuretic peptide showed no TNF-alpha-inhibitory effect. To investigate the underlying mechanism of ANP-mediated TNF-alpha inhibition, Northern blot was performed. ANP-treated macrophages displayed decreased TNF-alpha-mRNA levels. Besides the known inhibition of NF-kappaB activation, in this study we demonstrated that ANP also attenuates the activation of the proinflammatory transcription factor AP-1 (gel shift assay). ANP did not alter subunit composition of AP-1 complexes, as was shown by supershift assays applying anti-c-jun and anti-c-fos Abs. To get information on the ANP effect for human inflammatory processes, we investigated cytokine production in human LPS-activated blood. ANP significantly attenuated production of TNF-alpha and IL-1beta without affecting production of IL-10 and IL-1ra. In summary, ANP was shown to attenuate TNF-alpha production of LPS-activated macrophages via cGMP. The inhibition is suggested to involve transcriptional processes that are the result of reduced activation of responsible transcription factors.

Animals↗

PubMed2000

Atrial natriuretic peptide reduces expression of TNF-alpha mRNA during reperfusion of the rat liver upon decreased activation of NF-kappaB and AP-1.

BACKGROUND/AIMS: The cardiovascular hormone Atrial Natriuretic Peptide (ANP) attenuates activation of the pro-inflammatory transcription factor NF-kappaB in macrophages. ANP was also shown to protect from ischemia-reperfusion injury of the rat liver. This study aimed to investigate the effects of this immunomodulatory hormone and its second messenger cGMP on the activation of the two redox-sensitive transcription factors AP-1 and NF-kappaB and the expression of corresponding pro-inflammatory target genes during ischemia and reperfusion of the liver. The identification of the mechanisms underlying the protection by ANP should reveal new aspects concerning the pathomechanisms of ischemia/reperfusion injury. METHODS: Rat livers were perfused with and without ANP or 8-Br-cGMP preceding 24 h of cold storage in University of Wisconsin solution. During reperfusion NF-kappaB and AP-1 DNA binding activities were determined in freeze-clamped liver samples by electrophoretic mobility shift assay. Protein levels of p50, p65, and of IkappaB were determined by Western blot. mRNA coding for inducible nitric oxide synthase, cyclooxygenase-2, and TNF-alpha was determined by RT-PCR and Northern blot. RESULTS: After 45 min of reperfusion DNA binding activities of NF-kappaB were increased, whereas in ANP pre-treated livers this effect was markedly reduced. AP-1, another important redox-sensitive transcription factor, was activated and in the course of reperfusion the subunit composition of AP-1 changed as assessed by supershift assays. ANP markedly reduced binding activities of both forms of AP-1. 8-Br-cGMP mimicked the effects of ANP on NF-kappaB and AP-1. Neither inducible nitric oxide synthase nor cyclooxygenase-2 mRNA could be detected. In contrast, a profound expression of transcripts coding for TNF-alpha was detected in the course of reperfusion and ANP markedly reduced TNF-alpha mRNA expression. CONCLUSION: ANP seems to mediate its protective effect during ischemia and reperfusion by reducing the activation of NF-kappaB and AP-1 via cGMP. The reduced binding activity of these redox-sensitive transcription factors was accompanied by a diminished mRNA expression of TNF-alpha, a cytokine known to be involved in cellular damage in ischemia reperfusion injury.

Animals↗

PubMed2000

Structural requirements of sesquiterpene lactones to inhibit LPS-induced nitric oxide synthesis in RAW 264.7 macrophages.

Some sesquiterpene lactones were recently demonstrated to inhibit inducible nitric oxide synthase (iNOS)-dependent nitric oxide (NO) synthesis. The primary objective of the present study was, therefore, to find evidence for structural requirements of sesquiterpene lactones regarding their capability to inhibit iNOS-dependent NO synthesis. Sesquiterpene lactones 1-11 were examined for their influence on nitrite accumulation in cell culture supernatants of LPS-induced RAW 264.7 macrophages. Except the taraxinic acid beta-D-glucopyranosylester 8 all compounds showed a dose-dependent inhibition of nitrite accumulation in cell culture supernatants with IC50 values ranging from 0.5 to 36.8 microM. High activity seemed to be dependent on an alpha-methylene-gammalactone functionality. Cytotoxicity and the ability to inhibit activation of transcription factor NF-kappaB are further biological activities of sesquiterpene lactones. The second point of interest was, therefore, whether the structural requirements of sesquiterpene lactones for these activities may differ or be the same for those needed to inhibit iNOS-dependent NO synthesis. Using concentrations of 1-11 required to inhibit NO synthesis cell viability was determined and NF-kappaB binding activity was measured by gel-shift experiments. Interestingly, compounds almost equally effective in inhibiting nitrite accumulation did not show the same cytotoxic potential, and most sesquiterpene lactones inhibited nitrite accumulation at concentrations where inhibition of NF-kappaB activation was not significant. These results suggest that different biological activities of sesquiterpene lactones have different structural requirements.

Animals↗

PubMed1999

Anti-inflammatory activities of hypocretenolides from Leontodon hispidus.

Hypocretenolides, a small group of sesquiterpene lactones with an unusual ring structure, are constituents of a small number of species from the Lactuceae tribe (Asteraceae). Three biogenetically closely related 14-hypocretenolides from Leontodon hispidus L. were investigated for a putative anti-inflammatory activity. 14-Hydroxyhypocretenolide-beta-D-glucoside-4'-14"-hydroxyhypocr etenoate significantly exhibited in vivo anti-inflammatory activity in the croton oil-induced mouse ear edema. To obtain first information regarding the molecular targets which might be affected by this constituent, two in vitro bioassays were performed: (i) DNA binding activity of the transcription factor NF-kappa B was evaluated by electrophoretic mobility shift assay (EMSA) using TNF-alpha-activated Jurkat T cells and (ii) nitrite accumulation in cell culture supernatants of LPS-activated RAW 264.7 macrophages was determined as a parameter for inducible nitric oxide synthase (iNOS)-dependent nitric oxide release. In order to gain information about structure-activity relationships, additionally the aglycone 14-hydroxyhypocretenolide and its D-glycoside were investigated in these in vitro systems. 14-Hydroxyhypocretenolide-beta-D-glucoside-4'-14"-hydroxyhypocr etenoate as well as its aglycone exhibited activity in both test systems, whereas the D-glucoside was not or only weakly active.

Animals↗

PubMed1999

Prevention of Kupffer cell-induced oxidant injury in rat liver by atrial natriuretic peptide.

The generation of reactive oxygen species (ROS) by activated Kupffer cells contributes to liver injury following liver preservation, shock, or endotoxemia. Pharmacological interventions to protect liver cells against this inflammatory response of Kupffer cells have not yet been established. Atrial natriuretic peptide (ANP) protects the liver against ischemia-reperfusion injury, suggesting a possible modulation of Kupffer cell-mediated cytotoxicity. Therefore, we investigated the mechanism of cytoprotection by ANP during Kupffer cell activation in perfused rat livers of male Sprague-Dawley rats. Activation of Kupffer cells by zymosan (150 microgram/ml) resulted in considerable cell damage, as assessed by the sinusoidal release of lactate dehydrogenase and purine nucleoside phosphorylase. Cell damage was almost completely prevented by superoxide dismutase (50 U/ml) and catalase (150 U/ml), indicating ROS-related liver injury. ANP (200 nM) reduced Kupffer cell-induced injury via the guanylyl cyclase-coupled A receptor (GCA receptor) and cGMP: mRNA expression of the GCA receptor was found in hepatocytes, endothelial cells, and Kupffer cells, and the cGMP analog 8-bromo-cGMP (8-BrcGMP; 50 microM) was as potent as ANP in protecting from zymosan-induced cell damage. ANP and 8-BrcGMP significantly attenuated the prolonged increase of hepatic vascular resistance when Kupffer cell activation occurred. Furthermore, both compounds reduced oxidative cell damage following infusion of H2O2 (500 microM). In contrast, superoxide anion formation of isolated Kupffer cells was not affected by ANP and only moderately reduced by 8-BrcGMP. In conclusion, ANP protects the liver against Kupffer cell-related oxidant stress. This hormonal protection is mediated via the GCA receptor and cGMP, suggesting that the cGMP receptor plays a critical role in controlling oxidative cell damage. Thus ANP signaling should be considered as a new pharmacological target for protecting liver cells against the inflammatory response of activated Kupffer cells without eliminating the vital host defense function of these cells.

Animals↗

PubMed1998

Effect of chronic GH overproduction on cardiac ANP expression and circulating ANP levels.

Sodium and water retention are common in acromegaly and upon GH administration. The underlying mechanisms, however, have not been clearly characterized as yet. Therefore, the aim of this study was to examine possible alterations of atrial natriuretic peptide (ANP), an endogenous regulator of volume homeostasis, in response to chronic elevated GH. We used GH-transgenic mice (GH-TM) as a model for chronic hypersomatotropinemia and moreover investigated 7 and 27 week old animals, respectively, in order to discriminate between short and long term effects of GH overexpression. Hematocrit values were reduced in GH-TM compared to control animals and it is known that plasma volume is increased in these animals. Structural lesions of the kidney were found in the GH-TM, however, in the animals studied there were no signs of renal insufficiency as evidenced by serum creatinine and urea levels. The serum concentration of immunoreactive ANP (IR-ANP) determined by RIA was significantly (P < 0.005) elevated in the young GH-TM as compared to control littermates (81.7+/-13.3 vs. 50.9+/-10.8 fmol/ml). The increase in serum IR-ANP of 27 week old GH-TM, however did not reach the level of significance (57.13+/-16.3 vs. 50.25+/-16.4 fmol/ml). Serum samples of control mice as well as of the 7 week old animals mainly contained ANP 99-126, known to be the circulating form of ANP. In contrast, serum of 27 week old GH-TM predominantly showed the cardiac storage form of ANP, ANP 1-126. Cardiac expression of ANP was quantified by Northern blot analysis. mRNA coding for ANP was found 1.2- and 2-fold increased in the atria of 7 and 27 week old GH-TM, respectively. In parallel, a 2.2-fold (7 week) and 2-fold (27 week) increase of IR-ANP was observed in transgenic atria compared to tissue of control animals. In contrast, no significant difference of ANP mRNA expression or of content of IR-ANP was observed in the ventricles of both groups of animals. In conclusion, GH-TM show various alterations in their ANP status suggesting an influence of the peptide on the effect of GH in fluid retention.

Aging↗

PubMed1998

Autocrine regulation of inducible nitric-oxide synthase in macrophages by atrial natriuretic peptide.

Atrial natriuretic peptide (ANP), a cardiovascular hormone, has been shown to inhibit synthesis of nitric oxide in lipopolysaccharide (LPS)-activated mouse bone marrow-derived macrophages via activation of its guanylate cyclase-coupled receptor. The goal of the present study was to elucidate the potential sites of inducible nitric-oxide synthase (iNOS) regulation affected by ANP and revealed the following. 1) ANP and dibutyryl-cGMP did not inhibit catalytic iNOS activity measured by the conversion rate of L-[3H]arginine to L-[3H]citrulline in homogenates of LPS-treated cells. 2) Pretreatment of cells with ANP dose-dependently reduced the LPS-induced L-[3H]citrulline production that has been shown to be due to reduced iNOS protein levels detected by Western blot. 3) ANP does not alter the ratio of catalytically active iNOS dimer versus inactive iNOS monomer considered to be a major post-translational regulatory mechanism for the enzyme. 4) Macrophages exposed to ANP display decreased LPS-induced iNOS mRNA levels. 5) Importantly, two basic mechanisms seem to be responsible for this observation, i.e. ANP specifically induced acceleration of iNOS mRNA decay and ANP reduced binding activity of NF-kappaB, the transcription factor predominantly responsible for LPS-induced iNOS expression in murine macrophages. Moreover, 6) ANP acts via an autocrine mechanism since recently ANP was shown to be secreted by LPS-activated macrophages, and we demonstrated here that LPS-induced NO synthesis was increased after blocking the binding of endogenous ANP by a receptor antagonist. These observations suggest ANP as a new autocrine macrophage factor regulating NO synthesis both transcriptionally and post-transcriptionally. ANP may help to balance NO production of activated macrophages and thus may allow successful immune response without adverse effects on host cells.

Animals↗

PubMed1998

The guanylate cyclase-coupled natriuretic peptide receptor: a new target for prevention of cold ischemia-reperfusion damage of the rat liver.

The aim of our studies was to investigate hormonal prevention of hepatic preservation damage by the atrial natriuretic peptide (ANP) and the mechanisms involved. Isolated perfusion of rat livers was performed in a nonrecirculating fashion. Twenty minutes of preischemic perfusion was performed with or without different concentrations of ANP, followed by 24-hour storage in cold University of Wisconsin (UW) solution. Two hundred nanomoles of ANP prevented hepatocellular damage during a 2-hour reperfusion period as indicated by a marked attenuation of the sinusoidal efflux of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP), and by reduced Trypan blue uptake. Furthermore, postischemic bile flow as an indicator of liver function was significantly improved by about 60% with 200 nmol/L ANP. No protection was conveyed by 20 nmol/L ANP nor by pretreatment with 200 nmol/L ANP for only 10 minutes. The effects of ANP seemed to be mediated by the guanylate cyclase-coupled A (GC-A) receptor and cyclic guanosine monophosphate (cGMP): whereas expression of both GC-A and GC-B receptors as well as of the GC-C receptor was found, cGMP did protect from ischemia-reperfusion damage, but selective ligands of the B and C receptor did not. To begin to determine the mechanisms of ANP-mediated protection, different parameters were investigated: ANP had no effect on portal pressure as an indicator of hepatic circulation, nor on intracellular energy depletion determined by adenosine nucleotide concentration. However, the marked augmentation of nuclear factor kappaB (NF-kappaB) binding activity during reperfusion was prevented in ANP-pretreated livers. In conclusion, pretreatment with ANP protects the rat liver from cold ischemia-reperfusion damage. This effect is mediated via the GC-A receptor and cGMP, and may be linked to an influence of ANP on NF-kappaB activation. Thus, ANP signaling via the GC-A receptor should be considered as a new pharmacological target to prevent preservation injury of the liver.

Animals↗