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M Hecker

Publications and source records attributed to M Hecker.

At least 163 records · Page 9Linked to original sources
PubMed1993

Enzymic and nonenzymic release of NO accounts for the vasodilator activity of the metabolites of CAS 936, a novel long-acting sydnonimine derivative.

The molecular mechanism(s) underlying the vasodilator activity of CAS 936 (3-(cis-2,6-dimethylpiperidino)-N-(4-methoxybenzoyl)-sydn oni mine) and its metabolites 3-(cis-2,6-dimethylpiperidino)-sydnonimine (C87 3754) and N-(cis-2,6-dimethylpiperidino)-N-nitroso-2-aminoacetonitrile (C873786) was investigated. These compounds were tested for their relaxant activity in isolated rabbit arterial segments, activation of purified soluble guanylyl cyclase and release of nitric oxide (NO) in vitro and in vivo. C873754 and C873786 inhibited the noradrenaline-induced contraction and increased the cyclic GMP content of endothelium-denuded rabbit aortic and femoral segments, whereas CAS 936 was without effect. Similarly, both metabolites, but not CAS 936, activated purified soluble guanylyl cyclase (EC50 about 30 microM) and released NO in buffered aqueous solutions, as detected by electron spin resonance (esr) spectrometry. Both in vitro and in vivo an accumulation of NO was detected by esr spectrometry in vascular tissues exposed to the metabolites of CAS 936, whereas a significant release of NO from CAS 936 was only detected in the isolated rabbit liver, but not in vascular tissue. It is conceivable, therefore, that the metabolites of CAS 936 appearing in the systemic circulation after hepatic biotransformation induce vasodilatation by release of NO and activation of soluble guanylyl cyclase in vascular smooth muscle. Moreover, the activation of soluble guanylyl cyclase in vitro by the metabolites of CAS 936 was significantly enhanced by co-incubation with certain particulate fractions from bovine aortic endothelial and smooth muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Catabolite repression of beta-glucanase synthesis in Bacillus subtilis.

beta-Glucanase synthesis in Bacillus subtilis was repressed by glucose and other substrates of glycolysis. Experiments with different pts mutants showed that the phosphoenolpyruvate: sugar phosphotransferase system is not involved in carbon catabolite repression of beta-glucanase synthesis. Carbon catabolite repression of beta-glucanase synthesis was completely abolished in a ccpA mutant. An operator structure similar to those upstream of amyE and the xyl operon was found and was shown by site-directed mutagenesis to be the target for carbon catabolite repression of beta-glucanase synthesis. The presence of this operator on a multi-copy plasmid resulted in a reduced repression of both beta-glucanase and alpha-amylase synthesis. It seems likely that the gene encoding these enzymes are part of one regulon with respect to catabolite repression.

Bacillus subtilis↗
PubMed1993

Selective inhibition of agonist-induced but not shear stress-dependent release of endothelial autacoids by thapsigargin.

1. The effects of the Ca(2+)-ATPase inhibitor, thapsigargin, on the shear stress-dependent and on the agonist-stimulated release of endothelium-derived relaxing factor, i.e. nitric oxide (NO), and prostacyclin (PGI2) were studied in bovine and human cultured endothelial cells as well as in endothelium-intact arterial segments of the rabbit. 2. Preincubation with thapsigargin (1 microM for 10 min) had no effect on the shear stress-dependent release of NO from bovine aortic endothelial cells grown on beads, but abolished the release of NO induced by ADP, bradykinin, ionomycin or poly-L-lysine. Similarly, thapsigargin completely abrogated the agonist-stimulated PGI2 release from these cells, but had no effect on the shear stress-dependent release of PGI2. 3. The acetylcholine-induced release of NO from the luminally perfused thoracic aorta and femoral artery of the rabbit was suppressed by pretreatment with thapsigargin (1 microM). In contrast, thapsigargin did not affect the shear stress-dependent release of NO from the femoral artery. 4. Administration of thapsigargin to these vascular preparations or to cultured endothelial cells alone produced a substantial release of both NO and PGI2. This release declined towards previous values after washout of thapsigargin. 5. In human and bovine cultured endothelial cells, thapsigargin (1-1000 nM) caused a dose-dependent sustained rise in [Ca2+]i, an effect that was abolished in the absence of extracellular Ca2+. Stimulation of these cells with bradykinin, histamine, ADP or ionomycin after previous exposure to thapsigargin (30-1000 nM) no longer caused an increase in [Ca2+]i. of the release of these endothelial autacoids caused by shear stress or receptor-dependent and independent agonists.

Adenosine Diphosphate↗
PubMed1993

Vasoconstriction and increased flow: two principal mechanisms of shear stress-dependent endothelial autacoid release.

The mechanisms of which nitric oxide (NO) and prostacyclin (PGI2) are released from endothelium-intact rabbit femoral arteries under resting conditions and after stimulation by either shear stress or acetylcholine (ACh) were investigated. The concentration of NO in the effluate was determined by monitoring the NO-mediated stimulation of purified soluble guanylyl cyclase, and that of PGI2 was done using a specific radioimmunoassay for its stable hydrolysis product, 6-ketoprostaglandin F1 alpha, NO release under static (no-flow) conditions and in the absence of a stimulus accounted for 10-15% of the maximum release of NO from luminally perfused segments stimulated with ACh and was attenuated by removal of extracellular Ca2+. A six- to sevenfold increase in shear stress (from 0.15 to 1 dyn/cm2), generated either by vasoconstriction at constant flow or by an increase in flow at constant diameter, elicited a five- to sevenfold increase in NO release, which was correlated with increasing shear stress. The same increase in shear stress also enhanced the release of PGI2 from the femoral artery segments by 11- to 12-fold. Removal of extracellular Ca2+ abolished the shear stress-dependent PGI2 released but did not affect that of NO. In contrast, the ACh-stimulated NO release was strongly inhibited in the absence of extracellular Ca2+ (78% inhibition). Charybdotoxin, an inhibitor of Ca(2+)-activated K+ channels, and glibenclamide, an inhibitor of the ATP-sensitive K+ channel, had no effect on the shear stress-dependent release of NO.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1993

Endothelium-derived bradykinin: implications for angiotensin-converting enzyme-inhibitor therapy.

The effects of angiotensin-converting enzyme (ACE) inhibitors on endothelial autacoid formation were determined in human cultured endothelial cells and in endothelium-intact bovine coronary arteries under resting conditions and after stimulation with bradykinin. Incubation of cultured human endothelial cells with moexiprilat or ramiprilat (0.3 microM) caused a maintained increase in resting intracellular calcium [Ca2+]i, which was prevented by the selective B2-receptor antagonist Hoe 140 (0.1 microM). Both ACE inhibitors also significantly enhanced the increase in [Ca2+]i elicited by bradykinin (3 nM). In parallel with their effect on resting [Ca2+]i, moexiprilat and ramiprilat both induced an increase in intracellular cyclic GMP (cGMP). This increase was prevented by Hoe 140 (0.1 microM) and was abolished by NG-nitro-L-arginine (30 microM), indicating a kinin-induced nitric oxide (NO) formation in this response. The elevation in [Ca2+]i also led to an enhanced production of prostacyclin (PGI2), as indicated by an increase in the concentration of 6-keto prostaglandin F1 alpha (PGF1 alpha) in the cell supernatant. Similar effects of the ACE inhibitors on endothelial autacoid production were observed in endothelium-intact bovine coronary arteries. Like bradykinin (30 nM), moexiprilat (0.3 microM) elicited a nearly twofold increase in the cGMP content of these arteries, which was abolished by both NG-nitro-L-arginine and removal of the endothelium. The functional consequences of this ACE inhibitor-induced increase in vascular cGMP were reflected by a distinct relaxation of arteries preconstricted with PGF2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

6-Ketoprostaglandin F1 alpha↗
PubMed1993

Angiotensin-converting enzyme inhibitors unmask endogenous kinin production by bovine coronary artery endothelium.

The angiotensin converting enzyme (ACE) inhibitors, moexiprilat and ramiprilat, relaxed preconstricted endothelium-intact bovine coronary artery rings and enhanced the relaxant response to bradykinin. The relaxation was observed in the presence of a cyclooxygenase inhibitor and without previous exposure to bradykinin. ACE inhibitor-dependent relaxation was attenuated by the selective B2-kinin receptor antagonist, Hoe 140, and completely abolished by removal of the endothelium. Bradykinin or moexiprilat also significantly increased the cyclic guanosine monophosphate (cGMP) content of these coronary segments, an effect which was abolished by the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine (NNA), or by removal of the endothelium. NNA also diminished the relaxant response to moexiprilat, but only partially inhibited that to bradykinin, suggesting that the ACE inhibitor-induced relaxation was predominantly mediated by endothelial NO release, whereas bradykinin acted in part by another endothelium-dependent mechanism. These findings indicate that ACE inhibitors can elicit endothelium-dependent relaxations presumably by facilitating the accumulation of endothelium-derived kinins in or at the vessel wall. This local mechanism may significantly contribute to the antihypertensive action of these compounds in vivo.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1993

Signal transduction in endothelium-dependent vasodilatation.

Modulation of vascular tone is one important function of the endothelium. This can occur via two principal mechanisms: by modulating the local concentration of circulating vasoactive substances (e.g. adenine nucleotides, angiotensin II, biogenic amines, bradykinin), and by synthesizing and releasing vasoactive autacoids. The most important endothelium-derived vasodilator autacoids are nitric oxide (NO) and prostacyclin (PGI2). By counteracting neuro- and myogenic vasoconstriction, the continuous release of these autacoids from the vascular endothelium represents a sensitive and highly effective local system for maintaining an adequate blood flow to the organs. Impaired production of NO (and PGI2), either as a result of endothelial injury or dysfunction, has been implicated in the pathology of a variety of cardiovascular diseases, such as hypertension, hypercholesterolaemia, atherosclerosis and diabetes. Therefore, the prevention and/or reversal of the functional and morphological changes of the endothelium associated with these diseases is an important therapeutic goal. This brief overview covers current knowledge concerning the intracellular pathways that link endothelial activation by receptor-dependent and -independent stimuli to the formation of NO and PGI2.

Animals↗
PubMed1992

The two-step conversion of big endothelin 1 to endothelin 1 and degradation of endothelin 1 by subcellular fractions from human polymorphonuclear leukocytes.

The metabolism of big endothelin 1 (bET) and endothelin 1 (ET-1) by subcellular fractions from human polymorphonuclear leukocytes (PMNs) was investigated by bioassay and reversed-phase high-performance liquid chromatography. More than 80% of endothelin-converting activity was recovered from the cytosolic fraction, which in addition to ET-1 generated other peptides from bET. The processing of bET to all its metabolites including ET-1 was prevented by the serine protease inhibitor 3,4-dichloroisocoumarin (DCI; 50 microM) or the elastase inhibitor ONO-5046 (100 microM) but not by phenylmethylsulfonyl fluoride (PMSF; 143 microM), another serine protease inhibitor. Paradoxically, human leukocyte elastase, despite generating a bET fragmentation pattern similar to that of PMN cytosol, produced very little ET-1. However, subsequent treatment of the elastase-derived metabolites of bET with PMN cytosol in the presence of ONO-5046 dramatically increased the amount of ET-1 formed. The generation of ET-1 following this intervention was inhibited by DCI. The PMN membrane preparation degraded ET-1 to a major metabolite, similar to that produced from ET-1 by elastase, and several minor products, paralleled by a loss of its smooth muscle contracting activity. The degradation of ET-1 by PMN microsomes was prevented by DCI, PMSF, or ONO-5046. Our results suggest that an elastase-initiated serine protease cascade is responsible for the sequential conversion of bET to ET-1 by the PMN cytosol. Elastase also partly accounts for the ET-metabolizing properties of PMN microsomes.

Animals↗
PubMed1992

Determination of endothelin-converting enzyme activity by high-performance liquid chromatography-on-line radioactive flow monitoring.

A rapid method to investigate the metabolism of 125I-labelled or non-labelled human big endothelin to endothelin-1 by reversed-phase high-performance liquid chromatography (HPLC) and on-line radioactive flow monitoring and/or ultraviolet detection was developed. Samples were processed by solid-phase extraction (average recovery 70-80% for non-labelled and 20-25% for 125I-labelled big endothelin and endothelin-1) followed by HPLC analysis (total analysis time 20 min). The method was successfully employed to monitor the conversion of big endothelin to endothelin-1 by various blood-borne cells, such as human polymorphonuclear leukocytes or monocytes/macrophages.

Animals↗
PubMed1992

An expression vector system providing plasmid stability and conditional suicide of plasmid-containing cells.

A cloning vector system was constructed on the basis of the pBR322 derivative pEG1 by introducing the whole parB locus of plasmid R1 cloned behind the promoter of the alkaline phosphatase gene (phoA) of Escherichia coli. The parB locus in combination with the phoA promoter ensures both (i) plasmid stabilization due to the post-segregational killing of plasmid-free cells during growth and (ii) killing of the cells induced by the potential environmental signal phosphate limitation. This vector, therefore, appears to be a model system for increasing the stability of recombinant plasmids and for decreasing the potential risks in the application of recombinant bacteria in industrial fermentations.

Alkaline Phosphatase↗
PubMed1992

Characterization of serine protease-derived metabolites of big endothelin in the cytosolic fraction from human polymorphonuclear leukocytes.

We have characterized by bioassay and reversed-phase high-performance liquid chromatography analysis (rp-HPLC) the conversion of human big endothelin-1 (bET-1) to endothelin-1 (ET-1) by the cytosolic fraction from human polymorphonuclear leukocytes (PMNs). Either the general serine protease inhibitor 3,4-dichloroisocoumarin (DCI; 50 microM) or the selective elastase inhibitor ONO-5046 (100 microM) blocked the formation of ET-1 from bET-1. Interestingly, human leukocyte elastase formed some of the same products from bET-1 as the PMN cytosol, but generated negligible amounts of ET-1. However, coincubation of the elastase-derived fragments of bET-1 with the PMN cytosol in the presence of ONO-5046 resulted in a 17-fold increase in the formation of ET-1, indicating that an elastase-derived intermediate of bET-1 was subsequently cleaved by a soluble protease(s) to form mature ET-1. We have identified by electrospray-mass spectrometry (ESMS) analysis this intermediate as bET-1(1-22). Analysis of bET-1 digestion by human leukocyte cathepsin G revealed the formation of a biologically active metabolite chromatographically distinct from ET-1, identified as bET-1(1-31) by ESMS. These findings indicate the presence of complex enzyme systems in human PMNs capable of activating bET-1.

Cathepsin G↗
PubMed1992

Mechanisms of the endothelium-dependent relaxation induced by NG-hydroxy-L-arginine.

There are three mechanisms by which NG-hydroxy-L-arginine (L-HOArg) induces endothelium-dependent relaxations. L-HOArg is a substrate for the constitutive nitric oxide (NO) synthase present in endothelial cells (ECs). It reacts with NO released from EC to form a potent and more stable vasodilator. Moreover, it induces a relatively stable, EC-dependent relaxation that is not blocked by the inhibitors of NO synthesis. Subsequently, we have investigated the effects of hydroxyguanidine (HOG) on the biological activity of endothelium-derived relaxing factor (EDRF). HOG potentiated the relaxant responses of rabbit aortic strips to EDRF released from EC by adenosine diphosphate (ADP) or bradykinin as well as those induced by authentic NO. Importantly, it was not a substrate for NO synthesis and it did not affect the generation of prostacyclin by ECs. Thus, the effects of HOG were due to the chemical reaction of HOG with NO released from ECs and the formation of a more stable vasodilator. Moreover, HOG augmented not only agonist-triggered, but also flow-induced, EC-dependent relaxation and both effects of HOG were abolished by NG-nitro-L-arginine methyl ester (L-NO2-Arg). In contrast, the EC-dependent relaxation induced by L-HOArg was not inhibited by L-NO2Arg. Moreover, it was not affected by the removal of extracellular Ca2+, but was blocked by oxyhemoglobin and potentiated by superoxide dismutase. These results demonstrate the involvement of the hydroxyguanidino moiety of L-HOArg in its reaction with NO. Moreover, they strongly support the notion that nitrix oxide mediates both the agonist-triggered and flow-induced endothelium-dependent relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1992

Characterization of a microsomal calcium-dependent nitric oxide synthase in activated J774.2 monocyte/macrophages.

The nitric oxide (NO) synthase activity present in murine J774.2 monocyte/macrophages was characterized in terms of its intracellular localization, substrate specificity, and Ca2+ dependency. Traces of constitutive NO synthase activity were found in the microsomal fraction from noninduced J774.2 cells, whereas no NO synthase activity was detected in the cytosol. After 24 h in the presence of bacterial lipopolysaccharide and mouse interferon, NO synthase activity was substantially increased in both fractions with 51-60% of the total activity present in the cytosol. These activities, however, were clearly different, for the microsomal enzyme was Ca2+ dependent, whereas the cytosolic NO synthase was not. Moreover, NG-hydroxy-L-arginine (L-HOArg), L-homo-arginine, and several L-arginine (L-Arg)-containing dipeptides could replace L-Arg as substrates for the Ca(2+)-independent NO synthase, whereas the Ca(2+)-dependent enzyme accepted only L-Arg, L-HOArg, or L-Arg-L-Arg as substrates. Thus, a microsomal Ca(2+)-dependent NO synthase is induced in J774.2 monocyte/macrophages with a substrate specificity different from the inducible Ca(2+)-independent NO synthase as well as the constitutive NO synthase in, for example, endothelial cells. Irrespective of their intracellular localization, therefore, at least three isoforms of NO synthase exist, all of which can accommodate substrates different from L-Arg in size, charge, and hydrophobicity.

Amino Acid Oxidoreductases↗
PubMed1992

Stress proteins and cross-protection by heat shock and salt stress in Bacillus subtilis.

Bacillus subtilis induced a set of general stress proteins in response to a salt or heat stress. Cells subjected to a mild heat stress showed a protective response which enabled them to survive otherwise lethal temperatures (e.g. 52 degrees C). In a similar way bacteria were enabled to survive toxic concentrations of NaCl by pretreatment with lower salt concentrations. A mild heat shock induced a cross-protection against lethal salt stress. The pretreatment of cells with low salt, however, was less effective in the induction of thermotolerance than a preceding mild heat stress. Three stress proteins were identified on the basis of their N-terminal amino acid sequences as homologues of GroEL, DnaK and ClpP of Escherichia coli. The role of general and specific stress proteins in the induction of thermotolerance/salt tolerance and cross-protection is discussed.

ATP-Dependent Proteases↗
PubMed1992

Potentiation of the vasorelaxant activity of nitric oxide by hydroxyguanidine: implications for the nature of endothelium-derived relaxing factor.

1. We recently demonstrated that NG-hydroxy-L-arginine (L-HOArg) is a substrate for the constitutive nitric oxide (NO) synthase present in bovine aortic endothelial cells cultured on microcarrier beads (EC). Furthermore, L-HOArg reacts chemically with NO released from these cells to form a potent and more stable vasodilator. This is most likely through a reaction with the hydroxyguanidino group. 2. Here, we studied the interaction of a simpler molecule, hydroxyguanidine (HOG) with NO. 3. HOG (10 microM), like L-HOArg (10 microM) or NG-hydroxy-D-arginine (D-HOArg, 10 microM), potentiated and stabilized the relaxant activity of authentic NO. 4. When NO was bubbled through the solution of HOG, a new compound was formed. It had similar physicochemical properties to those of the previously described L-HOArg/NO adduct. It was also a potent vasodilator and its action was inhibited by oxyhaemoglobin (10 microM), indicating formation of a NO-containing substance. 5. Moreover, HOG (10 microM) was not a substrate for the constitutive NO synthase present in the microsomal fraction of EC and did not affect the flow-induced or bradykinin-stimulated generation of prostacyclin, as measured by 6-keto-PGF1 alpha. 6. We also studied the effect of HOG on the endothelium-derived relaxing factor (EDRF) released from the column of EC. HOG (10 microM) potentiated and stabilized the relaxations of rabbit aortic strips induced by EDRF released by bradykinin (5-20 pmol) or ADP (5-10 nmol). These relaxations were inhibited by NG-nitro-L-arginine methyl ester (L-NAME, 10 microM) and L-arginine (L-Arg, 1 mM) reversed the inhibitory effects of L-NAME. 7. HOG (10 iM) augmented the basal (flow-induced) EC-dependent relaxations which were also inhibited by L-NAME (10 1M) and the effects of L-NOArg were reversed by L-Arg (1 mM).8. Thus, the hydroxyguanidino moiety of L-HOArg is involved in the reaction with NO. Moreover, the comparable reaction of the hydroxyguanidino compounds with NO on the one hand and with flowinduced and agonist-triggered EDRF on the other, strongly supports their common identity.

Amino Acid Oxidoreductases↗
PubMed1992

Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis.

By using an internal part of the dnaK gene from Bacillus megaterium as a probe, a 5.2-kb HindIII fragment of chromosomal DNA of Bacillus subtilis was cloned. Downstream sequences were isolated by in vivo chromosome walking. Sequencing of 5,085 bp revealed four open reading frames in the order orf39-grpE-dnaK-dnaJ. orf39 encodes a 39-kDa polypeptide of unknown biological function with no noticeable homology to any other protein within the data bases. Alignment of the GrpE protein with those of three other bacterial species revealed a low overall homology, but a higher homology restricted to two regions which might be involved in interactions with other proteins. Alignment of the DnaK protein with six bacterial DnaK polypeptides revealed that a contiguous region of 24 amino acids is absent from the DnaK proteins of all known gram-positive species. Primer extension studies revealed three potential transcription start sites, two preceding orf39 (S1 and S2) and a third one in front of grpE (S3). S2 and S3 were activated at a high temperature. Northern (RNA) analysis led to the detection of three mRNA species of 4.9, 2.6, and 1.5 kb. RNA dot blot experiments revealed an at-least-fivefold increase in the amount of specific mRNA from 0 to 5 min postinduction and then a rapid decrease. A transcriptional fusion between dnaK and the amyL reporter gene exhibited a slight increase in alpha-amylase activity after heat induction. A 9-bp inverted repeat was detected in front of the coding region of orf39. This inverted repeat is present in a number of other heat shock operons in other microorganisms ranging from cyanobacteria to mycobacteria. The biological property of this inverted repeat as a putative key element in the induction of heat shock genes is discussed. The dnaK locus was mapped at about 223 degrees on the B. subtilis genetic map.

Amino Acid Sequence↗
PubMed1992

Cloning, sequencing, mapping, and transcriptional analysis of the groESL operon from Bacillus subtilis.

Using a gene probe of the Escherichia coli groEL gene, a 1.8-kb HindIII fragment of chromosomal DNA of Bacillus subtilis was cloned. Upstream sequences were isolated as a 3-kb PstI fragment. Sequencing of 2,525 bp revealed two open reading frames in the order groES groEL. Alignment of the GroES and GroEL proteins with those of eight other eubacteria revealed 50 to 65% and 72 to 84% sequence similarity, respectively. Primer extension studies revealed one potential transcription start site preceding the groESL operon (S) which was activated upon temperature upshift. Northern (RNA) analysis led to the detection of two mRNA species of 2.2 and 1.5 kb. RNA dot blot experiments revealed an at least 10-fold increase in the amount of specific mRNA from 0 to 5 min postinduction, remaining at this high level for 10 min and then decreasing. A 9-bp inverted repeat within the 5' leader region of the mRNA might be involved in regulation of the heat shock response. By using PBS1 transduction, the groESL operon was mapped at about 342 degrees.

Amino Acid Sequence↗