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Ralf Mrowka

Publications and source records attributed to Ralf Mrowka.

14 recordsLinked to original sources
PubMed2006

Adenosine restores angiotensin II-induced contractions by receptor-independent enhancement of calcium sensitivity in renal arterioles.

Adenosine is coupled to energy metabolism and regulates tissue blood flow by modulating vascular resistance. In this study, we investigated isolated, perfused afferent arterioles of mice, which were subjected to desensitization during repeated applications of angiotensin II. Exogenously applied adenosine restores angiotensin II-induced contractions by increasing calcium sensitivity of the arterioles, along with augmented phosphorylation of the regulatory unit of the myosin light chain. Adenosine restores angiotensin II-induced contractions via intracellular action, because inhibition of adenosine receptors do not prevent restoration, but inhibition of NBTI sensitive adenosine transporters does. Restoration was prevented by inhibition of Rho-kinase, protein kinase C, and the p38 mitogen-activated protein kinase, which modulate myosin light chain phosphorylation and thus calcium sensitivity in the smooth muscle. Furthermore, adenosine application increased the intracellular ATP concentration in LuciHEK cells. The results of the study suggest that restoration of the angiotensin II-induced contraction by adenosine is attributable to the increase of the calcium sensitivity by phosphorylation of the myosin light chain. This can be an important component of vascular control during ischemic and hypoxic conditions. Additionally, this mechanism may contribute to the mediation of the tubuloglomerular feedback by adenosine in the juxtaglomerular apparatus of the kidney.

Adenosine↗
PubMed2006

Translational control of collagen prolyl 4-hydroxylase-alpha(I) gene expression under hypoxia.

Hypoxia is a pro-fibrotic stimulus, which is associated with enhanced collagen synthesis, as well as with augmented collagen prolyl 4-hydroxylase (C-P4H) activity. C-P4H activity is controlled mainly by regulated expression of the alpha C-P4H subunit. In this study we demonstrate that the increased synthesis of C-P4H-alpha(I) protein in human HT1080 fibroblasts under long term hypoxia (36 h, 1% oxygen) is controlled at the translational level. This is mediated by an interaction of RNA-binding protein nucleolin (approximately 64 kDa form) at the 5'- and 3'-untranslated regions (UTR) of the mRNA. The 5'/3'-UTR-dependent mechanism elevates the C-P4H-alpha(I) expression rate 2.3-fold, and participates in a 5.3-fold increased protein level under long term hypoxia. The interaction of nucleolin at the 5'-UTR occurs directly and depends on the existence of an AU-rich element. Statistical evaluation of the approximately 64-kDa nucleolin/RNA interaction studies revealed a core binding sequence, corresponding to UAAAUC or AAAUCU. At the 3'-UTR, nucleolin assembles indirectly via protein/protein interaction, with the help of another 3'-UTR-binding protein, presumably annexin A2. The increased protein level of the approximately 64-kDa nucleolin under hypoxia can be attributed to an autocatalytic cleavage of a high molecular weight nucleolin form, without alterations in nucleolin mRNA concentration. Thus, the alteration of translational efficiency by nucleolin, which occurs through a hypoxia inducible factor independent pathway, is an important step in C-P4H-alpha(I) regulation under hypoxia.

3' Untranslated Regions↗
PubMed2006

Heterogeneous nuclear ribonucleoprotein-A2/B1 modulate collagen prolyl 4-hydroxylase, alpha (I) mRNA stability.

Collagen prolyl 4-hydroxylase (C-P4H) alpha-subunit is of regulatory importance in the assembling of C-P4H tetramers, which are necessary for the hydroxylation of procollagen chains. Change in collagen expression by hypoxia or iron diminishment is a significant issue in extracellular matrix remodeling. It was proposed that C-P4H-alpha (I) is regulated at the posttrancriptional level under these conditions. Here we report that the induction of C-P4H-alpha (I) in human fibrosarcoma cells HT1080 by the iron chelator 2,2-dipyridyl is predominantly caused by an enhancement of mRNA stability. This effect is mediated by an increased synthesis and binding of heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1, which interacts with a (U)(16) element located in the 3'-untranslated region of C-P4H-alpha (I) mRNA. Luciferase reporter gene assays depending on C-P4H-alpha (I) 3'-untranslated region and co-transfection with hnRNP-A2/B1 provide evidence that the (U)(16) element is necessary and sufficient for posttranscriptional control of C-P4H-alpha (I) synthesis under the analyzed conditions. Further indication for the significance of hnRNP-A2/B1 in C-P4H-alpha (I) induction was obtained by micro array experiments. In a data set representing 686 independent physiological conditions, we found a significant positive correlation between hnRNP-A2/B1 and C-P4H-alpha (I) mRNAs.

2,2'-Dipyridyl↗
PubMed2005

Influence of the adenosine A1 receptor on blood pressure regulation and renin release.

The present study was performed to investigate the role of adenosine A1 receptors in regulating blood pressure in conscious mice. Adenosine A1-receptor knockout (A1R-/-) mice and their wild-type (A1R+/+) littermates were placed on standardized normal-salt (NS), high-salt (HS), or salt-deficient (SD) diets for a minimum of 10 days before telemetric blood pressure and urinary excretion measurements in metabolic cages. On the NS diet, daytime and nighttime mean arterial blood pressure (MAP) was 7-10 mmHg higher in A1R-/- than in A1R+/+ mice. HS diet did not affect the MAP in A1R-/- mice, but the daytime and nighttime MAP of the A1R+/+ mice increased by approximately 10 mmHg, to the same level as that in the A1R-/-. On the SD diet, day- and nighttime MAP decreased by approximately 6 mmHg in both A1R-/- and A1R+/+ mice, although the MAP remained higher in A1R-/- than in A1R+/+ mice. Although plasma renin levels decreased with increased salt intake in both genotypes, the A1R-/- mice had an approximately twofold higher plasma renin concentration on all diets compared with A1R+/+ mice. Sodium excretion was elevated in the A1R-/- compared with the A1R+/+ mice on the NS diet. There was no difference in sodium excretion between the two genotypes on the HS diet. Even on the SD diet, A1R-/- mice had an increased sodium excretion compared with A1R+/+ mice. An abolished tubuloglomerular feedback response and reduced tubular reabsorption can account for the elevated salt excretion found in A1R-/- animals. The elevated plasma renin concentrations found in the A1R-/- mice could also result in increased blood pressure. Our results confirm that adenosine, acting through the adenosine A1 receptor, plays an important role in regulating blood pressure, renin release, and sodium excretion.

Animals↗
PubMed2005

Role of nucleolin in posttranscriptional control of MMP-9 expression.

Matrix-metalloproteinases (MMPs), which are able to degrade extra cellular matrix (ECM) components, are crucial in ECM-remodeling, under physiological (e.g., embryogenesis, wound healing, angiogenesis) or pathophysiological conditions (e.g., arthritis, cancer progression and metastasis, fibrosis). Treating HT1080 cells, a human fibrosarcoma cell line, with the iron chelator 2,2-Dipyridyl, which mimics certain aspects of hypoxia, leads to a 3-fold elevated Matrix-metalloproteinase-9 (MMP-9) protein level. This elevation occurs within 3 h, without any change of mRNA-concentration. The rapid increase in MMP-9 expression is caused by an enhancement of translational efficiency characterized by a recruitment of translationally inactive MMP-9 mRNP-complexes into the rough endoplasmatic reticulum (rER). Reporter gene assays, which depend on the untranslated regions (UTR) of MMP-9 mRNA, reveal that the posttranscriptional regulation is mainly attributed to the 3'UTR. RNA/protein interaction studies indicate that the elevated binding of nucleolin ( approximately 64 kDa form) to the 3'UTR may be of major importance for the increased efficiency of MMP-9 translation. The results show that MMP-9 expression can be regulated posttranscriptionally, affecting the efficiency of translation and localization of the mRNA.

2,2'-Dipyridyl↗
PubMed2004

Sequence-related human proteins cluster by degree of evolutionary conservation.

Gene duplication followed by adaptive evolution is thought to be a central mechanism for the emergence of novel genes. To illuminate the contribution of duplicated protein-coding sequences to the complexity of the human genome, we study the connectivity of pairwise sequence-related human proteins and construct a network (N) of linked protein sequences with shared similarities. We find that (i) the connectivity distribution P (k) for k sequence-related proteins decays as a power law P (k) approximately k(-gamma) with gamma approximately 1.2 , (ii) the top rank of N consists of a single large cluster of proteins ( approximately 70%) , while bottom ranks consist of multiple isolated clusters, and (iii) structural characteristics of N show both a high degree of clustering and an intermediate connectivity ("small-world" features). We gain further insight into structural properties of N by studying the relationship between the connectivity distribution and the phylogenetic conservation of proteins in bacteria, plants, invertebrates, and vertebrates. We find that (iv) the proportion of sequence-related proteins increases with increasing extent of evolutionary conservation. Our results support that small-world network properties constitute a footprint of an evolutionary mechanism and extend the traditional interpretation of protein families.

Chromosome Mapping↗
PubMed2004

AT1 receptors mediate angiotensin II-induced release of nitric oxide in afferent arterioles.

BACKGROUND: Recent studies have indicated that angiotensin II (Ang II) possibly activates the nitric oxide (NO) system. We investigated the role of AT receptor subtypes (AT-R) in mediating the Ang II-induced NO release in afferent arterioles (Af) of mice. METHODS: Isolated Af of mice were perfused, and the isotonic contraction measured. Further, NO release was determined using DAF-FM, a fluorescence indicator for NO. Moreover, we qualitatively assessed the expression of AT-R at the mRNA level using reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Ang II reduced luminal diameters dose dependently (67.3 +/- 6.3% at 10(-6) mol/L). Inhibition of AT2-R with PD123.319 did not change the Ang II contractile response. AT1-R blockade with ZD7155 inhibited contraction. Stimulation of AT2-R during AT1-R inhibition with ZD7155, and preconstriction with norepinephrine (NE) had no influence on the diameter. Drug application via the perfusion pipette changed flow and pressure, and enhanced NO fluorescence by DeltaF = 4.0 +/- 0.4% (N= 14, background). Luminal application of Ang II (10(-7) mol/L) increased the NO fluorescence by DeltaF = 9.9 +/- 1.2% (N= 8). AT1-R blockade blunted the increase to background levels (DeltaF to 4.0 +/- 0.3%, N= 6, P < 0.05), but AT2-R blockade did not (8.1 +/- 0.9%, N= 9). L-NAME nearly abolished the Ang II effect on the NO fluorescence (DeltaF = 1.6 +/- 0.5% (N= 8). NE did not increase NO release beyond the background levels. RT-PCR showed expression of both AT1-R and AT2-R. CONCLUSION: The results indicate an Ang II-induced NO release in Af of mice, which is mediated by AT1-R. Thus, Ang II balances its own constrictor action in Af. This control mechanism is very important in view of high renin and angiotensin II concentration in the juxtaglomerular apparatus.

Angiotensin II↗
PubMed2003

Directionality of coupling of physiological subsystems: age-related changes of cardiorespiratory interaction during different sleep stages in babies.

Activity of many physiological subsystems has a well-expressed rhythmic character. Often, a dependency between physiological rhythms is established due to interaction between the corresponding subsystems. Traditional methods of data analysis allow one to quantify the strength of interaction but not the causal interrelation that is indispensable for understanding the mechanisms of interaction. Here we present a recently developed method for quantification of coupling direction and apply it to an important problem. Namely, we study the mutual influence of respiratory and cardiovascular rhythms in healthy newborns within the first 6 mo of life in quiet and active sleep. We find an age-related change of the coupling direction: the interaction is nearly symmetric during the first days and becomes practically unidirectional (from respiration to heart rhythm) at the age of 6 mo. Next, we show that the direction of interaction is mainly determined by respiratory frequency. If the latter is less than approximately 0.6 Hz, the interaction occurs dominantly from respiration to heart. With higher respiratory frequencies that only occur at very young ages, the dominating direction is less pronounced or even abolished. The observed dependencies are not related to sleep stage, suggesting that the coupling direction is determined by system-inherent dynamical processes, rather than by functional modulations. The directional analysis may be applied to other interacting narrow band oscillatory systems, e.g., in the central nervous system. Thus it is an important step forward in revealing and understanding causal mechanisms of interactions.

Biological Clocks↗
PubMed2003

Control of renin synthesis.

Studies published recently have considerably enhanced our understanding of the mechanisms controlling renin production. With regard to the control of renin transcription, two enhancer regions have been identified that markedly augment renin synthesis in cell lines. In the absence of this enhancer activity, the basic promoter of the renin gene increases transcription only two- to threefold. The location of one (Jones CA, Sigmund CD, McGowan RA, Kane-Haas CM, and Gross KW. Mol Endocrinol 4: 375-383, 1990) transcription enhancer in the mouse gene is at about -2.7 kb and in humans at roughly -11 kb. A second important region has been identified in a chorionic cell line to be located approximately 5 kb upstream of the transcription start site in humans. Another potentially important regulatory region may lie within approximately 3.9 kb upstream of the -11 kb enhancer, as suggested by several conserved sequences among species in this region. In addition to the control of renin transcription, it seems that renin translation and the stability of renin mRNA are also effectively regulated. This occurs via the 3'-untranslated region, to which several proteins can bind. The binding proteins were identified as hnRNP K and E1, dynamin, nucleolin, MINT homologous protein, and Y-Box 1.

Animals↗
PubMed2003

An evolutionary approach for identifying potential transcription factor binding sites: the renin gene as an example.

Evolutionary pressure has resulted in the conservation of certain nucleotide sequences. These conserved regions are potentially important for certain functions. Here we give an example of a comparison between noncoding sequences combined with other independent database information to shed light onto the regulation of the renin gene, a gene that has great importance for cardiovascular and renal homeostasis. To combine the information regarding conservation and weight matrices of transcription factor (TF) binding sites, an algorithm was developed (TFprofile). Notably, a local peak in the resulting binding profile coincides with a previously experimentally identified regulatory region for the renin gene. The existence of further peaks in the binding profile in the conserved 3.9-kb-long hRENc DNA block upstream of the renin gene suggests additional regions of potential importance for gene regulation. The algorithm TFprofile may be used to integrate information on cross-species evolutionary conservation and aspects of TF binding characteristics to provide putative regulatory DNA regions for experimental verification.

Animals↗
PubMed2002

Identification of coupling direction: application to cardiorespiratory interaction.

We consider the problem of experimental detection of directionality of weak coupling between two self-sustained oscillators from bivariate data. We further develop the method introduced by Rosenblum and Pikovsky [Phys. Rev. E 64, 045202 (2001)], suggesting an alternative approach. Next, we consider another framework for identification of directionality, based on the idea of mutual predictability. Our algorithms provide directionality index that shows whether the coupling between the oscillators is unidirectional or bidirectional, and quantifies the asymmetry of bidirectional coupling. We demonstrate the efficiency of three different algorithms in determination of directionality index from short and noisy data. These techniques are then applied to analysis of cardiorespiratory interaction in healthy infants. The results reveal that the direction of coupling between cardiovascular and respiratory systems varies with the age within the first 6 months of life. We find a tendency to change from nearly symmetric bidirectional interaction to nearly unidirectional one (from respiration to the cardiovascular system).

Age Factors↗
PubMed2002

Oligodb--interactive design of oligo DNA for transcription profiling of human genes.

MOTIVATION: The increasing use of customized oligo-DNA-microarrays has created a demand for convenient design of oligo nucleotides. The oligo-nucleotides have to serve as specific templates in the hybridization reaction. RESULTS: We have devised a web-based system for interactive design of suitable oligos for transcription profiling experiments with user-defined parameters. Oligodb is based on predicted human gene transcripts from the ENSEMBL project. The web interface allows the user to retrieve oligos for human transcripts in a flexible manner. Criteria for selecting the oligos are low similarity to all other known human genes, the absence of stable secondary structures, the length of the oligo, and the absence of low complexity regions. Furthermore, the user may select oligos corresponding to the desired position in the 5'-3' direction of the transcript, and based on the melting temperature. All data and results are tightly web-linked to the corresponding data entries of ENSEMBL project.

Algorithms↗
PubMed2001

Interaction of angiotensin II and nitric oxide in isolated perfused afferent arterioles of mice.

The present study was performed to evaluate angiotensin II (Ang II)-nitric oxide (NO) interaction in afferent arterioles (Af) of wild-type mice and mice that are homozygous (-/-) for disruption of the endothelial NO synthase (eNOS) gene. Af were microperfused, and the dose responses were assessed for the NO precursor L-arginine (n = 4), NO inhibitor NG-nitro-L-arginine methyl ester (L-NAME, n = 5), L-NAME after pretreatment with L-arginine (n = 5), Ang II (n = 8), and Ang II after pretreatment with L-NAME (n = 7). Acute administration of L-arginine and L-NAME (both in doses from 10(-6) to 10(-3) mol/L) did not change arteriolar diameter. Moreover, pretreatment with L-arginine did not change the response to L-NAME. However, Ang II, applied in doses of 10(-12), 10(-10), 10(-8), and 10(-6) mol/L, significantly reduced the lumen to 66.5 +/- 7.0% and 62.2 +/- 8.0% at 10(-8) and 10(-6) mol/L Ang II, respectively. The contraction was augmented after L-NAME pretreatment (19.5 +/- 13.6% and 25.5 +/- 10.2% at 10(-8) and 10(-6) mol/L Ang II, respectively). In eNOS (-/-) mice (n = 8), the response to Ang II also was enhanced (9.1 +/- 6.0% and 11.2 +/- 8.2% at 10(-8) and 10(-6) mol/L Ang II, respectively). Female mice did not differ from male mice in their reactivity to Ang II (n = 9) and Ang II + L-NAME pretreatment (n = 11). The study shows that (1) it is feasible to microperfuse mouse Af, (2) the basal production of endothelial NO is very low and not inducible by L-arginine in Af of mice, and (3) a counteracting effect of NO is initiated by Ang II. High Ang II sensitivity in eNOS (-/-) mice underscores the considerable role of endothelial-derived NO to balance Ang II vasoconstriction in Af.

Analysis of Variance↗