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Michael Bader

Publications and source records attributed to Michael Bader.

At least 91 records · Page 5Linked to original sources
PubMed2004

Functional characterization of the human atrial essential myosin light chain (hALC-1) in a transgenic rat model.

Most patients with hypertrophic cardiomyopathy and congenital heart diseases express the atrial essential myosin light chains (ALC-1) in their ventricles, partially replacing the ventricular essential light chains (VLC-1). This VLC-1/ALC-1 isoform shift is correlated with an increase in cross-bridge cycling kinetics as measured using skinned fibers from the hypertrophied ventricles of human hearts. To study the functional importance of hALC-1 in the intact perfused heart, we generated a transgenic rat model (TGR) overexpressing hALC-1 in the heart. Twelve-week-old TGR rats expressed 17 +/- 4 microg hALC-1 per mg of whole SDS-soluble protein. Their perfused heart contractility parameters were evaluated using the Langendorff preparation. Expression of hALC-1 was accompanied by statistically significant improvements (P<0.001) in the contractile parameters of the hearts of the TGR compared to the age matched control (WKY) animals, represented by increases from 20.8 +/- 2.3 to 45.1 +/- 3.6 mmHg/g heart weight in the developed left ventricular pressure, 1,035.7 +/- 89.8 to 2,181 +/- 135.4 mmHg/s in the contraction rate, and 713 +/- 60.2 to 1,364 +/- 137.4 mmHg/s in the relaxation rate in the WKY and the TGR groups respectively. Characterizing the functional effects of hALC-1 at the whole organ level represents a step towards gene therapy of heart failure.

Amino Acid Sequence↗
PubMed2004

Role of bradykinin B2 and B1 receptors in the local, remote, and systemic inflammatory responses that follow intestinal ischemia and reperfusion injury.

The administration of bradykinin may attenuate ischemia and reperfusion (I/R) injury by acting on B(2)Rs. Blockade of B(2)R has also been shown to ameliorate lesions associated with I/R injury. In an attempt to explain these contradictory results, the objective of the present work was to investigate the role of and interaction between B(1) and B(2) receptors in a model of intestinal I/R injury in mice. The bradykinin B(2)R antagonist (HOE 140) inhibited reperfusion-induced inflammatory tissue injury and delayed lethality. After I/R, there was an increase in the expression of B(1)R mRNA that was prevented by HOE 140. In mice that were deficient in B(1)Rs (B(1)R(-/-) mice), inflammatory tissue injury was abrogated, and lethality was delayed and partially prevented. Pretreatment with HOE 140 reversed the protective anti-inflammatory and antilethality effects provided by the B(1)R(-/-) phenotype. Thus, B(2)Rs are a major driving force for B(1)R activation and consequent induction of inflammatory injury and lethality. In contrast, activation of B(2)Rs may prevent exacerbated tissue injury and lethality, an effect unmasked in B(1)R(-/-) mice and likely dependent on the vasodilatory actions of B(2)Rs. Blockade of B(1)Rs could be a more effective strategy than B(2) or B(1)/B(2) receptor blockade for the treatment of the inflammatory injuries that follow I/R.

Animals↗
PubMed2004

Efficiency of transgenic rat production is independent of transgene-construct and overnight embryo culture.

The aim of the present work was to study factors affecting the efficiency of transgenic technology in rats. We investigated the possible effects of pronuclear microinjection of buffer or different DNA-constructs on survival and development of rat zygotes in vitro and in vivo as well as the influence of overnight culture of these embryos before transfer into pseudopregnant foster mothers. The survival rate of zygotes and their development to the two-cell and morula stage was not affected by pronuclear microinjection with different DNA-constructs or buffer. However, the development to the blastocyst stage was impaired. Nevertheless, there was no difference in blastocyst development between zygotes injected with DNA-constructs or with buffer. Neither was there a difference in cell number in in vitro cultured blastocysts resulting from pronuclear microinjection of a transgene compared with non-injected controls. The survival rate to term was about 30% irrespective of whether microinjected embryos were transferred immediately after microinjection or after overnight culture in vitro. However, a reduction in the survival to term was observed for non-injected zygotes when they were developed in vitro to the two-cell stage before transfer to a pseudopregnant female. The percentage of transgenic rats that resulted from microinjected zygotes was similar in all groups regardless of the DNA-construct used (2.7-10.0%). In conclusion, the main detrimental factor in the microinjection of rat zygotes is the introduction of solution in the pronucleus. Overnight culture of zygotes between microinjection and oviduct transfer does not decrease the efficiency of transgenic rat generation.

Animals↗
PubMed2004

Serotonin regulates mammary gland development via an autocrine-paracrine loop.

Mammary gland development is controlled by a dynamic interplay between endocrine hormones and locally produced factors. Biogenic monoamines (serotonin, dopamine, norepinephrine, and others) are an important class of bioregulatory molecules that have not been shown to participate in mammary development. Here we show that mammary glands stimulated by prolactin (PRL) express genes essential for serotonin biosynthesis (tryptophan hydroxylase [TPH] and aromatic amine decarboxylase). TPH mRNA was elevated during pregnancy and lactation, and serotonin was detected in the mammary epithelium and in milk. TPH was induced by PRL in mammosphere cultures and by milk stasis in nursing dams, suggesting that the gene is controlled by milk filling in the alveoli. Serotonin suppressed beta-casein gene expression and caused shrinkage of mammary alveoli. Conversely, TPH1 gene disruption or antiserotonergic drugs resulted in enhanced secretory features and alveolar dilation. Thus, autocrine-paracrine serotonin signaling is an important regulator of mammary homeostasis and early involution.

Animals↗
PubMed2004

Prevention of cardiac fibrosis and left ventricular dysfunction in diabetic cardiomyopathy in rats by transgenic expression of the human tissue kallikrein gene.

Diabetic cardiomyopathy includes fibrosis. Kallikrein (KLK) can inhibit collagen synthesis and promote collagen breakdown. We investigated cardiac fibrosis and left ventricular (LV) function in transgenic rats (TGR) expressing the human kallikrein 1 (hKLK1) gene in streptozotocin (STZ) -induced diabetic conditions. Six weeks after STZ injection, LV function was determined in male Sprague-Dawley (SD) rats and TGR(hKLK1) (n=10/group) by a Millar tip catheter. Total collagen content (Sirius Red staining) and expression of types I, III, and VI collagen were quantified by digital image analysis. SD-STZ hearts demonstrated significantly higher total collagen amounts than normoglycemic controls, reflected by the concomitant increment of collagen types I, III, and VI. This correlated with a significant reduction of LV function vs. normoglycemic controls. In contrast, surface-specific content of the extracellular matrix, including collagen types I, III, and VI expression, was significantly lower in TGR(hKLK1)-STZ, not exceeding the content of SD and TGR(hKLK1) controls. This was paralleled by a preserved LV function in TGR(hKLK1)-STZ animals. The kallikrein inhibitor aprotinin and the bradykinin (BK) B2 receptor antagonist icatibant reduced the beneficial effects on LV function and collagen content in TGR(hKLK1)-STZ animals. Transgenic expression of hKLK1 counteracts the progression of LV contractile dysfunction and extracellular matrix remodeling in STZ-induced diabetic cardiomyopathy via a BK B2 receptor-dependent pathway.

Animals↗
PubMed2004

Plasma and kidney angiotensin II levels and renal functional responses to AT(1) receptor blockade in hypertensive Ren-2 transgenic rats.

OBJECTIVE: The first aim of the present study was to assess plasma and kidney angiotensin II (ANG II) levels and renal cortical ANG II receptor subtype 1A (AT1A) mRNA expression in hypertensive Ren-2 transgenic rats (TGR) and in normotensive Hannover Sprague-Dawley (HanSD) rats. The second aim was to investigate potential differences between TGR and HanSD in blood pressure (BP) and renal functional responses to either intravenous (i.v.), i.e. systemic, or intrarenal (i.r.) AT1 receptor blockade with candesartan. METHODS: Rats were anesthetized and prepared for clearance experiments. In series 1, ANG II concentrations were assayed by radioimmunoassay and renal cortical AT1A mRNA expression by semiquantitative reverse transcriptase-polyacrylamide gel electrophoresis. In series 2, BP and renal functional responses were evaluated after either i.v. or i.r. bolus administration of candesartan. RESULTS: Plasma and kidney ANG II levels were significantly lower in TGR than in HanSD (39 +/- 5 versus 107 +/- 19 fmol/ml and 251 +/- 41 versus 571 +/- 95 fmol/g, respectively, P < 0.05). Renal AT1A mRNA expression was not different between TGR and HanSD. Intravenous candesartan caused comparable decreases in BP in TGR and HanSD and did not change renal plasma flow (RPF) or absolute and fractional sodium excretion in HanSD. In contrast, i.v. candesartan significantly increased RPF (+27 +/- 6%, P < 0.05) and absolute and fractional sodium excretion (+49 +/- 10 and + 42 +/- 9%, respectively P < 0.05) in TGR without changing glomerular filtration rate (GFR). Acute i.r. candesartan increased RPF by +36 +/- 6% (P < 0.05) in TGR but not in HanSD with a greater rise in absolute and fractional sodium excretion in TGR (+124 +/-8 and 97 +/- 9%, respectively) than in HanSD (+81 +/- 9 and +69 +/- 8%, respectively) (P < 0.05). CONCLUSIONS: The enhanced responses of RPF and sodium excretion to AT1 receptor blockade in TGR suggest that renal hemodynamics and sodium excretion in TGR are under strong ANG II influence. The compromised ability of the kidney to respond to BP elevations by appropriate increases in sodium excretion may contribute to the maintenance of high BP in TGR. Thus, the present findings provide new insights into the pathophysiology of hypertension in this model.

Angiotensin II↗
PubMed2003

Serotonylation of small GTPases is a signal transduction pathway that triggers platelet alpha-granule release.

Serotonin is a neurotransmitter in the central nervous system. In the periphery, serotonin functions as a ubiquitous hormone involved in vasoconstriction and platelet function. Serotonin is synthesized independently in peripheral tissues and neurons by two different rate-limiting tryptophan hydroxylase (TPH) isoenzymes. Here, we show that mice selectively deficient in peripheral TPH and serotonin exhibit impaired hemostasis, resulting in a reduced risk of thrombosis and thromboembolism, although the ultrastructure of the platelets is not affected. While the aggregation of serotonin-deficient platelets in vitro is apparently normal, their adhesion in vivo is reduced due to a blunted secretion of adhesive alpha-granular proteins. In elucidating the mechanism further, we demonstrate that serotonin is transamidated to small GTPases by transglutaminases during activation and aggregation of platelets, rendering these GTPases constitutively active. Our data provides evidence for a receptor-independent signaling mechanism, termed herein as "serotonylation," which leads to alpha-granule exocytosis from platelets.

Animals↗
PubMed2003

Forced homodimerization by site-directed mutagenesis alters guanylyl cyclase activity of natriuretic peptide receptor B.

Natriuretic peptides mediate their physiologic effects through activation of membrane-bound, guanylyl cyclase-coupled receptors (NPRs). Receptor dimerization is an important feature of signal transduction. This study was aimed at characterizing structurally important residues of the extracellular ligand-binding domain of NPR-B for receptor dimerization and cGMP generation. Deletion mutagenesis was used to replace cysteine residues at positions 53 (C53S), 417 (C417S), and 426 (C426S) by serine. Receptor expression, dimerization, whole-cell cGMP response, and guanylyl cyclase activity of membrane fractions were determined in stably transfected COS-7 cells. C53S, C417S, and C426S mutants were expressed and found to form disulfide-bridged covalent dimers. In contrast to NPR-B and C53S, C417S and C426S mutants displayed constitutive activity in whole cells (C417S, 146+/-12%, P<0.01; C426S, 153+/-7% of ligand-independent NPR-B cGMP generation, P<0.01). The cGMP response of C417S and C426S mutants in whole cells was dose dependent and approximately 4 times lower than that in NPR-B, whereas it was blunted in C53S-transfected cells (1 micromol/L CNP, NPR-B 2868+/-436%; C53S, 206+/-16% of control, P<0.001 vs NPR-B, C417S, and C426S). Guanylyl cyclase assay in transfected cells confirmed the constitutive activity of C417S and C426S mutants. These data suggest that receptor dimerization by covalent disulfide bridges alters ligand-independent as well as ligand-dependent receptor activity. Localization of the crosslink in relation to the cell membrane is important for configuration of the extracellular domain and the consecutive signal transduction.

Animals↗
PubMed2003

A unique central tryptophan hydroxylase isoform.

Serotonin (5-hydroxytryptophan, 5-HT) is a neurotransmitter synthesized in the raphe nuclei of the brain stem and involved in the central control of food intake, sleep, and mood. Accordingly, dysfunction of the serotonin system has been implicated in the pathogenesis of psychiatric diseases. At the same time, serotonin is a peripheral hormone produced mainly by enterochromaffin cells in the intestine and stored in platelets, where it is involved in vasoconstriction, haemostasis, and the control of immune responses. Moreover, serotonin is a precursor for melatonin and is therefore synthesized in high amounts in the pineal gland. Tryptophan hydroxylase (TPH) catalyzes the rate limiting step in 5-HT synthesis. Until recently, only one gene encoding TPH was described for vertebrates. By gene targeting, we functionally ablated this gene in mice. To our surprise, the resulting animals, although being deficient for serotonin in the periphery and in the pineal gland, exhibited close to normal levels of 5-HT in the brain stem. This led us to the detection of a second TPH gene in the genome of humans, mice, and rats, called TPH2. This gene is predominantly expressed in the brain stem, while the classical TPH gene, now called TPH1, is expressed in the gut, pineal gland, spleen, and thymus. These findings clarify puzzling data, which have been collected over the last decades about partially purified TPH proteins with different characteristics and justify a new concept of the serotonin system. In fact, there are two serotonin systems in vertebrates, independently regulated and with distinct functions.

Amino Acid Sequence↗
PubMed2003

Delayed maturation of catecholamine phenotype in nucleus tractus solitarius of rats with glial angiotensinogen depletion.

Cerebral catecholamines and angiotensins are both involved in the regulation of cardiovascular function. Recent in vitro studies have suggested that angiotensin II modulates noradrenergic neurotransmission by controlling both the expression and neuritic trafficking of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. To assess the potential existence of such mechanisms in vivo, we compared TH phenotype ontogeny in the nucleus tractus solitarius (NTS), which is the first central relay of the baroreflex, between control Sprague-Dawley rats and TGR(ASrAOGEN) rats (TG) with glial specific angiotensinogen (AOGEN) depletion. TG displayed a delayed increase in both TH-mRNA and TH protein levels, which sharply rises in the NTS of control rats within the fourth week. The delayed maturation of TH phenotype also affected the presence of TH protein in the neuropil, not only within the NTS region but also within the ventrolateral medulla. This was evidenced by a large decrease in the density of TH-containing neuronal processes in TG at 4 weeks only, without noticeable modification of the labeling of the neuritic marker MAP2, suggesting that neuritic trafficking of TH protein was transiently altered. These results indicate that glial AOGEN is crucial to coordinate within the fourth week the mechanisms driving the maturation of NTS catecholaminergic neurons and suggest that impairment of the central angiotensinergic system early in development can lead to cardiovascular dysfunction related to altered maturation of catecholaminergic neurons located in both the dorsal and the ventrolateral medulla.

Angiotensinogen↗
PubMed2003

Angiotensin-(1-7) is an endogenous ligand for the G protein-coupled receptor Mas.

The renin-angiotensin system plays a critical role in blood pressure control and body fluid and electrolyte homeostasis. Besides angiotensin (Ang) II, other Ang peptides, such as Ang III [Ang-(2-8)], Ang IV [Ang-(3-8)], and Ang-(1-7) may also have important biological activities. Ang-(1-7) has become an angiotensin of interest in the past few years, because its cardiovascular and baroreflex actions counteract those of Ang II. Unique angiotensin-binding sites specific for this heptapeptide and studies with a selective Ang-(1-7) antagonist indicated the existence of a distinct Ang-(1-7) receptor. We demonstrate that genetic deletion of the G protein-coupled receptor encoded by the Mas protooncogene abolishes the binding of Ang-(1-7) to mouse kidneys. Accordingly, Mas-deficient mice completely lack the antidiuretic action of Ang-(1-7) after an acute water load. Ang-(1-7) binds to Mas-transfected cells and elicits arachidonic acid release. Furthermore, Mas-deficient aortas lose their Ang-(1-7)-induced relaxation response. Collectively, these findings identify Mas as a functional receptor for Ang-(1-7) and provide a clear molecular basis for the physiological actions of this biologically active peptide.

Angiotensin I↗
PubMed2003

Androgen receptor independent cardiovascular action of the antiandrogen flutamide.

We have previously shown that flutamide (specific antagonist of the androgen receptor) has antihypertensive effects. In the present study we examined the mechanisms of flutamide action in the vasculature. The vascular effects of flutamide were assayed in aortae isolated from male or female Sprague-Dawley rats and from rats or mice lacking a functional androgen receptor ( tfm, testicular feminization mutation). The effect of flutamide on coronary flow was tested in isolated hearts. In addition, male hypertensive rats with tfm mutation were treated with flutamide, and blood pressure was monitored. Flutamide induced a relaxation of rat aortae from all the strains used (maximum relaxation at 10 microM: 51.3+/-5.2% of phenylephrine contraction) and increased the coronary flow. The aortic relaxation to flutamide was abolished by endothelium removal, or by inhibition of nitric oxide synthase, guanylyl cyclase, and tyrosine kinase but not by calmodulin inhibition. Flutamide treatment attenuated the development of hypertension in mouse renin transgenic rats with the tfm mutation. Flutamide produces direct vasodilation by inducing release of NO from the endothelium and causes subsequent activation of soluble guanylyl cyclase in an active androgen receptor independent manner. This response may contribute to the observed antihypertensive actions of flutamide.

Androgen Antagonists↗
PubMed2003

Decreased liver fatty acid binding capacity and altered liver lipid distribution in mice lacking the liver fatty acid-binding protein gene.

Although liver fatty acid-binding protein (L-FABP) is an important binding site for various hydrophobic ligands in hepatocytes, its in vivo significance is not understood. We have therefore created L-FABP null mice and report here their initial analysis, focusing on the impact of this mutation on hepatic fatty acid binding capacity, lipid composition, and expression of other lipid-binding proteins. Gel-filtered cytosol from L-FABP null liver lacked the main fatty acid binding peak in the fraction that normally comprises both L-FABP and sterol carrier protein-2 (SCP-2). The binding capacity for cis-parinaric acid was decreased >80% in this region. Molar ratios of cholesterol/cholesterol ester, cholesteryl ester/triglyceride, and cholesterol/phospholipid were 2- to 3-fold greater, reflecting up to 3-fold absolute increases in specific lipid classes in the order cholesterol > cholesterol esters > phospholipids. In contrast, the liver pool sizes of nonesterified fatty acids and triglycerides were not altered. However, hepatic deposition of a bolus of intravenously injected [14C]oleate was markedly reduced, showing altered lipid pool turnover. An increase of approximately 75% of soluble SCP-2 but little or no change of other soluble (glutathione S-transferase, albumin) and membrane (fatty acid transport protein, CD36, aspartate aminotransferase, caveolin) fatty acid transporters was measured. These results (i) provide for the first time a quantitative assessment of the contribution of L-FABP to cytosolic fatty acid binding capacity, (ii) establish L-FABP as an important determinant of hepatic lipid composition and turnover, and (iii) suggest that SCP-2 contributes to the accumulation of cholesterol in L-FABP null liver.

Acetyl-CoA C-Acetyltransferase↗
PubMed2003

Transgenic rat model of Huntington's disease.

Huntington's disease (HD) is a late manifesting neurodegenerative disorder in humans caused by an expansion of a CAG trinucleotide repeat of more than 39 units in a gene of unknown function. Several mouse models have been reported which show rapid progression of a phenotype leading to death within 3-5 months (transgenic models) resembling the rare juvenile course of HD (Westphal variant) or which do not present with any symptoms (knock-in mice). Owing to the small size of the brain, mice are not suitable for repetitive in vivo imaging studies. Also, rapid progression of the disease in the transgenic models limits their usefulness for neurotransplantation. We therefore generated a rat model transgenic of HD, which carries a truncated huntingtin cDNA fragment with 51 CAG repeats under control of the native rat huntingtin promoter. This is the first transgenic rat model of a neurodegenerative disorder of the brain. These rats exhibit adult-onset neurological phenotypes with reduced anxiety, cognitive impairments, and slowly progressive motor dysfunction as well as typical histopathological alterations in the form of neuronal nuclear inclusions in the brain. As in HD patients, in vivo imaging demonstrates striatal shrinkage in magnetic resonance images and a reduced brain glucose metabolism in high-resolution fluor-deoxy-glucose positron emission tomography studies. This model allows longitudinal in vivo imaging studies and is therefore ideally suited for the evaluation of novel therapeutic approaches such as neurotransplantation.

Animals↗
PubMed2003

The vesicular monoamine content regulates VMAT2 activity through Galphaq in mouse platelets. Evidence for autoregulation of vesicular transmitter uptake.

Variations in the neurotransmitter content of secretory vesicles enable neurons to adapt to network changes. Vesicular content may be modulated by vesicle-associated Go(2), which down-regulates the activity of the vesicular monoamine transmitter transporters VMAT1 in neuroendocrine cells and VMAT2 in neurons. Blood platelets resemble serotonergic neurons with respect to transmitter storage and release. In streptolysin O-permeabilized platelets, VMAT2 activity is also down-regulated by the G protein activator guanosine 5'-(beta(i)gamma-imido)triphosphate (GMppNp). Using serotonin-depleted platelets from peripheral tryptophan hydroxylase knockout (Tph1-/-) mice, we show here that the vesicular filling initiates the G protein-mediated down-regulation of VMAT2 activity. GMppNp did not influence VMAT2 activity in naive platelets from Tph1-/- mice. GMppNp-mediated inhibition could be reconstituted, however, when preloading Tph1-/- platelets with serotonin or noradrenaline. Galpha(q) mediates the down-regulation of VMAT2 activity as revealed from uptake studies performed with platelets from Galpha(q) deletion mutants. Serotonergic, noradrenergic, as well as thromboxane A(2) receptors are not directly involved in the down-regulation of VMAT2 activity. It is concluded that in platelets the vesicle itself regulates transmitter transporter activity via its content and vesicle-associated Galpha(q).

Animals↗
PubMed2003

Tonin in rat heart with experimental hypertrophy.

The present study was undertaken to determine tonin expression and activity in rat heart presenting isoproterenol-induced hypertrophy. Renin, angiotensin-converting enzyme (ACE), and angiotensinogen (AG) expression were also determined. Wistar rats were treated with isoproterenol for 7 days (5 mg x kg(-1) x day(-1) sc). For untreated animals, the levels of tonin-specific activity in the atrium were 2.6- and 5.5-fold higher than those of the left and right ventricle, respectively. After treatment, the levels of tonin-specific activity increased twofold in the atrium but did not change in the ventricles. Renin expression was not detectable in these structures, and ACE expression levels did not change with treatment. AG expression was detected in the left ventricle at very low levels compared with the atrium and increased significantly only in the hypertrophied atrium (1.8-fold). Tonin mRNA was not detected in the ventricle but was found at low levels in the atrium, which increased after isoproterenol treatment. Our results permit us to conclude that tonin may play a role in the process of heart hypertrophy in the rat.

Adrenergic beta-Agonists↗
PubMed2003

Development of parthenogenetic rat embryos.

In an effort to establish cloning technology for the rat, we tested several methods (electric stimulation, treatment with ethanol or strontium) for the parthenogenetic activation of rat oocytes. We observed marked individual differences among rats of the outbred Wistar strain in their ability to yield activatable oocytes. These differences were independent of the activation protocol and may be due to a genetic predisposition that is crucial for the parthenogenetic activation of oocytes. The activation of oocytes was dependent upon the time between superovulation of the donor animal and the collection of the embryos. Aged oocytes (derived about 24 h after superovulation) were more prone to activation by each method than were younger oocytes, and some even underwent spontaneous activation without treatment and exhibited pronuclear formation and blastocyst development. All activation methods were effective in generating parthenogenetic rat embryos, and rat parthenotes developed until implantation. However, in general, short-term (15 min) and long-term (2 h) strontium treatment was superior to stimulation by ethanol or electric pulse for parthenogenetic activation. These results will be helpful in achieving successful cloning in the rat.

Animals↗