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- 5-HT1A (2)
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- Endothelbarriere (1)
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- Institut für Anatomie und Zellbiologie (11) (remove)
Background: The angiotensin II receptor subtype 2 (AT2 receptor) is ubiquitously and highly expressed in early postnatal life. However, its role in postnatal cardiac development remained unclear.
Methodology/Principal Findings: Hearts from 1, 7, 14 and 56 days old wild-type (WT) and AT2 receptor-deficient (KO) mice were extracted for histomorphometrical analysis as well as analysis of cardiac signaling and gene expression. Furthermore, heart and body weights of examined animals were recorded and echocardiographic analysis of cardiac function as well as telemetric blood pressure measurements were performed. Moreover, gene expression, sarcomere shortening and calcium transients were examined in ventricular cardiomyocytes isolated from both genotypes. KO mice exhibited an accelerated body weight gain and a reduced heart to body weight ratio as compared to WT mice in the postnatal period. However, in adult KO mice the heart to body weight ratio was significantly increased most likely due to elevated systemic blood pressure. At postnatal day 7 ventricular capillarization index and the density of \(\alpha\)-smooth muscle cell actin-positive blood vessels were higher in KO mice as compared to WT mice but normalized during adolescence. Echocardiographic assessment of cardiac systolic function at postnatal day 7 revealed decreased contractility of KO hearts in response to beta-adrenergic stimulation. Moreover, cardiomyocytes from KO mice showed a decreased sarcomere shortening and an increased peak Ca\(^{2+}\) transient in response to isoprenaline when stimulated concomitantly with angiotensin II.
Conclusion: The AT2 receptor affects postnatal cardiac growth possibly via reducing body weight gain and systemic blood pressure. Moreover, it moderately attenuates postnatal vascularization of the heart and modulates the beta adrenergic response of the neonatal heart. These AT2 receptor-mediated effects may be implicated in the physiological maturation process of the heart.
Local axonal function of STAT3 rescues axon degeneration in the pmn model of motoneuron disease
(2012)
Axonal maintenance, plasticity, and regeneration are influenced by signals from neighboring cells, in particular Schwann cells of the peripheral nervous system. Schwann cells produce neurotrophic factors, but the mechanisms by which ciliary neurotrophic factor (CNTF) and other neurotrophic molecules modify the axonal cytoskeleton are not well understood. In this paper, we show that activated signal transducer and activator of transcription-3 (STAT3), an intracellular mediator of the effects of CNTF and other neurotrophic cytokines, acts locally in axons of motoneurons to modify the tubulin cytoskeleton. Specifically, we show that activated STAT3 interacted with stathmin and inhibited its microtubule-destabilizing activity. Thus, ectopic CNTF-mediated activation of STAT3 restored axon elongation and maintenance in motoneurons from progressive motor neuronopathy mutant mice, a mouse model of motoneuron disease. This mechanism could also be relevant for other neurodegenerative diseases and provide a target for new therapies for axonal degeneration.
Multiple fluorescence in situ hybridization is the method of choice for studies aimed at determining simultaneous production of signal transduction molecules and neuromodulators in neurons. In our analyses of the monoamine receptor mRNA expression of peptidergic neurons in the rat telencephalon, double tyramide-signal-amplified fluorescence in situ hybridization delivered satisfactory results for coexpression analysis of neuropeptide Y (NPY) and serotonin receptor 2C (5-HT2C) mRNA, a receptor subtype expressed at high-to-moderate abundance in the regions analyzed. However, expression of 5-HT1A mRNA, which is expressed at comparatively low abundance in many telencephalic areas, could not be unequivocally identified in NPY mRNA-reactive neurons due to high background and poor signal-to-noise ratio in fluorescent receptor mRNA detections. Parallel chromogenic in situ hybridization provided clear labeling for 5-HT1A mRNA and additionally offered the possibility to monitor the chromogen deposition at regular time intervals to determine the optimal signal-to-noise ratio. We first developed a double labeling protocol combining fluorescence and chromogenic in situ hybridization and subsequently expanded this variation to combine double fluorescence and chromogenic in situ hybridization for triple labelings. With this method, we documented expression of 5-HT2C and/or 5-HT1A in subpopulations of telencephalic NPY-producing neurons. The method developed in the present study appears suitable for conventional light and fluorescence microscopy, combines advantages of fluorescence and chromogenic in situ hybridization protocols and thus provides a reliable non-radioactive alternative to previously published multiple labeling methods for coexpression analyses in which one mRNA species requires highly sensitive detection.
Aims
Cardiac atrial natriuretic peptide (ANP) participates in the maintenance of arterial blood pressure and intravascular volume homeostasis. The hypovolaemic effects of ANP result from coordinated actions in the kidney and systemic microcirculation. Hence, ANP, via its guanylyl cyclase-A (GC-A) receptor and intracellular cyclic GMP as second messenger, stimulates endothelial albumin permeability. Ultimately, this leads to a shift of plasma fluid into interstitial pools. Here we studied the role of caveolae-mediated transendothelial albumin transport in the hyperpermeability effects of ANP.
Methods and results
Intravital microscopy studies of the mouse cremaster microcirculation showed that ANP stimulates the extravasation of fluorescent albumin from post-capillary venules and causes arteriolar vasodilatation. The hyperpermeability effect was prevented in mice with conditional, endothelial deletion of GC-A (EC GC-A KO) or with deleted caveolin-1 (cav-1), the caveolae scaffold protein. In contrast, the vasodilating effect was preserved. Concomitantly, the acute hypovolaemic action of ANP was abolished in EC GC-A KO and Cav-1−/− mice. In cultured microvascular rat fat pad and mouse lung endothelial cells, ANP stimulated uptake and transendothelial transport of fluorescent albumin without altering endothelial electrical resistance. The stimulatory effect on albumin uptake was prevented in GC-A- or cav-1-deficient pulmonary endothelia. Finally, preparation of caveolin-enriched lipid rafts from mouse lung and western blotting showed that GC-A and cGMP-dependent protein kinase I partly co-localize with Cav-1 in caveolae microdomains.
Conclusion
ANP enhances transendothelial caveolae-mediated albumin transport via its GC-A receptor. This ANP-mediated cross-talk between the heart and the microcirculation is critically involved in the regulation of intravascular volume.
Aims/hypothesis
Several glucose-sensing pathways have been implicated in glucose-triggered secretion of glucagon-like peptide-1 (GLP-1) from intestinal L cells. One involves glucose metabolism and closure of ATP-sensitive K\(^+\) channels, and another exploits the electrogenic nature of Na\(^+\)-coupled glucose transporters (SGLTs). This study aimed to elucidate the role of these distinct mechanisms in glucose-stimulated GLP-1 secretion.
Methods
Glucose uptake into L cells (either GLUTag cells or cells in primary cultures, using a new transgenic mouse model combining proglucagon promoter-driven Cre recombinase with a ROSA26tdRFP reporter) was monitored with the FLII\(_{12}\)Pglu-700μδ6 glucose sensor. Effects of pharmacological and genetic interference with SGLT1 or facilitative glucose transport (GLUT) on intracellular glucose accumulation and metabolism (measured by NAD(P)H autofluorescence), cytosolic Ca\(^{2+}\) (monitored with Fura2) and GLP-1 secretion (assayed by ELISA) were assessed.
Results
L cell glucose uptake was dominated by GLUT-mediated transport, being abolished by phloretin but not phloridzin. NAD(P)H autofluorescence was glucose dependent and enhanced by a glucokinase activator. In GLUTag cells, but not primary L cells, phloretin partially impaired glucose-dependent secretion, and suppressed an amplifying effect of glucose under depolarising high K\(^+\) conditions. The key importance of SGLT1 in GLUTag and primary cells was evident from the impairment of secretion by phloridzin or Sglt1 knockdown and failure of glucose to trigger cytosolic Ca\(^{2+}\) elevation in primary L cells from Sglt1 knockout mice.
Conclusions/interpretation
SGLT1 acts as the luminal glucose sensor in L cells, but intracellular glucose concentrations are largely determined by GLUT activity. Although L cell glucose metabolism depends partially on glucokinase activity, this plays only a minor role in glucose-stimulated GLP-1 secretion.
The GTPase ARFRP1 controls the lipidation of chylomicrons in the Golgi of the intestinal epithelium
(2012)
The uptake and processing of dietary lipids by the small intestine is a multistep process that involves several steps including vesicular and protein transport. The GTPase ADP-ribosylation factor-related protein 1 (ARFRP1) controls the ARF-like 1 (ARL1)-mediated Golgi recruitment of GRIP domain proteins which in turn bind several Rab-GTPases. Here, we describe the essential role of ARFRP1 and its interaction with Rab2 in the assembly and lipidation of chylomicrons in the intestinal epithelium. Mice lacking Arfrp1 specifically in the intestine \((Arfrp1^{vil−/−})\) exhibit an early post-natal growth retardation with reduced plasma triacylglycerol and free fatty acid concentrations. \(Arfrp1^{vil−/−}\) enterocytes as well as Arfrp1 mRNA depleted Caco-2 cells absorbed fatty acids normally but secreted chylomicrons with a markedly reduced triacylglycerol content. In addition, the release of apolipoprotein A-I (ApoA-I) was dramatically decreased, and ApoA-I accumulated in the \(Arfrp1^{vil−/−}\) epithelium, where it predominantly co-localized with Rab2. The release of chylomicrons from Caco-2 was markedly reduced after the suppression of Rab2, ARL1 and Golgin-245. Thus, the GTPase ARFRP1 and its downstream proteins are required for the lipidation of chylomicrons and the assembly of ApoA-I to these particles in the Golgi of intestinal epithelial cells.
Multiple fluorescence in situ hybridization is the method of choice for studies aimed at determining simultaneous production of signal transduction molecules and neuromodulators in neurons. In our analyses of the monoamine receptor mRNA expression of peptidergic neurons in the rat telencephalon, double tyramide-signal-amplified fluorescence in situ hybridization delivered satisfactory results for coexpression analysis of neuropeptide Y (NPY) and serotonin receptor 2C (5-HT2C) mRNA, a receptor subtype expressed at high-to-moderate abundance in the regions analyzed. However, expression of 5-HT1A mRNA, which is expressed at comparatively low abundance in many telencephalic areas, could not be unequivocally identified in NPY mRNA-reactive neurons due to high background and poor signal-to-noise ratio in fluorescent receptor mRNA detections. Parallel chromogenic in situ hybridization provided clear labeling for 5-HT1A mRNA and additionally offered the possibility to monitor the chromogen deposition at regular time intervals to determine the optimal signal-to-noise ratio. We first developed a double labeling protocol combining fluorescence and chromogenic in situ hybridization and subsequently expanded this variation to combine double fluorescence and chromogenic in situ hybridization for triple labelings. With this method, we documented expression of 5-HT2C and/or 5-HT1A in subpopulations of telencephalic NPY-producing neurons. The method developed in the present study appears suitable for conventional light and fluorescence microscopy, combines advantages of fluorescence and chromogenic in situ hybridization protocols and thus provides a reliable non-radioactive alternative to previously published multiple labeling methods for coexpression analyses in which one mRNA species requires highly sensitive detection.
Reorganisation der Zellkontakte der Endothelbarriere bei der Stabilisierung durch cAMP und Rac1
(2012)
Zwischen Blutkompartiment und umliegenden Interstitium besteht eine Barriere, die durch eine einzelne Schicht aus Endothelzellen gebildet wird. Essentiell für diese Barriere, deren Funktion in der Begrenzung des Austausches von Flüssigkeit und gelösten Stoffen liegt, sind interzelluläre Junktionen, welche die Endothelzellen miteinander verbinden. Durch eine gestörte Funktion und Regulation der Endothelbarriere entstehen beim Menschen verschiedene Pathologien wie zum Beispiel Ödeme, hämorrhagischer Schlaganfall und vaskuläre Malformationen.
Es ist bekannt, dass cAMP die Endothelbarriere zum Teil durch Aktivierung der kleinen GTPase Rac1 stabilisiert. Trotz der großen medizinischen Relevanz dieses Signalweges, sind die damit einhergehenden Effekte auf die interzellulären Kontakte auf ultrastruktureller Ebene weitgehend unbekannt.
In mikrovaskulären Endothelzellkulturen kam es ähnlich wie in intakten Mikrogefäßen zur Stärkung der Barrierefunktion. So resultierte sowohl nach Behandlung mit Forskolin und Rolipram (F/R), welche zur Steigerung der intrazellulären cAMP-Spiegel führen, als auch nach Zugabe von 8-(4-chlorophenylthio)-2´-O-methyladenosin-3´,5´-cyclic monophosphorothioate (O-Me-cAMP), einem selektiven Aktivator des cAMP nachgeschalteten Epac/Rap1-Signalweges, ein Anstieg des TER; außerdem konnte durch beide Substanzen (F/R und O-Me-cAMP) die Aktivierung von Rac1 induziert werden. Desweiteren wurde eine verstärkte Intensität und Linearisierung des Immunfluoreszenzsignals der Zelljunktionsproteine VE-Cadherin und Claudin5 entlang der Zellgrenzen beobachtet.
In der ultrastrukturellen Analyse der interzellulären Kontaktzonen-Architektur zeigte sich unter F/R- oder O-Me-cAMP-Exposition ein signifikanter Anstieg an komplexen Interdigitationen. Diese komplexen Strukturen waren dadurch charakterisiert, dass sich die Membranen benachbarter Zellen, die durch zahlreiche endotheliale Junktionen stabilisiert wurden, über vergleichsweise lange Distanzen eng aneinanderlegten, so dass ein deutlich verlängerter Interzellularspalt resultierte. Die Inhibition der Rac1-Aktivierung durch NSC-23766 verminderte die Barrierefunktion und blockierte effektiv die O-Me-cAMP-vermittelte Barrierestabilisierung und Reorganisation der Kontaktzone einschließlich der Junktionsproteine.
Demgegenüber konnte die F/R-vermittelte Barrierestabilisierung durch NSC-23766 nicht beeinträchtigt werden.
Parallel dazu durchgeführte Experimente mit makrovaskulären Endothelien zeigten, dass es in diesem Zelltyp unter Bedingungen erhöhter cAMP-Konzentrationen weder zur Rac1-Aktivierung noch zur Barrierestärkung oder Kontaktzonen-Reorganisation kam.
Diese Ergebnisse deuten darauf hin, dass in mikrovaskulären Endothelien Rac1-vermittelte Änderungen der Kontaktzonen-Morphologie zur cAMP-induzierten Barrierestabilisierung beitragen.
Die organischen Kationentransporter der SLC22-Familie spielen eine Schlüsselrolle bei der Aufnahme, Ausscheidung und Verteilung vieler kationischer Medikamente und endogener Substanzen. Der erste klonierte organische Kationentransporter rOCT1 (OCT1 aus der Ratte) wurde bisher eingehend funktionell charakterisiert. rOCT1 ist elektrogen, transportiert organische Kationen unterschiedlicher Struktur wie z.B. Cholin, Tetraethylammonium (TEA) oder das Neurotoxin 1 Methyl-4-Phenylpyridinium (MPP) und wird durch verschiedene Substanzen wie beispielsweise Tetrabutylammonium (TBuA) inhibiert. Für die Entwicklung und Optimierung von Medikamenten ist ein besseres Verständnis der strukturellen Grundlage der polyspezifischen Substraterkennung und des Transportprozesses von entscheidender Bedeutung. Durch modellgestützte Mutagenese konnte für rOCT1 ein großer Spalt identifiziert werden, der von acht Transmembranhelices (TMHs) geformt wird und die putative Substratbindungstasche mit überlappenden Bindungsdomänen beinhaltet. Mittels der „Voltage-Clamp-Fluorometrie“ können Konformationsänderungen von rOCT1 während des Transportzyklus sichtbar gemacht werden. Unter Verwendung dieser Methode wurden spannungsabhängige Fluoreszenzänderungen in den Positionen 260, 380 und 483 der TMHs 5, 8 und 11 nachgewiesen. Interaktionen mit den Substraten Cholin und MPP sowie dem nicht transportierten Inhibitor TBuA von außen wirkten sich unterschiedlich auf die Bewegungen in den drei Positionen aus. Diese Ergebnisse demonstrieren, dass rOCT1 spannungsabhängige Konformationen einnimmt, bei deren Änderungen sich mindestens drei Transmembrandomänen (TMH 5, TMH 8 und TMH 11) bewegen und dass in Gegenwart von organischen Kationen die Spannungsabhängigkeit der Transporterkonformation beeinflusst wird. Des Weiteren wurde eine kritische Position innerhalb oder nahe der Substratbindungstasche von rOCT1 identifiziert, mit deren Hilfe der Transportweg irreversibel blockiert werden kann. In Position 478 wurde das Glycin durch ein Cystein ersetzt, das mittels des SH Gruppenreagenzes [2-(Trimethylammonium)ethyl] methanethiosulfonat Bromid (MTSET) kovalent modifiziert werden konnte. Diese Modifikation bewirkte eine starke Hemmung des Transports verschiedener Substrate wie z.B. Cholin, TEA oder MPP. Anhand von Bindungsstudien konnte gezeigt werden, dass die Bindung von MPP durch die MTSET Modifizierung in der nach außen gerichteten Konformation verhindert wurde. Die Einführung des Cysteins in Position 478 erhöhte die Affinität von TBuA und beeinflusste außerdem die substrat- und spannungsabhängigen Konformationsänderungen. Hierbei zeigte sich, dass in zwei der drei Positionen (260 und 483) die Fluoreszenzantwort des leeren Transporters verändert wurde. Neben den Fluoreszenzen im Gleichgewichtszustand wurden auch die Zeitkonstanten der Fluoreszenzantworten durch die Position 478 beeinflusst. Durch die Einführung eines Serins oder Threonins in diese Position konnten die Effekte des Cysteins 478 in Position 483 nachgeahmt werden. Die Blockierung des Transportwegs durch MTSET veränderte die Bewegungen des leeren Transporters in Position 260 und 483 kaum, während in Position 380 eine deutliche Reduktion der Fluoreszenzantwort gemessen wurde. Auch die substratabhängigen Fluoreszenzänderungen wurden in der Position 483 deutlich reduziert. Insgesamt weisen diese Daten darauf hin, dass rOCT1 Konformationsänderungen durchläuft, die spannungs- und substratabhängig sind und durch die Position 478 beeinflusst werden.
During stroke the blood–brain barrier (BBB) is damaged which can result in vasogenic brain edema and inflammation. The reduced blood supply leads to decreased delivery of oxygen and glucose to affected areas of the brain. Oxygen and glucose deprivation (OGD) can cause upregulation of glucose uptake of brain endothelial cells. In this letter, we investigated the influence of MK801, a non-competitive inhibitor of the NMDA-receptor, on the regulation of the glucose uptake and of the main glucose transporters glut1 and sglt1 in murine BBB cell line cerebEND during OGD. mRNA expression of glut1 was upregulated 68.7- fold after 6 h OGD, which was significantly reduced by 10 μM MK801 to 28.9-fold. Sglt1 mRNA expression decreased during OGD which was further reduced by MK801. Glucose uptake was significantly increased up to 907% after 6 h OGD and was still higher (210%) after the 20 h reoxygenation phase compared to normoxia. Ten micromolar MK801 during OGD was able to reduce upregulated glucose uptake after OGD and reoxygenation significantly. Presence of several NMDAR subunits was proven on the mRNA level in cerebEND cells. Furthermore, it was shown that NMDAR subunit NR1 was upregulated during OGD and that this was inhibitable by MK801. In conclusion, the addition of MK801 during the OGD phase reduced significantly the glucose uptake after the subsequent reoxygenation phase in brain endothelial cells.