Structural and functional characterization of a transgenic rat model of the human endothelin type A receptor

Frontpage Acknowledgements Table of Contents Abbreviations Introduction Material and Methods Results Discussion BibliographyThis dissertation work reports phenotypic findings in two independent lines of a tg rat model designed to express the human ETA receptor specifically in VSMCs. Based on mRNA expression data, both tg lines represent different vascular-bed specificity of ETA overexpression, with L6351 rats showing more pronounced expression in large arteries, whereas L6878 rats showed more pronounced increase in ETA expression in the mesenteric vascular bed. According to this expression pattern, the moderate, but significant increase in the media of large arteries (aorta, carotid artery) in L6351 was consistent with blood-pressure independent hypertrophic ETA effects. In contrast, the hypertensive phenotype in L6878 was associated with mesenteric artery wall hypertrophy and lumen loss. The analysis of this tg model was significantly hampered by the rapid decrease in transgene expression soon after birth (as evidenced from RNA expression data) which compelled us to analyze the phenotype in a very short time window of age of around one month. Preliminary results of receptor binding studies suggest that ETA transcript levels may underestimate the level of protein expression. This is supported by functional analysis of isolated-perfused 1st order mesenteric arteries showing no apparent association of ET-1 induced contractility with the level of ETA transcripts detected in homogenates of the superior mesenteric artery vascular bed. Moreover, as expected contractile response to ET-1 was less pronounced when compared to the increase in ETA transcript level (L6878) suggesting inhibitory posttranslational modulation of ETA function. It is worth noting that our (currently still limited) knowledge of ET receptor binding in this model is suggestive of a posttranslational ETA/ETB receptor interaction. Furthermore, demonstration of significant endothelial dysfunction (i.e., decreased ACh-induced vasorelaxation) in tg mesenteric arteries can be regarded as indirect functional evidence of increased ETA function. A standard protocol of injection anesthesia based on coadministration of the NMDA antagonist S-ketamine with the alpha2-adrenergic agonist xylazine, revealed induction of hypertension as a distinct secondary phenotype. According to our data, the exaggerated release of catecholamines by S-ketamine provides a plausible explanation. First, K/X-anesthetized tg rats showed significantly decreased blood pressure response to alpha1-adrenergic stimulation. Second, K/X-induced hypertension was immediately abolished by the alpha1-adrenergic antagonist prazosin. Third, isolated perfused mesenteric arteries of tg rats showed attenuated response to PE-induced constriction. Taken together, the phenotypic data presented in this dissertation provide the first direct in vivo evidence of a pathophysiological role of ETA in vascular hypertrophy and are also suggestive of a role for mesenteric artery ETA in high blood pressure induction. Furthermore, the data suggest an interaction of the ETA receptor with the adrenergic system.In dieser Dissertation werden die Phänotypen von zwei unabhängig generierten transgenen Rattenlinien beschrieben, die mit dem Ziel der gefäßmuskelspezifischen Überexpression des humanen Endothelin A Rezeptors (ETA) generiert wurden. Analysen der mRNA-Expression zeigten eine unterschiedliche Muster der vaskulären ETA-Überexpression mit einer ausgeprägteren Expression in großen Arterien in Linie L6351, während in Linie L6878 eine Überexpression in mesenterialen Gefäßbett gefunden wurde. Übereinstimmend mit diesem Expressionsmuster zeigte Linie L6351 eine signifikante, wenn auch moderat ausgeprägte blutdruckunabhängge Mediahypertrophie in Aorta und A. carotis. In der moderat hypertensiven Linie L6878 wurde hingegen auch in kleinen mesenterialen Arterien eine signifikante Gefäßwandhypertrophie assoziiert mit Lumenverlust gefunden. Die phänotypische Analyse war erschwert durch die postnatale Abregulation der Expression des Transgens, die Untersuchungen an sehr jungen Tieren (Alter ca. 1 Monat) erforderlich machten. Unsere noch vorläufigen Ergebnisse aus Rezeptorbindungsstudien deuten darauf hin, dass der relative Anstieg der mRNA- Spiegel geringer sein könnte als die Zunahme der Proteinexpression. Diese Annahme wird gestützt durch funktionelle Untersuchungen an isoliert- perfundierten Ästen der A. mesenterica der Linie L6351, weche eine signifikante Zunahme der Kontraktilität auf ET-1 bei nur geringer Zunahme der Transkriptspiegel zeigten. Die im Vergleich deutlich höhere mRNA-Expression des ETA in der mesenterialen Zirkulation der Linie L6878 war hingegen nicht mit einer im Vergleich zu L6351 erwarteten deutlich verstärkten kontraktilen Antwort isoliert-perfunidierter Mesenterialarterien assoziiert, was auch auf eine funktionelle Inhibition des ETA hinweisen könnte. Unsere bisherigen Rezeptorbindungsdaten weisen zudem auf eine posttranslationale Interaktion zwischen ETA und ETB. Zudem interpretieren wir die beobachtete endotheliale Dysfunktion in isoliert perfundierten transgenen Mesenterialarterien als funktionellen Ausdruck der ETA-Übexpression. Ein nicht erwarteter sekundärer Phänotyp zeigte sich durch Induktion eines ausgeprägten arteriellen Hypertonus nach Injektion des Anästhetikums S-Ketamin in Kombination mit dem Alpha 2-Agonisten Xylazin (K/X). Nach den vorliegenden Ergebnissen nehmen wir eine exzessive Freisetzung von Katecholaminen bei den transgenenTieren als Ursache an: Erstens zeigten mit K/X anästhesierte Tiere eine stark abgeschwächte Blutdruckantwort auf den Alpha 1-Agoinsten Phenylephrin (PE), zweitens wurde der K/X-induzierte Hypertonus durch Injektion des Alpha 1-Antagonisten Prazosin sofort normalisiert und drittens zeigten isoliert perfundierte Mesenterialarterien eine dramatisch abgeschwächte kontraktile Antwort auf PE. Zusammengefasst belegen die in dieser Dissertation erarbeiteten phänotypischen Ergebnisse erstmals einen direkten pathophysiologischen Zusammenhang zwischen dem ETA-Rezeptor und der vaskulären Hypertrophie in vivo und liefern Hinweise auf eine unmittelbare hypertensive Rolle dieses Rezeptors sowie auf eine funktionelle Interaktion mit dem adrenergen System

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