Yingbi Zhou

AT1b Receptor Predominantly Mediates Contractions in Major Mouse Blood Vessels

Abstract— In rodents, angiotensin (Ang) II type-1 (AT1) receptors exist as two pharmacologically identical subtypes: AT1a and AT1b. Recent studies have utilized mouse models with specific subtype receptor deletions to differentiate the functional difference between AT1 subtypes. However, little information is available on AT1 subtype expression in mouse vasculature. Therefore, in this study, AT1a−/− mice and wild-type littermates (AT1a+/+) were used to examine AT1 subtype expression and its functional relevance in mouse arterial vessels. Using RT-PCR and restriction enzyme digestion, we showed that AT1b accounts for most of the total AT1 mRNA in mouse abdominal aorta and femoral artery. In contrast, AT1a is the predominant subtype in kidney. To study the functional role of AT1 subtypes, we measured the in vitro contractility in vessels from AT1a−/− and AT1a+/+ mice. The Ang II concentration response curves in abdominal aorta and femoral artery were comparable between the two mouse strains. Furthermore, the Ang II response in AT1a−/− mouse vessels was completely antagonized by losartan, an AT1 antagonist. These results demonstrate that AT1b receptor is a major mediator for Ang II contractile response in mouse vessels, such as abdominal aorta and femoral artery.

Increased Expression of Cyclooxygenase-2 Mediates Enhanced Contraction to Endothelin ETA Receptor Stimulation in Endothelial Nitric Oxide Synthase Knockout Mice

The aim of this study was to determine whether prolonged loss of NO activity, in endothelial NO synthase knockout (eNOS−/−) mice, influences endothelin (ET) ETA receptor–mediated smooth muscle contraction and, if so, to define the underlying mechanism(s). In isolated endothelium-denuded abdominal aortas, contractions to the selective ETA receptor agonist ET-1(1-31) were significantly increased in aortas from eNOS−/− compared with wild-type (WT) mice. In contrast, contractions to the &agr;1-adrenergic agonist phenylephrine or the thromboxane (TX) A2 analog U-46619 were similar between eNOS−/− and WT mice. Immunofluorescent and Western blot analysis demonstrated that the aortic expression of ETA receptors was decreased in eNOS−/− compared with WT mice. Contractions evoked by ET-1(1-31), but not phenylephrine, were reduced by inhibition of cyclooxygenase-2 (COX-2) (indomethacin or celecoxib) or of TXA2/prostaglandin H2 receptors (SQ-29548). After COX inhibition, contractions to ET-1(1-31) were no longer increased and were actually decreased in eNOS−/− compared with WT aortas. Western blot analysis revealed that endothelium-denuded abdominal aortas express COX-2, but not COX-1, and that expression of COX-2 was significantly increased in eNOS−/− compared with WT mice. Contractions to the COX substrate arachidonic acid were also increased in eNOS−/− aortas. Furthermore, ET-1(1-31) but not phenylephrine stimulated production of the TXA2 metabolite TXB2, which was increased in eNOS−/− compared with WT aortas. Therefore, COX-2 plays a crucial and selective role in ETA-mediated smooth muscle contraction. Furthermore, COX-2 expression is increased in eNOS−/− mice, which overcomes a reduced expression of ETA receptors and enables a selective increase in contraction to ETA receptor stimulation.

Ablation of smooth muscle myosin heavy chain SM2 increases smooth muscle contraction and results in postnatal death in mice

The physiological relevance of smooth muscle myosin isoforms SM1 and SM2 has not been understood. In this study we generated a mouse model specifically deficient in SM2 myosin isoform but expressing SM1, using an exon-specific gene targeting strategy. The SM2 homozygous knockout (SM2−/−) mice died within 30 days after birth, showing pathologies including segmental distention of alimentary tract, retention of urine in renal pelvis, distension of bladder, and the development of end-stage hydronephrosis. In contrast, the heterozygous (SM2+/−) mice appeared normal and reproduced well. In SM2−/− bladder smooth muscle the loss of SM2 myosin was accompanied by a concomitant down-regulation of SM1 and a reduced number of thick filaments. However, muscle strips from SM2−/− bladder showed increased contraction to K+ depolarization or in response to M3 receptor agonist Carbachol. An increase of contraction was also observed in SM2−/− aorta. However, the SM2−/− bladder was associated with unaltered regulatory myosin light chain (MLC20) phosphorylation. Moreover, other contractile proteins, such as α-actin and tropomyosin, were not altered in SM2−/− bladder. Therefore, the loss of SM2 myosin alone could have induced hypercontractility in smooth muscle, suggesting that distinctly from SM1, SM2 may negatively modulate force development during smooth muscle contraction. Also, because SM2−/− mice develop lethal multiorgan dysfunctions, we propose this regulatory property of SM2 is essential for normal contractile activity in postnatal smooth muscle physiology.