Desensitization and Internalization of Endothelin Receptor A: IMPACT OF G PROTEIN-COUPLED RECEPTOR KINASE 2 (GRK2)-MEDIATED PHOSPHORYLATION*

Endothelin receptor A (ET_A), a G protein-coupled receptor, mediates endothelin signaling, which is regulated by GRK2. Three Ser and seven Thr residues recently proven to be phosphoacceptor sites are located in the C-terminal extremity (CTE) of the receptor following its palmitoylation site. We created various phosphorylation-deficient ET_A mutants. The phospholipase C activity of mutant receptors in HEK-293 cells was analyzed during continuous endothelin stimulation to investigate the impact of phosphorylation sites on ET_A desensitization. Total deletion of phosphoacceptor sites in the CTE affected proper receptor regulation. However, proximal and distal phosphoacceptor sites both turned out to be sufficient to induce WT-like desensitization. Overexpression of the Gα_q coupling-deficient mutant GRK2-D110A suppressed ET_A-WT signaling but failed to decrease phospholipase C activity mediated by the phosphorylation-deficient mutant ET_A-6PD. In contrast, GRK2-WT acted on both receptors, whereas the kinase-inactive mutant GRK2-D110A/K220R failed to inhibit signaling of ET_A-WT and ET_A-6PD. This demonstrates that ET_A desensitization involves at least two autonomous GRK2-mediated components: 1) a phosphorylation-independent signal decrease mediated by blocking of Gα_q and 2) a mechanism involving phosphorylation of Ser and Thr residues in the CTE of the receptor in a redundant fashion, able to incorporate either proximal or distal phosphoacceptor sites. High level transfection of GRK2 variants influenced signaling of ET_A-WT and ET_A-6PD and hints at an additional phosphorylation-independent regulatory mechanism. Furthermore, internalization of mRuby-tagged receptors was observed with ET_A-WT and the phosphorylation-deficient mutant ET_A-14PD (lacking 14 phosphoacceptor sites) and turned out to be based on a phosphorylation-independent mechanism.     

低氧对高原鼠兔和大鼠血液NO与ET-1的影响

将Wistar大鼠暴露于3 780 m低氧环境,分别于24 h、2 wk及3 wk后采用酶联免疫法和硝酸还原酶法测定血液中的ET~(-1)和NO的含量,计算NO/ET~(-1)值,并与高原鼠兔比较,探讨低氧条件下大鼠与高原鼠兔血液中NO与ET~(-1)含量的变化趋势。结果表明,低氧24 h后,大鼠血液中NO和ET~(-1)的含量显著高于同海拔的高原鼠兔(P<0·01),而NO/ET~(-1)值无显著差异(P>0·05)。随着大鼠在高海拔停留时间的延长,血液中NO含量呈减少趋势,而ET~(-1)则有上升趋势,二者呈显著的负相关(r2=0·2416,P<0·01)。高原鼠兔NO/ET~(-1)值约为大鼠低氧2 wk和3 wk的2倍(P<0·01)。说明不同低氧暴露时间,高原鼠兔和大鼠的NO、ET~(-1)及NO/ET~(-1)值有显著差异,提示NO/ET~(-1)值可以作为有机体是否适应高原低氧环境的一个指标。     

EDNRB基因编码区点突变及多态性在浙江地区散发性先天性巨结肠症中的检测与分析

本实验应用聚合酶链反应-单链构象多态性(single strand confbrmation polymorphism analysis of polymerase chain reaction products,PCR-SSCP)和DNA直接测序技术,对75例浙江地区散发性先天性巨结肠病例EDNRB基因编码区的全部7个外显子,进行了点突变与单核苷酸多态性的检测与分析,探讨浙江地区先天性巨结肠患者EDNRB基因的突变特征,阐明EDNRB基因与散发性先天性巨结肠症发病之间可能存在的关系。结果有6例患者在第4外显子上检测到密码子277位点 CTG→CTA的置换,导致亮氨酸的同义突变(L277L),属于单核苷酸多态性,发生率为8％(6/75)。有 2例患者在第2外显子上检测到密码子185位点GTG→ATG的置换,导致缬氨酸到蛋氨酸的错义突变(V185M),此突变型未在国内外文献中报道过,认为是新的基因突变型,突变率2．7％(2/75)。研究结果表明,浙江地区先天性巨结肠群体可发生EDNRB基因的杂合性突变,提示EDNRB基因与先天性巨结肠症的发病存在一定程度的关联。     

螺内酯治疗老年高血压的临床疗效及对血清炎性因子水平的影响

目的:探讨螺内酯治疗老年高血压的临床疗效及对血清炎性因子水平的影响。方法:选择2017年6月至2018年12月我院收治的144例老年高血压患者,根据治疗方法的不同将其分为观察组80例与对照组64例。对照组给予硝苯地平治疗,观察组给予螺内酯治疗,两组均治疗观察4周,对比两组治疗前后的血清内皮素(Endothelin,ET)-1和血栓素(Thromboxane,TX)-B2含量、白介素(Interleukin,IL)-6与可溶性细胞间粘附分子(Soluble intercellular adhesion molecule,s ICAM-1)水平的变化及临床疗效。结果:所有患者完成治疗,无严重不良反应发生。治疗后,观察组的总有效率为98.8%,显著高于对照组(85.9%,P0.05)。两组治疗后24 h收缩压(Systolic blood pressure,SBP)和舒张压(Diastolic bloodpressure,DBP)、血清ET-1、TX-B2、IL-6与s ICAM-1水平均显著低于治疗前(P0.05),且观察组以上指标均明显低于对照组(P0.05)。结论:螺内酯治疗老年高血压的临床疗效明显优于硝苯地平治疗,可能与其明显抑制血清与ET-1、TX-B2、IL-6与s ICAM-1水平有关。     

即刻电针介入对IBS模型大鼠血浆ET,CGRP水平及结肠ETR-A,CGRPmRNA表达的影响

目的：比较即刻电针天枢穴、足三里穴对肠易激综合征（ms）模型大鼠血浆降钙基因相关肽（CGRP）、内皮素（ET）水平及结肠组织中内皮素受体A（ETR－A）、CGRPmRNA表达的影响,旨在探讨电针即刻治疗IBS的部分机制。方法：采用WISTAR幼鼠制备肠易激综合征模型,随机分为空白对照组、模型组、天枢组、足三里组,每组8只。空白对照组不作任何处理,模型组只束缚不针刺,天枢组和足三里组在实验第8周电针治疗一次,留针20min。治疗结束后处死大鼠,取大鼠血浆及部分结肠组织进行生物活性物质检测。采用酶联免疫法检测血浆中CGRP、ET、结肠组织中ETR—A的含量,采用RT—PCR法检测结肠组织中CGRPmRNA表达。结果：（1）即刻电针对IBS模型大鼠血浆CGRP、ET水平的影响：与空白对照组比较,模型组CGRP水平明显降低（P〈0．01）;与模型组比较,天枢组、足三里组CGRP水平明显升高（P〈0．01）。与空白对照组比较,模型组ET水平升高（P〈0．05）;与模型组比较,天枢组ET水平明显降低（P〈0．01）。（2）即刻电针对IBS模型大鼠结肠组织ETR—A水平的影响：与空白对照组比较,模型组ETR—A水平明显升高（P〈0．01）;与模型组比较,足三里组ETR-A水平明显降低（P〈0．01）。（3）即刻电针对IBS模型大鼠结肠组织CGRPmRNA表达的影响：与空白对照组比较,模型组、天枢组、足三里组CGRPmRNA表达明显增强（P〈0．01,P〈0．05）;与模型组比较,足三里组CGRPmRNA表达减弱（P〈0．05）。结论：即刻电针介入后,能够调节机体的内环境紊乱和CGRP、ET的平衡失调。这种调节作用因穴位不同而具有不同的特点,天枢穴对血浆中CGRP、ET调节作用较强,足三里穴在受体和基因表达方面作用明显。     

彩色多普勒超声诊断不同病程老年2型糖尿病下肢血管病变的临床价值及与血清ET、NO的关系研究

摘要 目的：探讨彩色多普勒超声诊断不同病程老年2型糖尿病(T2DM)下肢血管病变(LEADDP)的临床价值及与血清内皮素(ET)、一氧化氮(NO)的关系。方法：选取我院于2019年5月~2020年4月期间收治的80例老年T2DM合并LEADDP患者为观察组,根据不同病程分为3组,<10年组30例,10~20年组33例,>20年组17例,另选取同时期我院收治的50名老年单纯T2DM患者为对照组,所有受检者均接受彩色多普勒超声检查,并检测血清ET、NO水平。对比观察组与对照组足背动脉狭窄发生率,对比不同病程患者LEADDP检出率,对比不同病程患者及对照组的动脉血管内径、动脉血流量及血清ET、NO水平,Pearson相关性分析动脉血管内径、动脉血流量与血清ET和NO水平的相关性。结果：观察组足背动脉狭窄发生率明显高于对照组(P<0.05);不同病程患者LEADDP检出率随着病程延长而增加(P<0.05);不同病程患者动脉血管内径、动脉血流量、NO水平随着病程的的延长而降低,ET水平随着病程的延长而升高(P<0.05);Pearson相关性分析显示,动脉血管内径、动脉血流量与血清ET水平呈负相关,与NO水平呈正相关(P<0.05)。结论：老年T2DM合并LEADDP患者存在明显的下肢动脉管径狭窄和血流量缓慢,病变程度随着病程延长而增加,与血清ET、NO水平有密切关系,彩色多普勒超声诊断可有效评价其病变程度。     

Cell-free synthesis of membrane proteins: Tailored cell models out of microsomes

Incorporation of proteins in biomimetic giant unilamellar vesicles (GUVs) is one of the hallmarks towards cell models in which we strive to obtain a better mechanistic understanding of the manifold cellular processes. The reconstruction of transmembrane proteins, like receptors or channels, into GUVs is a special challenge. This procedure is essential to make these proteins accessible to further functional investigation. Here we describe a strategy combining two approaches: cell-free eukaryotic protein expression for protein integration and GUV formation to prepare biomimetic cell models. The cell-free protein expression system in this study is based on insect lysates, which provide endoplasmic reticulum derived vesicles named microsomes. It enables signal-induced translocation and posttranslational modification of de novo synthesized membrane proteins. Combining these microsomes with synthetic lipids within the electroswelling process allowed for the rapid generation of giant proteo-liposomes of up to 50 μm in diameter. We incorporated various fluorescent protein-labeled membrane proteins into GUVs (the prenylated membrane anchor CAAX, the heparin-binding epithelial growth factor like factor Hb-EGF, the endothelin receptor ETB, the chemokine receptor CXCR4) and thus presented insect microsomes as functional modules for proteo-GUV formation. Single-molecule fluorescence microscopy was applied to detect and further characterize the proteins in the GUV membrane. To extend the options in the tailoring cell models toolbox, we synthesized two different membrane proteins sequentially in the same microsome. Additionally, we introduced biotinylated lipids to specifically immobilize proteo-GUVs on streptavidin-coated surfaces. We envision this achievement as an important first step toward systematic protein studies on technical surfaces.     

VIP and endothelin receptor antagonist: An effective combination against experimental pulmonary arterial hypertension

Background

Pulmonary Arterial Hypertension (PAH) remains a therapeutic challenge, and the search continues for more effective drugs and drug combinations. We recently reported that deletion of the vasoactive intestinal peptide (VIP) gene caused the spontaneous expression of a PH phenotype that was fully corrected by VIP. The objectives of this investigation were to answer the questions: 1) Can VIP protect against PH in other experimental models? and 2) Does combining VIP with an endothelin (ET) receptor antagonist bosentan enhance its efficacy?

Methods

Within 3 weeks of a single injection of monocrotaline (MCT, s.c.) in Sprague Dawley rats, PAH developed, manifested by pulmonary vascular remodeling, lung inflammation, RV hypertrophy, and death within the next 2 weeks. MCT-injected animals were either untreated, treated with bosentan (p.o.) alone, with VIP (i.p.) alone, or with both together. We selected this particular combination upon finding that VIP down-regulates endothelin receptor expression which is further suppressed by bosentan. Therapeutic outcomes were compared as to hemodynamics, pulmonary vascular pathology, and survival.

Results

Treatment with VIP, every other day for 3 weeks, begun on the same day as MCT, almost totally prevented PAH pathology, and eliminated mortality for 45 days. Begun 3 weeks after MCT, however, VIP only partially reversed PAH pathology, though more effectively than bosentan. Combined therapy with both drugs fully reversed the pathology, while preventing mortality for at least 45 days.

Conclusions

1) VIP completely prevented and significantly reversed MCT-induced PAH; 2) VIP was more effective than bosentan, probably because it targets a wider range of pro-remodeling pathways; and 3) combination therapy with VIP plus bosentan was more effective than either drug alone, probably because both drugs synergistically suppressed ET-ET receptor pathway.     

Induction of 5-aminolevulinate synthase by activators of steroid biosynthesis

Different cytochromes P450 are involved in steroid biosynthesis. These cytochromes have heme as the prosthetic group. We previously reported that ACTH, an activator of glucocorticoid biosynthesis in adrenal, requires heme biosynthesis for a maximal response. In the present study, we investigated the effect of ACTH, and the effect of two activators of the adrenal mineralocorticoid synthesis, endothelin-1 and low sodium diet on 5-aminolevulinate-synthase (ALA-s) mRNA. ALA-s is the rate-limiting enzyme in heme biosynthesis. It was found that infusion of rats with ACTH for 1 h caused an increase of adrenal ALA-s mRNA and activity accompanied by an increase in plasma corticosterone. CYP21, a cytochrome involved in the synthesis of both corticosterone and aldosterone, was not modified at the RNA level in adrenal glands by 1 h of ACTH infusion. Consistently, infusion of endothelin-1 for 1 h increased ALA-s mRNA and aldosterone content in adrenal gland without modifying CYP21 mRNA levels. To study if ALA-s is also regulated by the main physiological stimuli that increase adrenal mineralocorticoid secretion, we fed rats with low salt diet for 2 or 15 days. Low salt diet treatment increased adrenal gland ALA-s mRNA levels. On the other hand, the rapid stimulation of ALA-s mRNA by ACTH which acts through cyclic AMP was confirmed in H295R human adrenocortical cells, the only human adrenal cell line that has a steroid secretion pattern and regulation similar to primary cultures of adrenal cells. Our findings suggest that the acute activation of adrenal steroidogenic cytochromes by trophic hormones involves an increase in heme biosynthesis which will favor the production of active cytochromes.