beta(2)-Adrenergic receptor signaling complexes in cardiomyocyte caveolae/lipid rafts

The traditional notion that catecholamine actions are mediated by the predominant beta(1)-adrenergic receptor (beta(1)-AR) subtype linked to the activation of adenylyl cyclase and the accumulation of cyclic adenosine 3',5'-monophosphate (cAMP) in cardiomyocytes has been challenged by recent studies showing that cardiomyocytes co-express pharmacologically distinct beta(2)-AR subtypes that activate a more broad range of downstream effectors. While beta(1)- and beta(2)-ARs exert largely functionally equivalent cellular actions in heterologous expression systems, signaling by endogenous beta-AR subtypes in highly differentiated cells such as cardiomyocytes can be strikingly different. There is growing evidence that certain features of the signaling phenotypes for beta-AR subtypes in cardiomyocytes are inconsistent with traditional models that attribute signaling specificity to high-affinity protein-protein interactions between receptors, G-proteins, and effectors freely mobile on surface membranes. This chapter summarizes recent studies that focus on membrane microdomains (such as caveolae or lipid rafts) as sites that differentially localizing individual beta-AR subtypes as well as the downstream signaling machinery that generates, propagates, and downregulates the cAMP-protein kinase A signaling pathway. To the extent that this mechanism calibrates beta-AR responses in cardiomyocytes, it would be expected to be pertinent to the pathogenesis of heart failure, where chronic/persistent beta-AR signaling contributes to ventricular remodeling and impacts on long-term survival.     

Compartmentalisation of cAMP-dependent signalling by caveolae in the adult cardiac myocyte

Cyclic AMP exhibits local (sarcolemmal) and global (cytosolic) patterns of signalling, allowing receptor-specific signals to be generated by a single second messenger. Here we determine whether caveolae, invaginated lipid rafts, are responsible for confining the β₂ adrenoceptor (AR) cAMP signal to the sarcolemmal compartment. Myocytes were treated with the cholesterol-depleting agent methyl-β-cyclodextrin (MβC) to disrupt caveolae. Caveolae-containing membrane fractions were isolated by detergent-free sucrose gradient fractionation. Cell shortening and phosphorylation of the sarcoplasmic reticular protein phospholamban (PLB) and the myofilament protein troponin I (TnI) were measured in response to β₂ AR stimulation (with salbutamol in the presence of 1 μM atenolol). Ser¹⁶ phosphorylation of PLB (pPLB), Ser^22,23 phosphorylation of TnI (pTnI), and positive lusitropy were used as indices of global cAMP signals. The ability of MβC to disrupt caveolae was confirmed by selective depletion of the buoyant membrane fractions of cholesterol and caveolin 3, the 2 essential components of caveolae. In control cells, no change in pPLB, pTnI or time to half relaxation was recorded with β₂ AR stimulation (P > 0.05), but following caveolar disruption a 60–70% increase in phosphorylation of both proteins was seen, accompanied by positive lusitropy (P < 0.05). These data show for the first time that disrupting caveolae converts the sarcolemmal-confined cAMP signal associated with β₂ AR stimulation to a global signal that targets proteins of the sarcoplasmic reticulum and myofilaments, with functional sequelae. The role of caveolae in spatial control of cAMP may be relevant to perturbation of β AR signalling in cardiovascular disease.     

Viral entry,lipid rafts and caveosomes

Lipid rafts and caveolae are detergent‐insoluble plasma membrane microdomains, involved in cellular endocytic processes and signalling. Several viruses, including a human pathogen, echovirus 1, and an extensively studied simian virus 40 utilize these domains for internalization into the host cells. Interaction of viruses with receptors on the cell surface triggers specific conformational changes of the virus particle and can give rise to signalling events, which determine the mechanisms of virus entry. After internalization via cell surface lipid rafts or caveolae, virus‐containing vesicles can fuse with caveosomes, pre‐existing cytoplasmic organelles, or dock on other intracellular organelles. These pathways may deliver viruses further to different cellular destinations, where the viral replication cycle then takes place. The information concerning the viral entry processes is important for understanding the details of the infections, for finding new targets for antiviral therapy and for elucidating the cellular internalization pathways in general.     

脂肪细胞细胞膜穴样凹陷的研究进展

细胞膜穴样凹陷是细胞膜表面特异性内陷结构,小窝蛋白是其结构蛋白。细胞膜穴样凹陷分布广泛,脂肪细胞是含量最丰富的细胞。新近的研究表明,脂肪细胞细胞膜穴样凹陷是营养物质进入的门户,也是营养物质代谢转化的平台。本文对脂肪细胞细胞膜穴样凹陷特有的功能加以综述。     

Benzo[a]pyrene induces intercellular adhesion molecule-1 through a caveolae and aryl hydrocarbon receptor mediated pathway

Toxicologic and epidemiologic studies have linked benzo[a]pyrene (B[a]P) exposure with cardiovascular diseases such as atherosclerosis. The mechanisms of action leading to these diseases have not been fully understood. One key step in the development of atherosclerosis is vascular endothelial dysfunction, which is characterized by increased adhesiveness. To determine if B[a]P could lead to increased endothelial adhesiveness, the effects of B[a]P on human endothelial cell intercellular adhesion molecule-1 (ICAM-1) expression was investigated. B[a]P was able to increase ICAM-1 protein only after pretreatment with the aryl hydrocarbon receptor (AhR) agonist β-naphthoflavone (β-NF). Knockdown of AhR by siRNA or treatment with AhR antagonist α-naphthoflavone (α-NF) eliminated the induction of ICAM-1 from B[a]P, confirming the necessity of AhR in this process. Likewise, B[a]P only increased monocyte adhesion to the vascular endothelium when cells were pretreated with β-NF. Experiments were done to define a signaling mechanism. B[a]P increased phosphorylation of MEK and p38-MAPK, and inhibitors to these proteins blunted the ICAM-1 induction. B[a]P was also able to increase AP-1 DNA binding and phosphorylation of cJun. Phosphorylation of cJun was disrupted by MEK and p38-MAPK inhibitors linking the signaling cascade. Finally, the importance of membrane microdomains, caveolae, was demonstrated by knockdown of the structural protein caveolin-1. Disruption of caveolae eliminated the B[a]P-induced ICAM-1 expression. These data suggest a possible pro-inflammatory mechanism of action of B[a]P involving caveolae, leading to increased vascular endothelial adhesiveness, and this inflammation may be a critical step in the development of B[a]P-induced atherosclerosis.     

Caveolin-1 is related to lipid droplet formation in hepatic stellate cells in human liver

Caveolins (CAVs) regulate intracellular cholesterol transport by a complex process involving caveolae, endoplasmic reticulum (ER), and the Golgi network. Hepatic stellate cells (HSCs) are the central site for retinoid storage in the liver and indeed the entire body. Herein, we attempted to elucidate the ultrastructural localization and expression of caveolin-1 (CAV-1) in human HSCs during the progression of liver cirrhosis (LC). Normal and hepatitis C-related cirrhotic liver samples were prepared using a modified perfusion-fixation method to fix organelle structures and molecules in their in vivo positions, and examined using immunoelectron microscopy. In control liver specimens, CAV-1 was minimally associated with low electron density lipid droplets (LDs) segregated around zones 1―2, and specifically associated with membranes surrounding LDs. CAV-1 was segregated in high-density LDs, consistent with the formation of membrane-enclosed lipid-rich vesicular structures, as well as caveolae on plasma membranes around zones 2―3. In cirrhotic liver specimens, CAV-1 molecules were inserted into the cytoplasmic leaflets of ER membranes for transportation to LDs. Thus, CAV-1 transport to LDs might represent an intracellular pathway from the ER in cirrhotic liver tissue.     

Novel mechanism for sudden infant death syndrome: Persistent late sodium current secondary to mutations in caveolin-3

BACKGROUND: Sudden infant death syndrome (SIDS) is one of the leading causes of death during the first year of life. Long QT syndrome (LQTS)-associated mutations may be responsible for 5% to 10% of SIDS cases. We recently established CAV3-encoded caveolin-3 as a novel LQTS-associated gene with mutations producing a gain-of-function, LQT3-like molecular/cellular phenotype. OBJECTIVE: The purpose of this study was to determine the prevalence and functional properties of CAV3 mutations in SIDS. METHODS: Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing, postmortem genetic testing of CAV3 was performed on genomic DNA isolated from frozen necropsy tissue on a population-based cohort of unrelated cases of SIDS (N = 134, 57 females, average age = 2.7 months). CAV3 mutations were engineered using site-directed mutagenesis and heterologously expressed in HEK293 cell lines stably expressing the SCN5A-encoded cardiac sodium channel. RESULTS: Overall, three distinct CAV3 mutations (V14L, T78M, and L79R) were identified in three of 50 black infants (6-month-old male, 2-month-old female, and 8 month-old female), whereas no mutations were detected in 83 white infants (P <.05). CAV3 mutations were more likely in decedents 6 months or older (2/12) than in infants who died before 6 months (1/124, P = .02). Voltage clamp studies showed that all three CAV3 mutations caused a significant fivefold increase in late sodium current compared with controls. CONCLUSION: This study provides the first molecular and functional evidence implicating CAV3 as a pathogenic basis of SIDS. The LQT3-like phenotype of increased late sodium current supports an arrhythmogenic mechanism for some cases of SIDS.     

Caveolin-1、Caveolae和动脉粥样硬化

Caveolae是细胞表面直径为50—100nm的胞膜穴样内陷,主要由胆固醇、糖基鞘磷脂、鞘磷脂和一些结构蛋白如Caveolin组成。Caveolin-1是Caveolae的主要组成成分,它与细胞骨架蛋白间接偶联来维持Caveolae的内陷形态。Caveolae和Caveolin-1在参与动脉粥样硬化的各种细胞如内皮细胞、巨噬细胞、平滑肌细胞表面都有表达,它们通过对各种细胞功能和多个信号转导途径的调节参与对动脉粥样硬化的形成和发展。     

Caveolin-1在大肠癌组织中的表达及其临床生物学意义

目的探讨Caveolin-1蛋白在人大肠癌发生、发展过程中表达的变化及其临床生物学意义。方法应用免疫组化S-P法检测92例大肠癌、27例腺瘤及22例癌旁正常黏膜组织中Caveolin-1蛋白的表达情况,并探讨Caveolin-1蛋白表达与大肠癌各临床病理学参数之间的关系。结果Caveolin-1蛋白在大肠癌、腺瘤与癌旁正常黏膜组织中阳性表达率的差异有统计学意义（P〈0．01）,分别为29．35％、59．26％和86．36％。Caveolin-1蛋白在不同组织学类型癌组织中阳性表达率的差异无统计学意义（P〉0．05）,但与大肠癌的分化程度、浸润深度及区域淋巴结有无转移有关（P〈0．05）。在有无远处转移的比较中显示Caveolin-1的阳性表达率无统计学差异（P〉0．05）。结论Caveolin-1蛋白在癌旁正常黏膜、腺瘤及癌组织中呈渐进性低表达,可能在大肠癌的发生发展中发挥作用,在大肠癌的恶性演进和转移中具有重要意义,可能作为大肠癌重要的生物学标志物。     

Caveolae蛋白与胃癌发生发展的表观遗传学关系

胃癌是我国最常见的恶性肿瘤之一,其死亡率占我国恶性肿瘤死亡率之首。胃癌的病因复杂,虽经数十年的研究,至今仍无明确定论。目前普遍认为其发生是遗传和环境因素共同作用的结果。随着对表观遗传学认识的深入,遗传因子在疾病中的作用被不断发现,对疾病的诊断、治疗和预防起到了一定的作用。本文结合Caveolae蛋白的研究现状综述其与胃癌发生发展的表观遗传学关系,希望对胃癌发病机制的研究提供新的思路。