灵长类大脑前额叶NE Alpha-2Ⅰ亚型受体参与记忆过程的证据

灵长类大脑前额皮质与感觉联合皮质和皮质下结构有广泛的纤维联系, 可广 泛接受来自体内外的感觉信息。前额皮质与注意和短时记忆过程都有关, 可能 主沟区与短时空间记忆的关系更为密切, 北区的去甲肾上腺素能系统参与短时记 忆过程, alpha-2Ⅰ去甲肾上腺素亚型受体与增强记忆有关。参23

Measurement of the Interaction Between Recombinant I-domain from Integrin alpha 2 beta 1 and a Triple Helical Collagen Peptide with the GFOGER Binding Motif Using Molecular Force Spectroscopy.

The role of the collagen-platelet interaction is of crucial importance to the haemostatic response during both injury and pathogenesis of the blood vessel wall. Of particular interest is the high affinity interaction of the platelet transmembrane receptor, alpha 2 beta 1, responsible for firm attachment of platelets to collagen at and around injury sites. We employ single molecule force spectroscopy (SMFS) using the atomic force microscope (AFM) to study the interaction of the I-domain from integrin alpha 2 beta 1 with a synthetic collagen related triple-helical peptide containing the high-affinity integrin-binding GFOGER motif, and a control peptide lacking this sequence, referred to as GPP. By utilising synthetic peptides in this manner we are able to study at the molecular level subtleties that would otherwise be lost when considering cell-to-collagen matrix interactions using ensemble techniques. We demonstrate for the first time the complexity of this interaction as illustrated by the complex multi-peaked force spectra and confirm specificity using control blocking experiments. In addition we observe specific interaction of the GPP peptide sequence with the I-domain. We propose a model to explain these observations.

树（左鼠右句）前额叶皮层神经元信息处理功能的alpha-2去甲肾上腺素能受体机制研究

本研究采用微电极胞外记录技术和微电泳技术探讨了树（左鼠右句）前额叶神经元的电生理及其alpha-2去甲肾上腺素能受体的性质发现：1.树（左鼠右句）前额叶神经元的自发放电以中频(1-10次/秒)为主。2.树（左鼠右句）前额叶存在多模和单模感觉相关神经元。3.树（左鼠右向）前额叶存在clonidine敏感神经元。4.在树（左鼠右向）前额叶多模神经元中，有较高比例的clonidine敏感神经元。本研究提示：树（左鼠右句）前额叶的clonidine敏感神经元大多为单模和多模神经元。推测这些感觉相关神经元的信息处理功能可能与alpha-2受体有关。此外，本研究表明树（左鼠右句）前额叶神经元电活动具有前额叶的特征。

Sol-gel-derived alpha(2)-K7P2W17VO62/graphite/organoceramic composite as the electrode material for a renewable amperometric hydrogen peroxide sensor

The conductive alpha (2)-K7P2W17VO62/graphite/organoceramic composite was prepared by dispersing alpha (2)-K7P2W17VO62 and graphite powder in a propyltrimethoxysilane-based sol-gel solution; it was used as the electrode material for an amperometric hydrogen peroxide sensor. The modified electrode had a homogeneous mirror-like surface and showed well defined cyclic voltammograms. Square-wave voltammetry was employed to study the pH-dependent electrochemical behavior of c alpha (2)-K7P2W17VO62 doped in the graphite organoceramic matrix, and the experiment showed that both protons and sodium cations participated in the odor process. A hydrodynamic voltammetric experiment was performed to characterize the electrode as an amperometric sensor for the determination of hydrogen peroxide. The sensor can be renewed easily in a repeatable manner by a mechanical polishing step and has a long operational lifetime. (C) 2000 Elsevier Science B.V. All rights reserved.

Coupling of beta2-adrenoceptor to Gi proteins and its physiological relevance in murine cardiac myocytes.

-Transgenic mouse models have been developed to manipulate beta-adrenergic receptor (betaAR) signal transduction. Although several of these models have altered betaAR subtypes, the specific functional sequelae of betaAR stimulation in murine heart, particularly those of beta2-adrenergic receptor (beta2AR) stimulation, have not been characterized. In the present study, we investigated effects of beta2AR stimulation on contraction, [Ca2+]i transient, and L-type Ca2+ currents (ICa) in single ventricular myocytes isolated from transgenic mice overexpressing human beta2AR (TG4 mice) and wild-type (WT) littermates. Baseline contractility of TG4 heart cells was increased by 3-fold relative to WT controls as a result of the presence of spontaneous beta2AR activation. In contrast, beta2AR stimulation by zinterol or isoproterenol plus a selective beta1-adrenergic receptor (beta1AR) antagonist CGP 20712A failed to enhance the contractility in TG4 myocytes, and more surprisingly, beta2AR stimulation was also ineffective in increasing contractility in WT myocytes. Pertussis toxin (PTX) treatment fully rescued the ICa, [Ca2+]i, and contractile responses to beta2AR agonists in both WT and TG4 cells. The PTX-rescued murine cardiac beta2AR response is mediated by cAMP-dependent mechanisms, because it was totally blocked by the inhibitory cAMP analog Rp-cAMPS. These results suggest that PTX-sensitive G proteins are responsible for the unresponsiveness of mouse heart to agonist-induced beta2AR stimulation. This was further corroborated by an increased incorporation of the photoreactive GTP analog [gamma-32P]GTP azidoanilide into alpha subunits of Gi2 and Gi3 after beta2AR stimulation by zinterol or isoproterenol plus the beta1AR blocker CGP 20712A. This effect to activate Gi proteins was abolished by a selective beta2AR blocker ICI 118,551 or by PTX treatment. Thus, we conclude that (1) beta2ARs in murine cardiac myocytes couple to concurrent Gs and Gi signaling, resulting in null inotropic response, unless the Gi signaling is inhibited; (2) as a special case, the lack of cardiac contractile response to beta2AR agonists in TG4 mice is not due to a saturation of cell contractility or of the cAMP signaling cascade but rather to an activation of beta2AR-coupled Gi proteins; and (3) spontaneous beta2AR activation may differ from agonist-stimulated beta2AR signaling.

Enhancement of cardiac function after adenoviral-mediated in vivo intracoronary beta2-adrenergic receptor gene delivery.

Exogenous gene delivery to alter the function of the heart is a potential novel therapeutic strategy for treatment of cardiovascular diseases such as heart failure (HF). Before gene therapy approaches to alter cardiac function can be realized, efficient and reproducible in vivo gene techniques must be established to efficiently transfer transgenes globally to the myocardium. We have been testing the hypothesis that genetic manipulation of the myocardial beta-adrenergic receptor (beta-AR) system, which is impaired in HF, can enhance cardiac function. We have delivered adenoviral transgenes, including the human beta2-AR (Adeno-beta2AR), to the myocardium of rabbits using an intracoronary approach. Catheter-mediated Adeno-beta2AR delivery produced diffuse multichamber myocardial expression, peaking 1 week after gene transfer. A total of 5 x 10(11) viral particles of Adeno-beta2AR reproducibly produced 5- to 10-fold beta-AR overexpression in the heart, which, at 7 and 21 days after delivery, resulted in increased in vivo hemodynamic function compared with control rabbits that received an empty adenovirus. Several physiological parameters, including dP/dtmax as a measure of contractility, were significantly enhanced basally and showed increased responsiveness to the beta-agonist isoproterenol. Our results demonstrate that global myocardial in vivo gene delivery is possible and that genetic manipulation of beta-AR density can result in enhanced cardiac performance. Thus, replacement of lost receptors seen in HF may represent novel inotropic therapy.

Alterations in cardiac adrenergic signaling and calcium cycling differentially affect the progression of cardiomyopathy.

The medical treatment of chronic heart failure has undergone a dramatic transition in the past decade. Short-term approaches for altering hemodynamics have given way to long-term, reparative strategies, including beta-adrenergic receptor (betaAR) blockade. This was once viewed as counterintuitive, because acute administration causes myocardial depression. Cardiac myocytes from failing hearts show changes in betaAR signaling and excitation-contraction coupling that can impair cardiac contractility, but the role of these abnormalities in the progression of heart failure is controversial. We therefore tested the impact of different manipulations that increase contractility on the progression of cardiac dysfunction in a mouse model of hypertrophic cardiomyopathy. High-level overexpression of the beta(2)AR caused rapidly progressive cardiac failure in this model. In contrast, phospholamban ablation prevented systolic dysfunction and exercise intolerance, but not hypertrophy, in hypertrophic cardiomyopathy mice. Cardiac expression of a peptide inhibitor of the betaAR kinase 1 not only prevented systolic dysfunction and exercise intolerance but also decreased cardiac remodeling and hypertrophic gene expression. These three manipulations of cardiac contractility had distinct effects on disease progression, suggesting that selective modulation of particular aspects of betaAR signaling or excitation-contraction coupling can provide therapeutic benefit.

Enhanced myocardial relaxation in vivo in transgenic mice overexpressing the beta2-adrenergic receptor is associated with reduced phospholamban protein.

To assess the effect of targeted myocardial beta-adrenergic receptor (AR) stimulation on relaxation and phospholamban regulation, we studied the physiological and biochemical alterations associated with overexpression of the human beta2-AR gene in transgenic mice. These mice have an approximately 200-fold increase in beta-AR density and a 2-fold increase in basal adenylyl cyclase activity relative to negative littermate controls. Mice were catheterized with a high fidelity micromanometer and hemodynamic recordings were obtained in vivo. Overexpression of the beta2-AR altered parameters of relaxation. At baseline, LV dP/dt(min) and the time constant of LV pressure isovolumic decay (Tau) in the transgenic mice were significantly shorter compared with controls, indicating markedly enhanced myocardial relaxation. Isoproterenol stimulation resulted in shortening of relaxation velocity in control mice but not in the transgenic mice, indicating maximal relaxation in these animals. Immunoblotting analysis revealed a selective decrease in the amount of phospholamban protein, without a significant change in the content for either sarcoplasmic reticulum Ca2+ ATPase or calsequestrin, in the transgenic hearts compared with controls. This study indicates that myocardial relaxation is both markedly enhanced and maximal in these mice and that conditions associated with chronic beta-AR stimulation can result in a selective reduction of phospholamban protein.

beta-Arrestin 1 and 2 differentially regulate heptahelical receptor signaling and trafficking.

The two widely coexpressed isoforms of beta-arrestin (termed beta arrestin 1 and 2) are highly similar in amino acid sequence. The beta-arrestins bind phosphorylated heptahelical receptors to desensitize and target them to clathrin-coated pits for endocytosis. To better define differences in the roles of beta-arrestin 1 and 2, we prepared mouse embryonic fibroblasts from knockout mice that lack one of the beta-arrestins (beta arr1-KO and beta arr2-KO) or both (beta arr1/2-KO), as well as their wild-type (WT) littermate controls. These cells were analyzed for their ability to support desensitization and sequestration of the beta(2)-adrenergic receptor (beta(2)-AR) and the angiotensin II type 1A receptor (AT(1A)-R). Both beta arr1-KO and beta arr2-KO cells showed similar impairment in agonist-stimulated beta(2)-AR and AT(1A)-R desensitization, when compared with their WT control cells, and the beta arr1/2-KO cells were even further impaired. Sequestration of the beta(2)-AR in the beta arr2-KO cells was compromised significantly (87% reduction), whereas in the beta arr1-KO cells it was not. Agonist-stimulated internalization of the AT(1A)-R was only slightly reduced in the beta arr1-KO but was unaffected in the beta arr2-KO cells. In the beta arr1/2-KO cells, the sequestration of both receptors was dramatically reduced. Comparison of the ability of the two beta-arrestins to sequester the beta(2)-AR revealed beta-arrestin 2 to be 100-fold more potent than beta-arrestin 1. Down-regulation of the beta(2)-AR was also prevented in the beta arr1/2-KO cells, whereas no change was observed in the single knockout cells. These findings suggest that sequestration of various heptahelical receptors is regulated differently by the two beta-arrestins, whereas both isoforms are capable of supporting receptor desensitization and down-regulation.

A C-terminal motif found in the beta2-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Na+/H+ exchanger regulatory factor family of PDZ proteins.

The Na+/H+ exchanger regulatory factor (NHERF) binds to the tail of the beta2-adrenergic receptor and plays a role in adrenergic regulation of Na+/H+ exchange. NHERF contains two PDZ domains, the first of which is required for its interaction with the beta2 receptor. Mutagenesis studies of the beta2 receptor tail revealed that the optimal C-terminal motif for binding to the first PDZ domain of NHERF is D-S/T-x-L, a motif distinct from those recognized by other PDZ domains. The first PDZ domain of NHERF-2, a protein that is 52% identical to NHERF and also known as E3KARP, SIP-1, and TKA-1, exhibits binding preferences very similar to those of the first PDZ domain of NHERF. The delineation of the preferred binding motif for the first PDZ domain of the NHERF family of proteins allows for predictions for other proteins that may interact with NHERF or NHERF-2. For example, as would be predicted from the beta2 receptor tail mutagenesis studies, NHERF binds to the tail of the purinergic P2Y1 receptor, a seven-transmembrane receptor with an intracellular C-terminal tail ending in D-T-S-L. NHERF also binds to the tail of the cystic fibrosis transmembrane conductance regulator, which ends in D-T-R-L. Because the preferred binding motif of the first PDZ domain of the NHERF family of proteins is found at the C termini of a variety of intracellular proteins, NHERF and NHERF-2 may be multifunctional adaptor proteins involved in many previously unsuspected aspects of intracellular signaling.

beta2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein.

G protein-coupled receptor activation leads to the membrane recruitment and activation of G protein-coupled receptor kinases, which phosphorylate receptors and lead to their inactivation. We have identified a novel G protein-coupled receptor kinase-interacting protein, GIT1, that is a GTPase-activating protein (GAP) for the ADP ribosylation factor (ARF) family of small GTP-binding proteins. Overexpression of GIT1 leads to reduced beta2-adrenergic receptor signaling and increased receptor phosphorylation, which result from reduced receptor internalization and resensitization. These cellular effects of GIT1 require its intact ARF GAP activity and do not reflect regulation of GRK kinase activity. These results suggest an essential role for ARF proteins in regulating beta2-adrenergic receptor endocytosis. Moreover, they provide a mechanism for integration of receptor activation and endocytosis through regulation of ARF protein activation by GRK-mediated recruitment of the GIT1 ARF GAP to the plasma membrane.

Ligand-induced overexpression of a constitutively active beta2-adrenergic receptor: pharmacological creation of a phenotype in transgenic mice.

Transgenic overexpression (40- to 100-fold) of the wild-type human beta2-adrenergic receptor in the hearts of mice leads to a marked increase in cardiac contractility, which is apparently due to the low level of spontaneous (i.e., agonist-independent) activity inherent in the receptor. Here we report that transgenic mice expressing a mutated constitutively active form of the receptor (CAM) show no such phenotype, owing to its modest expression (3-fold above endogenous cardiac beta-adrenergic receptor levels). Surprisingly, treatment of the animals with a variety of beta-adrenergic receptor ligands leads to a 50-fold increase in CAM beta2-adrenergic receptor expression, by stabilizing the CAM beta2-adrenergic receptor protein. Receptor up-regulation leads in turn to marked increases in adenylate cyclase activity, atrial tension determined in vitro, and indices of cardiac contractility determined in vivo. These results illustrate a novel mechanism for regulating physiological responses, i.e., ligand-induced stabilization of a constitutively active but inherently unstable protein.

Potentiation of beta-adrenergic signaling by adenoviral-mediated gene transfer in adult rabbit ventricular myocytes.

Our laboratory has been testing the hypothesis that genetic modulation of the beta-adrenergic signaling cascade can enhance cardiac function. We have previously shown that transgenic mice with cardiac overexpression of either the human beta2-adrenergic receptor (beta2AR) or an inhibitor of the beta-adrenergic receptor kinase (betaARK), an enzyme that phosphorylates and uncouples agonist-bound receptors, have increased myocardial inotropy. We now have created recombinant adenoviruses encoding either the beta2AR (Adeno-beta2AR) or a peptide betaARK inhibitor (consisting of the carboxyl terminus of betaARK1, Adeno-betaARKct) and tested their ability to potentiate beta-adrenergic signaling in cultured adult rabbit ventricular myocytes. As assessed by radioligand binding, Adeno-beta2AR infection led to approximately 20-fold overexpression of beta-adrenergic receptors. Protein immunoblots demonstrated the presence of the Adeno-betaARKct transgene. Both transgenes significantly increased isoproterenol-stimulated cAMP as compared to myocytes infected with an adenovirus encoding beta-galactosidase (Adeno-betaGal) but did not affect the sarcolemmal adenylyl cyclase response to Forskolin or NaF. beta-Adrenergic agonist-induced desensitization was significantly inhibited in Adeno-betaARKct-infected myocytes (16+/-2%) as compared to Adeno-betaGal-infected myocytes (37+/-1%, P < 0.001). We conclude that recombinant adenoviral gene transfer of the beta2AR or an inhibitor of betaARK-mediated desensitization can potentiate beta-adrenergic signaling.

An alpha 2-macroglobulin-like protein is the cue to gregarious settlement of the barnacle Balanus amphitrite

Many benthic marine invertebrates, like barnacles, have a planktonic larval stage whose primary purpose is dispersal. How these species colonize suitable substrata is fundamental to understanding their evolution, population biology, and wider community dynamics. Unlike larval dispersal, settlement occurs on a relatively small spatial scale and involves larval behavior in response to physical and chemical characteristics of the substratum. Biogenic chemical cues have been implicated in this process. Their identification, however, has proven challenging, no more so than for the chemical basis of barnacle gregariousness, which was first described >50 years ago. We now report that a biological cue to gregarious settlement, the settlement-inducing protein complex (SIPC), of the major fouling barnacle Balanus amphitrite is a previously undescribed glycoprotein. The SIPC shares a 30% sequence homology with the thioester-containing family of proteins that includes the alpha sub(2)-macroglobulins. The cDNA (5.2 kb) of the SIPC encodes a protein precursor comprising 1,547 aa with a 17-residue signal peptide region. A number of structural characteristics and the absence of a thioester bond in the SIPC suggest that this molecule is a previously undescribed protein that may have evolved by duplication from an ancestral alpha sub(2)-macroglobulin gene. Although the SIPC is regarded as an adult cue that is recognized by the cyprid at settlement, it is also expressed in the juvenile and in larvae, where it may function in larva-larva settlement interactions.