live-imaging-assay-for-assessing-roles-ca2-sphingomyelinase-repair

Live Imaging Assay for Assessing the Roles of Ca<sup>2+</sup> and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

University of Maryland 

Pathogenic yersiniae secrete 14 Yop proteins via the type III pathway. Synthesis of YopQ occurs when the type III machinery is activated by a low-calcium signal, but not when the calcium concentration is above 100 microM. To characterize the mechanism that regulates the expression of yopQ, mutants that permit synthesis of YopQ in the presence of calcium were isolated. Yersiniae bearing deletion mutations in yopN, tyeA, sycN, or yscB synthesized and secreted YopQ in both the presence and the absence of calcium. In contrast, yersiniae with a deletion in yopD or lcrH synthesized YopQ in the presence of calcium but did not secrete the polypeptide. These variants displayed no defect in YopQ secretion under low-calcium conditions, revealing that yopD and lcrH are required for the regulation of yopQ expression. Experiments with transcriptional and translational fusions to the npt reporter gene suggest that yopD and lcrH regulate yopQ expression at a posttranscriptional step. YopD and LcrH form a complex in the bacterial cytosol and bind yopQ mRNA. Models that can account for posttranscriptional regulatory mechanisms of yop expression are discussed.

YopD and LcrH regulate expression of Yersinia enterocolitica YopQ by a posttranscriptional mechanism and bind to yopQ RNA.

Esigma(E) RNA polymerase transcribes a regulon of folding factors for the bacterial envelope and is induced by physical and chemical stresses. The RseA anti-sigma factor inhibits the activity of Esigma(E) RNA polymerase. It is shown here that the N-terminal portion of sigma(E), residues 1-153, binds core RNA polymerase. RseA interacts with residues 154-191 of sigma(E), a site that is homologous to region 4, the sigma factor binding site for promoter DNA. Mutations that reduce transcription of Esigma(E) RNA polymerase map to sigma(E) residues 178, 181, and 183. Variant sigma(E) proteins with amino acid substitutions at residues 178, 181, or 183 do not associate with RseA. A regulatory mechanism is proposed whereby RseA binds to a C-terminal peptide of sigma(E) and inhibits the transcription of Esigma(E) RNA polymerase by blocking promoter recognition.

Interaction of the conserved region 4.2 of sigma(E) with the RseA anti-sigma factor.

The potent neuropeptide vasodilator, calcitonin gene-related peptide (CGRP), and the vasoactive peptide adrenomedullin (AM) are structurally related. Evidence from our laboratory has demonstrated that these peptides have potent microvascular actions of relevance to cardiovascular and inflammatory effects in health and disease. We wish to further investigate the actions of these peptides through studies in genetically modified mice. We have developed techniques to enable the quantitative analysis of CGRP and AM responses in the mouse microvasculature. A mouse isolated mesentery system was developed that measures changes in perfusion pressure used as an index of microvascular relaxation in the precontracted mesenteric microvascular bed. Bolus injections of CGRP and AM caused dose-dependent decreases in perfusion pressure that were proportional to vascular relaxation. An in vivo mouse skin assay was also used in which agents were injected intradermally into the dorsal skin. The effects of these agents was assessed by the extravascular accumulation of intravenously injected 125I-albumin for their ability to potentiate plasma extravasation induced by a mediator of increased microvascular permeability. CGRP and AM are not directly active in this assay, because it does not directly measure blood flow. However, the vasodilators acted in a potent and dose-dependent manner to significantly potentiate edema formation. The results demonstrate the potent activity of CGRP and the activity (although 100- to 300-fold less potent) of AM. Furthermore, the results demonstrate the increased potency of CGRP in the microvasculature when compared with the structurally distinct peptide VIP and PGE1.

The assessment of vasoactive properties of CGRP and adrenomedullin in the microvasculature: a study using in vivo and in vitro assays in the mouse.

The envelope of Escherichia coli is composed of an asymmetric lipid bilayer containing lipopolysaccharide, phospholipid and outer membrane proteins (OMPs). Physical and chemical stresses impact on the integrity of the outer membrane envelope and trigger the sigma(E)-dependent response, whereby E. coli activates the expression of genes that increase its capacity for folding OMPs and synthesizing lipopolysaccharide (LPS). While it has already been appreciated that misfolded OMPs induce the sigma(E) response, a role for LPS in activating this pathway was hitherto unknown. Here we show that ammonium metavandate (NH4VO3) induces multiple changes in E. coli LPS structure and activates the sigma(E)-dependent response without altering OMP. One such NH4VO3-mediated LPS decoration, the CrcA/PagP-catalysed addition of palmitate to lipid A, appeared to be alone sufficient to activate transcription at sigma(E)-dependent promoters. Furthermore, reduced acylation of LPS, caused by htrB or msbB mutations, also resulted in a constitutive expression of the sigma(E) regulon above wild-type levels. Production of these aberrant outer membrane lipids did not noticeably affect the composition or the amount of OMPs. A model is proposed whereby structural intermediates of the LPS biosynthetic pathway or modified LPS molecules may function as signals that activate the sigma(E) response.

Changes in lipopolysaccharide structure induce the sigma(E)-dependent response of Escherichia coli.

Bacillus anthracis, a spore forming Gram-positive microbe, is the causative agent of anthrax. Although plasmid encoded factors such as lethal toxin (LeTx), edema toxin (EdTx), and gamma-poly-d-glutamic acid (PGA) capsule are known to be required for disease pathogenesis, B. anthracis genes that enable spore invasion, phagosomal escape and macrophage replication are still unknown. To establish transposon mutagenesis as a tool for the characterization of anthrax genes, we employed the mariner-based mini-transposon Bursa aurealis in B. anthracis strain Sterne 7702. B. aurealis carrying an erythromycin resistance cassette and its cognate transposase were delivered by transformation of two plasmids. B. aurealis transposition can be selected for by temperature shift to prevent plasmid replication and by screening colonies for erythromycin resistance. Using inverse polymerase chain reaction, DNA fragments of 129 random erythromycin-resistant transposon mutants were amplified and submitted to DNA sequence analysis. These studies demonstrate that B. aurealis inserts randomly into the genome of B. anthracis and can therefore be employed for finding genes involved in virulence.

Transposon mutagenesis of Bacillus anthracis strain Sterne using Bursa aurealis.

The cutaneous vasculature plays a key role in the pathophysiology of inflammatory skin diseases. The vascular activity is under the control of the peripheral nervous system that includes locally released neuropeptides. Recently, we detected receptors for the neuropeptide galanin in association with dermal blood vessels, suggesting a role of the galanin-peptide-family in the regulation of the cutaneous microvasculature. Therefore, we have investigated galanin and galanin-like peptide (GALP), a neuropeptide previously only considered to be involved in metabolism and reproduction in the central nervous system, for vaso-modulatory activity in the murine skin in vivo. Picomole amounts of intradermally injected galanin and GALP decreased cutaneous blood flow and inhibited inflammatory edema formation. Both the full-length GALP (1-60) and the putative smaller proteolytic fragment GALP (3-32) showed similar effects. These activities are most likely mediated by galanin receptors galanin receptor subtype 2 (GalR2) and/or galanin receptor subtype 3 (GalR3), because reverse transcription-PCR analysis of murine skin revealed messenger RNA (mRNA) expression of GalR2 and GalR3 but not of galanin receptor subtype 1. The lack of galanin receptor mRNAs in endothelial and smooth muscle cells indicates a neuronal localization of these receptors around the vessels. These results indicate functional activity of GALP in the periphery in vivo and suggest a potential role as an inflammatory modulator.

Galanin-like peptides exert potent vasoactive functions in vivo.

Sulphonamide-derived medications are widely used, although not always recognized as such. We report the occurrence of bilateral angle closure glaucoma with sulphonamide-derived medications and highlight features of the presentation and treatment to assist in early diagnosis and management.

Bilateral angle closure glaucoma induced by sulphonamide-derived medications.

Ca2+ influx through plasma membrane lesions triggers a rapid repair process that was previously shown to require the exocytosis of lysosomal organelles (Reddy, A., E. Caler, and N. Andrews. 2001. Cell. 106:157-169). However, how exocytosis leads to membrane resealing has remained obscure, particularly for stable lesions caused by pore-forming proteins. In this study, we show that Ca2+-dependent resealing after permeabilization with the bacterial toxin streptolysin O (SLO) requires endocytosis via a novel pathway that removes SLO-containing pores from the plasma membrane. We also find that endocytosis is similarly required to repair lesions formed in mechanically wounded cells. Inhibition of lesion endocytosis (by sterol depletion) inhibits repair, whereas enhancement of endocytosis through disruption of the actin cytoskeleton facilitates resealing. Thus, endocytosis promotes wound resealing by removing lesions from the plasma membrane. These findings provide an important new insight into how cells protect themselves not only from mechanical injury but also from microbial toxins and pore-forming proteins produced by the immune system.

Repair of injured plasma membrane by rapid Ca2+-dependent endocytosis.

Ca(2+) influx through plasma membrane wounds triggers a rapid-repair response that is essential for cell survival. Earlier studies showed that repair requires the exocytosis of intracellular vesicles. Exocytosis was thought to promote resealing by 'patching' the plasma membrane lesion or by facilitating bilayer restoration through reduction in membrane tension. However, cells also rapidly repair lesions created by pore-forming proteins, a form of injury that cannot be resealed solely by exocytosis. Recent studies indicate that, in cells injured by pores or mechanical abrasions, exocytosis is followed by lesion removal through endocytosis. Describing the relationship between wound-induced exocytosis and endocytosis has implications for the understanding of muscular degenerative diseases that are associated with defects in plasma membrane repair.

Two-way traffic on the road to plasma membrane repair.

&#x8840;&#x7BA1;&#x8212;&#x5F20;&#x548C;&#x80BE;&#x4E0A;&#x817A;&#x9AD3;&#x8D28;&#x7D20;&#x76D1;&#x7BA1;&#x8840;&#x7BA1;&#x80BD; (AM)&#x3001; &#x80BD;&#x3001; &#x964D;&#x9499;&#x7D20;&#x57FA;&#x56E0;&#x76F8;&#x5173;&#x80BD;&#x5BB6;&#x65CF;&#x7684;&#x6210;&#x5458;&#xFF0C;&#x9884;&#x8BA1;&#x5728;&#x5FC3;&#x8840;&#x7BA1;&#x529F;&#x80FD;&#x969C;&#x788D;&#x4E2D;&#x53D1;&#x6325;&#x4E86;&#x5173;&#x952E;&#x7684;&#x4FDD;&#x62A4;&#x4F5C;&#x7528;&#x3002;&#x4E0A;&#x5348; (AM1) &#x53D7;&#x4F53;&#x7684;&#x539F;&#x5219;&#x7531; G &#x86CB;&#x767D;&#x4E0E;&#x964D;&#x9499;&#x7D20;&#x53D7;&#x4F53;&#x6837;&#x53D7;&#x4F53;&#x548C;&#x53D7;&#x4F53;&#x6D3B;&#x6027;&#x4FEE;&#x6539;&#x86CB;&#x767D; (&#x53D7;&#x4F53;&#x6D3B;&#x6027;&#x4FEE;&#x9970;&#x86CB;&#x767D; 2) &#x7EC4;&#x6210;&#x3002;&#x6709;&#x77E5;&#x4E4B;&#x751A;&#x5C11;&#x7684;&#x53D7;&#x4F53;&#x901A;&#x8FC7;&#x4E0A;&#x5348;&#x5728;&#x75BE;&#x75C5;&#x4E2D;&#x7684;&#x884C;&#x4E3A;&#x3002;&#x6211;&#x4EEC;&#x6709;&#x9488;&#x5BF9;&#x6027;&#x7684;&#x5E73;&#x6ED1;&#x808C;&#x53D7;&#x4F53;&#x6D3B;&#x6027;&#x4FEE;&#x6539; 2 &#x86CB;&#x767D;&#x8F6C;&#x57FA;&#x56E0;&#x5C0F;&#x9F20;&#x4F7F;&#x7528;&#x529F;&#x80FD; AM1 &#x53D7;&#x4F53;&#x7684;&#x8840;&#x7BA1;&#x5BC6;&#x5EA6;&#x7684;&#x63D0;&#x9AD8;&#xFF0C;&#x8868;&#x73B0;&#x51FA;&#x53D7;&#x4F53;&#x6D3B;&#x6027;&#x4FEE;&#x9970;&#x86CB;&#x767D; 2 &#x8F6C;&#x57FA;&#x56E0;&#x5C0F;&#x9F20;&#x4E0D;&#x53D7;&#x8840;&#x7BA1;&#x7D27;&#x5F20;&#x7D20; II &#x8BF1;&#x5BFC;&#x9AD8;&#x8840;&#x538B;&#x6216;&#x5FC3;&#x810F;&#x80A5;&#x5927;&#x3002;&#x7136;&#x800C;&#xFF0C;&#x8840;&#x7BA1;&#x589E;&#x751F;&#xFF0C;&#x8840;&#x7BA1;&#x7EC6;&#x80DE;&#x7C98;&#x9644;&#x5206;&#x5B50; 1 &#x548C;&#x5355;&#x6838;&#x7EC6;&#x80DE;&#x8D8B;&#x5316;&#x86CB;&#x767D; 1 &#x7684;&#x8868;&#x8FBE;&#xFF0C;&#x4F1A;&#x5927;&#x5927;&#x964D;&#x4F4E;&#x7684;&#x8F6C;&#x57FA;&#x56E0;&#x5C0F;&#x9F20;&#xFF0C;&#x6240;&#x786E;&#x5B9A;&#x7684;&#x7EC4;&#x7EC7;&#x5B66;&#x6280;&#x672F;&#x4E3B;&#x52A8;&#x8109;&#x58C1;&#x5185;&#x3002;&#x8FD9;&#x8868;&#x660E; AM1 &#x8840;&#x7BA1;&#x5E73;&#x6ED1;&#x808C;&#x53D7;&#x4F53;&#x80FD;&#x591F;&#x8C03;&#x8282;&#x4F53;&#x5185;&#x7684;&#x5730;&#x65B9;&#x4FDD;&#x62A4;&#x3002;&#x8FD9;&#x662F;&#x7531;&#x5E73;&#x6ED1;&#x808C;&#x7EC6;&#x80DE;&#x7684;&#x589E;&#x6B96;&#x7814;&#x7A76;&#x652F;&#x6301;&#x3002;&#x76F8;&#x6BD4;&#x4E4B;&#x4E0B;&#xFF0C;&#x4F4E;&#x8840;&#x538B;&#x548C;&#x4F11;&#x514B;&#x6A21;&#x578B;&#x4E2D;&#x7684;&#x708E;&#x75C7;&#x7A0B;&#x5EA6;&#x662F;&#x7C7B;&#x4F3C;&#x4E8E;&#x90A3;&#x4E9B;&#x91CE;&#x751F;&#x578B;&#x5C0F;&#x9F20;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x65E0;&#x6CD5;&#x68C0;&#x6D4B;&#x8840;&#x7BA1;&#x6D3B;&#x6027;&#x7EC4;&#x4EF6;&#x4E2D; AM1 &#x53D7;&#x4F53;&#x7684;&#x4F5C;&#x7528;&#xFF0C;&#x5E76;&#x63D0;&#x4F9B;&#x8BC1;&#x636E;&#x663E;&#x793A;&#x5BF9;&#x4E0A;&#x5348;&#x7684;&#x964D;&#x538B;&#x53CD;&#x5E94;&#x6709;&#x8131;&#x654F;&#x4F53;&#x5185;&#x3002;&#x5728;&#x5730;&#x65B9;&#x4E00;&#x7EA7;&#xFF0C;&#x4EE5;&#x9632;&#x8BF1;&#x53D1;&#x9AD8;&#x8840;&#x538B;&#x8840;&#x7BA1;&#x589E;&#x751F;&#x8840;&#x7BA1;&#x5E73;&#x6ED1;&#x808C; AM1 &#x53D7;&#x4F53;&#x884C;&#x4E3A;&#x548C;&#x708E;&#x75C7;&#x53CD;&#x5E94;&#x63D0;&#x4F9B;&#x4E86;&#x8BC1;&#x636E;&#xFF0C;&#x9488;&#x5BF9;&#x8FD9;&#x79CD;&#x53D7;&#x4F53;&#x7684;&#x53D1;&#x73B0;&#x53EF;&#x80FD;&#x662F;&#x4E00;&#x79CD;&#x6709;&#x76CA;&#x7684;&#x6CBB;&#x7597;&#x65B9;&#x6CD5;&#x3002;

&#x8840;&#x7BA1;&#x7D27;&#x5F20;&#x7D20; II &#x8BF1;&#x5BFC;&#x8840;&#x7BA1;&#x5E73;&#x6ED1;&#x808C;&#x9488;&#x5BF9;&#x53D7;&#x4F53;&#x6D3B;&#x6027;&#x4FEE;&#x9970;&#x86CB;&#x767D; 2 &#x8F6C;&#x57FA;&#x56E0;&#x5C0F;&#x9F20;&#x8840;&#x7BA1;&#x80A5;&#x539A;&#x7684;&#x4FDD;&#x62A4;&#x3002;

The vasodilator and vascular regulatory peptide adrenomedullin (AM), a member of the calcitonin gene-related peptide family of peptides, is predicted to play a pivotal protective role in cardiovascular dysfunction. The principle AM (AM1) receptor is composed of a G protein-linked calcitonin receptor-like receptor and a receptor activity-modifying protein (receptor activity-modifying protein 2). There is little knowledge of the receptors via which AM acts in diseases. Using smooth muscle-targeted receptor activity-modifying protein 2 transgenic mice with increased vascular density of functional AM1 receptors, we demonstrate that receptor activity-modifying protein 2 transgenic mice are not protected against angiotensin II-induced hypertension or cardiac hypertrophy. However, vascular hypertrophy, together with vascular cell adhesion molecule 1 and monocyte chemotactic protein 1 expression, is significantly reduced in the aortic walls of transgenic mice, as determined by histological techniques. This indicates that the AM1 vascular smooth muscle receptor can mediate local protection in vivo. This is supported by proliferation studies in cultured smooth muscle cells. By comparison, levels of hypotension and inflammation in a shock model were similar to those in wild-type mice. Thus, a role of the AM1 receptor in the vasoactive component could not be detected, and evidence is provided to show that the hypotensive response to AM is subject to desensitization in vivo. The finding that the vascular smooth muscle AM1 receptor acts at a local level to protect against hypertension-induced vascular hypertrophy and inflammation provides evidence that targeting this receptor may be a beneficial therapeutic approach.

Protection of angiotensin II-induced vascular hypertrophy in vascular smooth muscle-targeted receptor activity-modifying protein 2 transgenic mice.

Le vasodilatateur et peptide r&eacute;gulation vasculaire adr&eacute;nom&eacute;dulline (AM), un membre de la famille de peptide li&eacute; au g&egrave;ne de la calcitonine des peptides, est cens&eacute; pour jouer un r&ocirc;le protecteur en trouble cardiovasculaire. Le principe du r&eacute;cepteur AM (AM1) est compos&eacute; d&#39;un r&eacute;cepteur de comme r&eacute;cepteur calcitonine li&eacute;e &agrave; la prot&eacute;ine G et une r&eacute;cepteur-modification d&#39;activit&eacute; prot&eacute;ine (r&eacute;cepteur-modification d&#39;activit&eacute; 2). Il y a peu de connaissances des r&eacute;cepteurs par l&#39;interm&eacute;diaire de quels actes AM dans les maladies. &Agrave; l&#39;aide de souris transg&eacute;niques r&eacute;cepteurs ax&eacute;s sur le muscle lisse-modification d&#39;activit&eacute; prot&eacute;ine 2 avec une densit&eacute; vasculaire accrue des r&eacute;cepteurs fonctionnels de AM1, nous d&eacute;montrons que les souris transg&eacute;niques receptor-modification d&#39;activit&eacute; prot&eacute;ine 2 ne sont pas prot&eacute;g&eacute;es contre l&#39;angiotensine II-induced hypertension ou une hypertrophie cardiaque. Cependant, l&#39;hypertrophie vasculaire, ainsi que de la mol&eacute;cule d&#39;adh&eacute;sion cellulaire vasculaire 1 et expression de la prot&eacute;ine chimiotactique 1 monocyte, est consid&eacute;rablement r&eacute;duit dans les parois aortiques de souris transg&eacute;niques, tel que d&eacute;termin&eacute; par des techniques histologiques. Cela indique que le r&eacute;cepteur du muscle lisse vasculaire AM1 peut servir de m&eacute;diateur locale protection in vivo. Ceci est soutenu par des &eacute;tudes de prolif&eacute;ration de cellules musculaires lisses cultiv&eacute;es. &Agrave; titre de comparaison, les niveaux de l&#39;hypotension et l&#39;inflammation dans un mod&egrave;le de choc &eacute;taient semblables &agrave; celles des souris de type sauvage. Ainsi, un r&ocirc;le du r&eacute;cepteur AM1 dans le composant vasomoteur n&#39;a pas pu &ecirc;tre d&eacute;tect&eacute;, et la preuve est apport&eacute;e pour montrer que la r&eacute;ponse hypotensive &agrave; AM est soumis &agrave; une d&eacute;sensibilisation in vivo. La conclusion que le muscle lisse vasculaire AM1 r&eacute;cepteur agit au niveau local pour se prot&eacute;ger contre l&#39;hypertrophie vasculaire induite par l&#39;hypertension art&eacute;rielle et l&#39;inflammation apporte la preuve que ciblant ce r&eacute;cepteur peut &ecirc;tre une approche th&eacute;rapeutique b&eacute;n&eacute;fique.

Protection de l&#39;angiotensine II vasculaire hypertrophie induite chez des souris transg&eacute;niques r&eacute;cepteur ax&eacute;s sur le muscle lisse vasculaire modifiant l&#39;activit&eacute; prot&eacute;ine 2.

Die gef&auml;&szlig;erweiternden und vaskul&auml;re regulatorische Peptide Adrenomedullin (AM), ein Mitglied der Familie der Calcitonin-Gene-related Peptide Peptide, wird voraussichtlich eine entscheidende sch&uuml;tzende Rolle in kardiovaskul&auml;re Dysfunktion. Das Prinzip (AM1) AM Rezeptor besteht aus ein G-Protein-chromosomale Calcitonin-Rezeptor-Like-Rezeptor und eine Aktivit&auml;t &auml;ndern Protein Verband (Rezeptor-Aktivit&auml;t &auml;ndern Protein 2). Es gibt wenig Wissen &uuml;ber die Rezeptoren &uuml;ber welche AM Handlungen in Krankheiten. Mit Rezeptor Glattmuskel-bezogene Aktivit&auml;t &auml;ndern Protein 2 transgene M&auml;use mit erh&ouml;hte vaskul&auml;re Dichte an funktionalen AM1-Rezeptoren, zeigen wir, dass Rezeptor-Aktivit&auml;t &auml;ndern Protein 2 transgene M&auml;use nicht gegen Angiotensin II-induzierte Hypertonie oder Herzhypertrophie gesch&uuml;tzt sind. Jedoch ist vaskul&auml;rer Hypertrophie, zusammen mit vaskul&auml;ren Zelladh&auml;sionsmolek&uuml;l 1 und Monozyte chemotaktische Protein 1 Ausdruck, in die Aorta W&auml;nde der transgenen M&auml;usen, festgelegt durch histologische Techniken deutlich reduziert. Dies bedeutet, dass der AM1-vaskul&auml;rer Glatter Muskelzellen-Rezeptor in-vivo lokalen Schutz vermitteln kann. Unterst&uuml;tzt wird dies durch Verbreitung Studien in kultivierten glatten Muskelzellen. Im Vergleich dazu waren die Hypotonie und Entz&uuml;ndung in einem Schock-Modell &auml;hnlich denen in Wildtyp M&auml;usen. So eine Rolle des Rezeptors AM1 in der vasoaktive Komponente konnte nicht erkannt werden, und nachgewiesen ist um zu zeigen, dass die hypotone Reaktion auf AM vorbehaltlich der in-vivo Desensibilisierung. Die Feststellung, dass der vaskul&auml;rer Glatter Muskelzellen AM1-Rezeptor auf lokaler Ebene zum Schutz gegen Bluthochdruck-induzierter vaskul&auml;rer Hypertrophie wirkt und Entz&uuml;ndung Beweise liefert, dass die Ausrichtung dieser Rezeptor m&ouml;glicherweise einen positiven therapeutischen Ansatz.

Schutz der Angiotensin II-induzierter vaskul&auml;rer Hypertrophie vaskul&auml;rer Glatter Muskelzellen ausgerichtete Rezeptor-Aktivit&auml;t &auml;ndern Protein 2 transgenen M&auml;usen.

Il vasodilatatore e vascolare del peptide normativo Adrenomedullina (AM), un membro della famiglia del peptide correlato al gene della calcitonina di peptidi, &egrave; previsto a giocare un ruolo protettivo nelle disfunzioni cardiovascolari. Il principio del ricevitore AM (AM1) &egrave; composto da un recettore receptor-like di calcitonina legati a proteine G e una modifica della attivit&agrave; proteina recettore (Modifica attivit&agrave; proteina recettore 2). C&#39;&egrave; poca conoscenza dei recettori tramite quali atti AM in malattie. Utilizzando mirati muscolo liscio recettore modifica attivit&agrave; proteina 2 topi transgenici con maggiore densit&agrave; vascolare dei recettori AM1 funzionali, dimostriamo che il recettore modifica attivit&agrave; proteina 2 topi transgenici non sono protetti contro angiotensina II-ipertensione indotta o ipertrofia cardiaca. Tuttavia, l&#39;ipertrofia vascolare, insieme a 1 la molecola di adesione delle cellule vascolari e monocito proteina chemiotattica 1 espressione, &egrave; significativamente ridotta nelle pareti dell&#39;aorta di topi transgenici, come determinato dalle tecniche istologiche. Questo indica che il recettore della muscolatura liscia vascolare AM1 pu&ograve; mediare protezione locale in vivo. Questo &egrave; supportato da studi di proliferazione di cellule muscolari lisce coltivate. In confronto, i livelli di infiammazione in un modello di shock e ipotensione erano simili a quelli nei topi wild-type. Cos&igrave;, un ruolo del recettore AM1 nel componente vasoattivo potrebbe non essere rilevato, ed elementi di prova sufficienti a dimostrare che la risposta ipotensiva a AM &egrave; soggetta a desensibilizzazione in vivo. La constatazione che il recettore della muscolatura liscia vascolare AM1 agisce a livello locale a proteggere contro l&#39;ipertensione indotta ipertrofia vascolare e infiammazione fornisce la prova che questo recettore di targeting pu&ograve; essere un utile approccio terapeutico.

Protezione di ipertrofia dell&#39;angiotensina II-indotta vascolare in vascular targeting muscolo liscio recettore modifica attivit&agrave; proteina 2 topi transgenici.

&#x8840;&#x7BA1;&#x62E1;&#x5F35;&#x85AC;&#x3068;&#x8840;&#x7BA1;&#x8ABF;&#x7BC0;&#x30DA;&#x30D7;&#x30C1;&#x30C9; &#x30A2;&#x30C9;&#x30EC;&#x30CE;&#x30E1;&#x30C7;&#x30E5;&#x30EA;&#x30F3; &#xFF08;&#x5348;&#x524D;&#xFF09;&#x3001;&#x30DA;&#x30D7;&#x30C1;&#x30C9;&#x3001;&#x30AB;&#x30EB;&#x30B7;&#x30C8;&#x30CB;&#x30F3;&#x907A;&#x4F1D;&#x5B50;&#x95A2;&#x9023;&#x30DA;&#x30D7;&#x30C1;&#x30C9; &#x30D5;&#x30A1;&#x30DF;&#x30EA;&#x306E;&#x30E1;&#x30F3;&#x30D0;&#x30FC;&#x3092;&#x4E88;&#x6E2C;&#x306B;&#x304A;&#x3051;&#x308B;&#x5FC3;&#x8840;&#x7BA1;&#x7CFB;&#x306E;&#x6A5F;&#x80FD;&#x969C;&#x5BB3;&#x4FDD;&#x8B77;&#x306E;&#x6975;&#x3081;&#x3066;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x518D;&#x751F;&#x3057;&#x307E;&#x3059;&#x3002;&#x539F;&#x5247;&#x5348;&#x524D; &#xFF08;AM1) &#x53D7;&#x5BB9;&#x4F53;&#x306F;&#x3001;G &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x30EA;&#x30F3;&#x30AF; &#x30AB;&#x30EB;&#x30B7;&#x30C8;&#x30CB;&#x30F3;&#x53D7;&#x5BB9;&#x4F53;&#x69D8;&#x53D7;&#x5BB9;&#x4F53;&#x3068;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D3;&#x30C6;&#x30A3;&#x3092;&#x5909;&#x66F4;&#x3059;&#x308B;&#x53D7;&#x5BB9;&#x4F53;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA; (&#x53D7;&#x5BB9;&#x4F53;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D3;&#x30C6;&#x30A3;&#x3092;&#x5909;&#x66F4;&#x3059;&#x308B;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA; 2&#xFF09; &#x306E;&#x3067;&#x69CB;&#x6210;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x5C11;&#x3057;&#x306E;&#x77E5;&#x8B58;&#x306E;&#x53D7;&#x5BB9;&#x4F53;&#x3092;&#x4ECB;&#x3057;&#x305F;&#x65E5;&#x6A5F;&#x80FD;&#x75BE;&#x60A3;&#x3067;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x53D7;&#x5BB9;&#x4F53;&#x306E;&#x5E73;&#x6ED1;&#x7B4B;&#x3092;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8; &#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D3;&#x30C6;&#x30A3;&#x3092;&#x5909;&#x66F4;&#x3059;&#x308B;&#x86CB;&#x767D;&#x8CEA; 2 &#x306E;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30B8;&#x30A7;&#x30CB;&#x30C3;&#x30AF; &#x30DE;&#x30A6;&#x30B9;&#x6A5F;&#x80FD; AM1 &#x53D7;&#x5BB9;&#x4F53;&#x306E;&#x8840;&#x7BA1;&#x5BC6;&#x5EA6;&#x306E;&#x5897;&#x52A0;&#x306B;&#x4F34;&#x3063;&#x3066;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3001;&#x6211;&#x3005; &#x53D7;&#x5BB9;&#x4F53;&#x86CB;&#x767D;&#x8CEA; 2 &#x306E;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30B8;&#x30A7;&#x30CB;&#x30C3;&#x30AF; &#x30DE;&#x30A6;&#x30B9;&#x304C;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D3;&#x30C6;&#x30A3;&#x3092;&#x5909;&#x66F4;&#x3059;&#x308B;&#x30A2;&#x30F3;&#x30B8;&#x30AA;&#x30C6;&#x30F3;&#x30B7;&#x30F3; II &#x9AD8;&#x8840;&#x5727;&#x3084;&#x5FC3;&#x81D3;&#x80A5;&#x5927;&#x306B;&#x5BFE;&#x3057;&#x3066;&#x4FDD;&#x8B77;&#x3055;&#x308C;&#x3066;&#x3044;&#x306A;&#x3044;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3057;&#x304B;&#x3057;&#x3001;&#x5927;&#x52D5;&#x8108;&#x58C1;&#x306E;&#x7D44;&#x7E54;&#x5B66;&#x7684;&#x6280;&#x8853;&#x306B;&#x3088;&#x3063;&#x3066;&#x6C7A;&#x5B9A;&#x3068;&#x3057;&#x3066;&#x306E;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30B8;&#x30A7;&#x30CB;&#x30C3;&#x30AF; &#x30DE;&#x30A6;&#x30B9;&#x8840;&#x7BA1;&#x7D30;&#x80DE;&#x63A5;&#x7740;&#x5206;&#x5B50; 1 &#x3068;&#x5358;&#x7403;&#x306E;&#x8D70;&#x5316;&#x6027;&#x86CB;&#x767D;&#x8CEA; 1 &#x306E;&#x8868;&#x73FE;&#x3001;&#x4E00;&#x7DD2;&#x306E;&#x8840;&#x7BA1;&#x80A5;&#x5927;&#x304C;&#x5927;&#x5E45;&#x306B;&#x524A;&#x6E1B;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x306F;&#x3001;AM1 &#x8840;&#x7BA1;&#x5E73;&#x6ED1;&#x7B4B;&#x306E;&#x53D7;&#x5BB9;&#x4F53;&#x304C; in vivo &#x3067;&#x306E;&#x5730;&#x65B9;&#x306E;&#x4FDD;&#x8B77;&#x3092;&#x4EF2;&#x4ECB;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x306F;&#x57F9;&#x990A;&#x5E73;&#x6ED1;&#x7B4B;&#x7D30;&#x80DE;&#x5897;&#x6B96;&#x7814;&#x7A76;&#x306B;&#x3088;&#x3063;&#x3066;&#x30B5;&#x30DD;&#x30FC;&#x30C8;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x6BD4;&#x8F03;&#x3067;&#x306F;&#x3001;&#x4F4E;&#x8840;&#x5727;&#x75C7;&#x3068;&#x30B7;&#x30E7;&#x30C3;&#x30AF; &#x30E2;&#x30C7;&#x30EB;&#x306B;&#x304A;&#x3051;&#x308B;&#x708E;&#x75C7;&#x306E;&#x30EC;&#x30D9;&#x30EB;&#x306F;&#x91CE;&#x751F;&#x578B;&#x30DE;&#x30A6;&#x30B9;&#x306B;&#x4F3C;&#x3066;&#x3044;&#x305F;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;AM1 &#x53D7;&#x5BB9;&#x4F53;&#x8840;&#x7BA1;&#x4F5C;&#x52D5;&#x6027;&#x306E;&#x30B3;&#x30F3;&#x30DD;&#x30FC;&#x30CD;&#x30F3;&#x30C8;&#x306E;&#x5F79;&#x5272;&#x3092;&#x691C;&#x51FA;&#x3067;&#x304D;&#x307E;&#x305B;&#x3093;&#x3067;&#x3057;&#x305F;&#x3068;&#x4F4E;&#x8840;&#x5727;&#x5FDC;&#x7B54;&#x5348;&#x524D;&#x306B;&#x8131;&#x611F;&#x4F5C;&#x4E88;&#x544A; in vivo &#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3059;&#x306B;&#x8A3C;&#x62E0;&#x304C;&#x63D0;&#x4F9B;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x8840;&#x7BA1;&#x5E73;&#x6ED1;&#x7B4B; AM1 &#x53D7;&#x5BB9;&#x4F53;&#x9AD8;&#x8840;&#x5727;&#x306B;&#x3088;&#x308B;&#x8840;&#x7BA1;&#x80A5;&#x5927;&#x306B;&#x5BFE;&#x3057;&#x3066;&#x4FDD;&#x8B77;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x30ED;&#x30FC;&#x30AB;&#x30EB; &#x30EC;&#x30D9;&#x30EB;&#x3067;&#x6A5F;&#x80FD;&#x3057;&#x3001;&#x708E;&#x75C7;&#x8A3C;&#x62E0;&#x3092;&#x63D0;&#x4F9B;&#x3059;&#x308B;&#x3053;&#x306E;&#x53D7;&#x5BB9;&#x4F53;&#x3092;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x3092;&#x898B;&#x3064;&#x3051;&#x308B;&#x3053;&#x3068;&#x306F;&#x3001;&#x6709;&#x76CA;&#x306A;&#x6CBB;&#x7642;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;

&#x8840;&#x7BA1;&#x5E73;&#x6ED1;&#x7B4B;&#x3092;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x53D7;&#x5BB9;&#x4F53;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D3;&#x30C6;&#x30A3;&#x3092;&#x5909;&#x66F4;&#x3059;&#x308B;&#x86CB;&#x767D;&#x8CEA; 2 &#x306E;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30B8;&#x30A7;&#x30CB;&#x30C3;&#x30AF; &#x30DE;&#x30A6;&#x30B9;&#x306B;&#x304A;&#x3051;&#x308B;&#x30A2;&#x30F3;&#x30B8;&#x30AA;&#x30C6;&#x30F3;&#x30B7;&#x30F3; II &#x306B;&#x3088;&#x308B;&#x8840;&#x7BA1;&#x80A5;&#x5927;&#x306E;&#x4FDD;&#x8B77;&#x3002;

Vasodilator &#xBC0F; &#xD608;&#xAD00; &#xADDC;&#xC81C; &#xD3A9; &#xD2F0; &#xB4DC; adrenomedullin (&#xC624;&#xC804;), &#xD3A9; &#xD2F0; &#xB4DC;, &#xCE7C; &#xC2DC; &#xD1A0; &#xB2CC; &#xC720;&#xC804;&#xC790; &#xAD00;&#xB828; &#xD3A9; &#xD2F0; &#xB4DC; &#xC81C;&#xD488;&#xAD70; &#xC2EC;&#xD608; &#xAD00; &#xAE30;&#xB2A5; &#xC7A5;&#xC560;&#xC5D0; &#xC911;&#xCD94;&#xC801;&#xC778; &#xBCF4;&#xD638; &#xC5ED;&#xD560;&#xC744; &#xD560; &#xC804;&#xB9DD; &#xC774;&#xB2E4;. &#xC6D0;&#xCE59; &#xC624;&#xC804; (AM1) &#xC218;&#xC6A9; &#xCCB4; G &#xB2E8;&#xBC31;&#xC9C8; &#xC5F0;&#xACB0; &#xB41C; &#xCE7C; &#xC2DC; &#xD1A0; &#xB2CC; &#xC218;&#xC6A9; &#xCCB4;&#xC640; &#xAC19;&#xC740; &#xC218;&#xC6A9; &#xCCB4;&#xC640; &#xC218;&#xC6A9; &#xCCB4; &#xD65C;&#xB3D9;&#xC744; &#xC218;&#xC815; &#xB2E8;&#xBC31;&#xC9C8; (&#xC218;&#xC6A9; &#xCCB4; &#xD65C;&#xB3D9;&#xC744; &#xC218;&#xC815; &#xB2E8;&#xBC31;&#xC9C8; 2)&#xC73C;&#xB85C; &#xAD6C;&#xC131; &#xB429;&#xB2C8;&#xB2E4;. &#xC9C8;&#xBCD1;&#xC5D0;&#xC11C; &#xC624;&#xC804; &#xC791;&#xB3D9;&#xC744; &#xD1B5;&#xD574; &#xC218;&#xC6A9; &#xCCB4;&#xC758; &#xC57D;&#xAC04;&#xC758; &#xC9C0;&#xC2DD;&#xC774; &#xC788;&#xB2E4;. &#xBD80;&#xB4DC;&#xB7EC;&#xC6B4; &#xADFC;&#xC721; &#xD0C0;&#xAC9F;&#xD305; &#xC218;&#xC6A9; &#xCCB4; &#xD65C;&#xB3D9;&#xC744; &#xC218;&#xC815; &#xB2E8;&#xBC31;&#xC9C8; 2 &#xC720;&#xC804;&#xC790; &#xBCC0;&#xD615; &#xC950;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xAE30;&#xB2A5; AM1 &#xC218;&#xC6A9; &#xCCB4;&#xC758; &#xC99D;&#xAC00; &#xD608;&#xAD00; &#xBC00;&#xB3C4;, &#xC218;&#xC6A9; &#xCCB4; &#xD65C;&#xB3D9;&#xC744; &#xC218;&#xC815; &#xB2E8;&#xBC31;&#xC9C8; 2 &#xC720;&#xC804;&#xC790; &#xBCC0;&#xD615; &#xC0DD;&#xC950; angiotensin II &#xC720;&#xBC1C; &#xACE0;&#xD608;&#xC555; &#xB610;&#xB294; &#xC2EC;&#xC7A5; &#xBE44; &#xB300;&#xC5D0; &#xB300; &#xD55C; &#xBCF4;&#xD638; &#xB418;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xD608;&#xAD00; &#xC138;&#xD3EC; &#xC811;&#xCC29; &#xBD84;&#xC790; 1 &#xBC0F; monocyte &#xD608; &#xB2E8;&#xBC31;&#xC9C8; 1 &#xC2DD;, &#xD608;&#xAD00; &#xBE44; &#xB300; &#xC870;&#xC9C1;&#xD559; &#xAE30;&#xBC95;&#xC5D0; &#xC758;&#xD574; &#xD655;&#xC778; &#xB41C; &#xC720;&#xC804;&#xC790; &#xBCC0;&#xD615; &#xC950;&#xC758; &#xB300;&#xB3D9;&#xB9E5; &#xBCBD;&#xC5D0; &#xD06C;&#xAC8C; &#xC904;&#xC5B4;&#xB4ED;&#xB2C8;&#xB2E4;. AM1 &#xD608;&#xAD00; &#xBD80;&#xB4DC;&#xB7EC;&#xC6B4; &#xADFC;&#xC721; &#xC218;&#xC6A9; &#xCCB4; &#xB85C;&#xCEEC; &#xBCF4;&#xD638; vivo&#xC5D0;&#xC11C; &#xC911;&#xC7AC;&#xD560; &#xC218; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;. &#xC774; &#xAD50;&#xC591;&#xB41C; &#xBD80;&#xB4DC;&#xB7EC;&#xC6B4; &#xADFC;&#xC721; &#xC138;&#xD3EC;&#xC758; &#xD655;&#xC0B0; &#xC5F0;&#xAD6C;&#xC5D0; &#xC758;&#xD574; &#xC9C0;&#xC6D0; &#xB429;&#xB2C8;&#xB2E4;. &#xBE44;&#xAD50; &#xD558; &#xC5EC;, &#xC218;&#xC900;&#xC758; &#xC800; &#xD608;&#xC555; &#xBC0F; &#xC1FC;&#xD06C; &#xBAA8;&#xB378;&#xC758; &#xC5FC;&#xC99D; &#xC57C;&#xC0DD;-&#xD0C0;&#xC785; &#xB9C8;&#xC6B0;&#xC2A4;&#xC758; &#xAC83;&#xACFC; &#xC720;&#xC0AC; &#xD588;&#xB2E4;. &#xB530;&#xB77C;&#xC11C;, vasoactive &#xAD6C;&#xC131; &#xC694;&#xC18C;&#xC5D0;&#xC11C; AM1 &#xC218;&#xC6A9; &#xCCB4;&#xC758; &#xC5ED;&#xD560;&#xC744; &#xAC80;&#xC0C9; &#xD558;&#xC9C0; &#xBABB;&#xD588;&#xC2B5;&#xB2C8;&#xB2E4;, &#xADF8;&#xB9AC;&#xACE0; &#xC99D;&#xAC70; &#xC624;&#xC804; hypotensive &#xC751;&#xB2F5; &#xB454;&#xAC10; &#xC0DD;&#xCCB4; &#xC870;&#xAC74;&#xC5D0; &#xB530;&#xB77C; &#xD45C;&#xC2DC; &#xB418;&#xB294;. &#xD608;&#xAD00; &#xBD80;&#xB4DC;&#xB7EC;&#xC6B4; &#xADFC;&#xC721; AM1 &#xC218;&#xC6A9; &#xCCB4; &#xD608;&#xAD00; &#xBE44; &#xB300; &#xACE0;&#xD608;&#xC555; &#xC720;&#xBC1C; &#xB85C;&#xBD80;&#xD130; &#xBCF4;&#xD638; &#xD558;&#xAE30; &#xC704;&#xD574; &#xB85C;&#xCEEC; &#xC218;&#xC900;&#xC5D0;&#xC11C; &#xC5ED;&#xD560; &#xBC0F; &#xC5FC;&#xC99D;&#xC774; &#xC218;&#xC6A9;&#xC774; &#xCCB4;&#xB97C; &#xB300;&#xC0C1;&#xC73C;&#xB85C; &#xADF8; &#xC99D;&#xAC70;&#xB97C; &#xC81C;&#xACF5; &#xCC3E;&#xB294; &#xC720;&#xC775;&#xD55C; &#xCE58;&#xB8CC; &#xC811;&#xADFC; &#xC788;&#xC744; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

Angiotensin II &#xC720;&#xB3C4; &#xD608;&#xAD00; &#xBE44; &#xD608;&#xAD00; &#xBD80;&#xB4DC;&#xB7EC;&#xC6B4; &#xADFC;&#xC721; &#xD0C0;&#xAC9F;&#xD305; &#xC218;&#xC6A9; &#xCCB4; &#xD65C;&#xB3D9;&#xC744; &#xC218;&#xC815; &#xB2E8;&#xBC31;&#xC9C8; 2 &#xC720;&#xC804;&#xC790; &#xBCC0;&#xD615; &#xC0DD;&#xC950;&#xC5D0;&#xC11C;&#xC758; &#xBCF4;&#xD638;.

El vasodilatador y p&eacute;ptido regulador vascular adrenomedulina (AM), un miembro de la familia de p&eacute;ptido relacionado con el gen de la calcitonina de p&eacute;ptidos, se prev&eacute; que desempe&ntilde;an un papel fundamental de la protecci&oacute;n en disfunci&oacute;n cardiovascular. El principio del receptor de AM (AM1) se compone de un receptor de receptor-como calcitonina vinculados con prote&iacute;na G y una prote&iacute;na de modificadores de la actividad de receptor (modificadores de la actividad del receptor prote&iacute;na 2). Hay poco conocimiento de los receptores v&iacute;a que act&uacute;a de AM en enfermedades. Utilizando ratones transg&eacute;nicos receptor orientada por m&uacute;sculo liso modificadores de la actividad 2 de la prote&iacute;na con mayor densidad vascular de receptores funcionales de AM1, demostramos que ratones transg&eacute;nicos receptor modificadores de la actividad prote&iacute;na 2 no est&aacute;n protegidas contra la hipertensi&oacute;n inducida por el II de angiotensina o hipertrofia cardiaca. Sin embargo, hipertrofia vascular, junto con la mol&eacute;cula de adhesi&oacute;n celular vascular 1 y expresi&oacute;n de la prote&iacute;na quimiot&aacute;ctica 1 de monocitos, se reduce significativamente en las paredes de la aorta de ratones transg&eacute;nicos, seg&uacute;n lo determinado por t&eacute;cnicas histol&oacute;gicas. Esto indica que el receptor del m&uacute;sculo liso vascular AM1 puede mediar la protecci&oacute;n local en vivo. Esto es apoyada por estudios de proliferaci&oacute;n de las c&eacute;lulas cultivadas del m&uacute;sculo liso. En comparaci&oacute;n, los niveles de la hipotensi&oacute;n y la inflamaci&oacute;n en un modelo de choque fueron similares a los ratones de tipo salvaje. Por lo tanto, no se pudo detectar un papel del receptor AM1 en el componente vasoactivo, y se proporciona evidencia para demostrar que la respuesta hipotensa a AM sufre de desensibilizaci&oacute;n en vivo. El hallazgo de que el receptor del m&uacute;sculo liso vascular AM1 act&uacute;a a nivel local para proteger contra la hipertrofia vascular inducida por la hipertensi&oacute;n e inflamaci&oacute;n proporciona la evidencia la orientaci&oacute;n de este receptor puede ser un enfoque terap&eacute;utico beneficioso.

Protecci&oacute;n de la hipertrofia de la vascular de la angiotensina II-inducida en ratones transg&eacute;nicos receptor orientada por m&uacute;sculo liso vascular modificadores de la actividad 2 de la prote&iacute;na.

Rapid plasma membrane resealing is essential for cellular survival. Earlier studies showed that plasma membrane repair requires Ca(2+)-dependent exocytosis of lysosomes and a rapid form of endocytosis that removes membrane lesions. However, the functional relationship between lysosomal exocytosis and the rapid endocytosis that follows membrane injury is unknown. In this study, we show that the lysosomal enzyme acid sphingomyelinase (ASM) is released extracellularly when cells are wounded in the presence of Ca(2+). ASM-deficient cells, including human cells from Niemann-Pick type A (NPA) patients, undergo lysosomal exocytosis after wounding but are defective in injury-dependent endocytosis and plasma membrane repair. Exogenously added recombinant human ASM restores endocytosis and resealing in ASM-depleted cells, suggesting that conversion of plasma membrane sphingomyelin to ceramide by this lysosomal enzyme promotes lesion internalization. These findings reveal a molecular mechanism for restoration of plasma membrane integrity through exocytosis of lysosomes and identify defective plasma membrane repair as a possible component of the severe pathology observed in NPA patients.

Exocytosis of acid sphingomyelinase by wounded cells promotes endocytosis and plasma membrane repair.

Upon host cell contact, the protozoan parasite Trypanosoma cruzi triggers cytosolic Ca(2+) transients that induce exocytosis of lysosomes, a process required for cell invasion. However, the exact mechanism by which lysosomal exocytosis mediates T. cruzi internalization remains unclear. We show that host cell entry by T. cruzi mimics a process of plasma membrane injury and repair that involves Ca(2+)-dependent exocytosis of lysosomes, delivery of acid sphingomyelinase (ASM) to the outer leaflet of the plasma membrane, and a rapid form of endocytosis that internalizes membrane lesions. Host cells incubated with T. cruzi trypomastigotes are transiently wounded, show increased levels of endocytosis, and become more susceptible to infection when injured with pore-forming toxins. Inhibition or depletion of lysosomal ASM, which blocks plasma membrane repair, markedly reduces the susceptibility of host cells to T. cruzi invasion. Notably, extracellular addition of sphingomyelinase stimulates host cell endocytosis, enhances T. cruzi invasion, and restores normal invasion levels in ASM-depleted cells. Ceramide, the product of sphingomyelin hydrolysis, is detected in newly formed parasitophorous vacuoles containing trypomastigotes but not in the few parasite-containing vacuoles formed in ASM-depleted cells. Thus, T. cruzi subverts the ASM-dependent ceramide-enriched endosomes that function in plasma membrane repair to infect host cells.

Trypanosoma cruzi subverts the sphingomyelinase-mediated plasma membrane repair pathway for cell invasion.

Cells permeabilized by the bacterial pore-forming toxin streptolysin O (SLO) reseal their plasma membrane in a Ca(2+) -dependent manner. Resealing involves Ca(2+) -dependent exocytosis of lysosomes, release of acid sphingomyelinase and rapid formation of endosomes that carry the transmembrane pores into the cell. The intracellular fate of the toxin-carrying endocytic vesicles, however, is still unknown. Here, we show that SLO pores removed from the plasma membrane by endocytosis are sorted into the lumen of lysosomes, where they are degraded. SLO-permeabilized cells contain elevated numbers of total endosomes, which increase gradually in size while transitioning from endosomes with flat clathrin coats to large multivesicular bodies (MVBs). Under conditions that allow endocytosis and plasma membrane repair, SLO is rapidly ubiquitinated and gradually degraded, in a process sensitive to inhibitors of lysosomal hydrolysis but not of proteasomes. The endosomes induced by SLO permeabilization become increasingly acidified and promote SLO degradation under normal conditions, but not in cells silenced for expression of Vps24, an ESCRT-III complex component required for the release of intraluminal vesicles into MVBs. Thus, cells dispose of SLO transmembrane pores by ubiquitination/ESCRT-dependent sorting into the lumen of late endosomes/lysosomes.

Christina Tam

Department of Cell Biology and Molecular Genetics

University of Maryland

Live Imaging Assay for Assessing the Roles of Ca²⁺ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Christina Tam

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cell Biology and Molecular Genetics

University of Maryland

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Department of Cell Biology and Molecular Genetics

Live Imaging Assay for Assessing the Roles of Ca²⁺ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds