automated-acoustic-dispensing-for-serial-dilution-peptide-agonists

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

MedImmune

TRPC calcium channels are emerging as a ubiquitous feature of vertebrate cells, but understanding of them is hampered by limited knowledge of the mechanisms of activation and identity of endogenous regulators. We have revealed that one of the TRPC channels, TRPC5, is strongly activated by common endogenous lysophospholipids including lysophosphatidylcholine (LPC) but, by contrast, not arachidonic acid. Although TRPC5 was stimulated by agonists at G-protein-coupled receptors, TRPC5 activation by LPC occurred downstream and independently of G-protein signaling. The effect was not due to the generation of reactive oxygen species or because of a detergent effect of LPC. LPC activated TRPC5 when applied to excised membrane patches and thus has a relatively direct action on the channel structure, either because of a phospholipid binding site on the channel or because of sensitivity of the channel to perturbation of the bilayer by certain lipids. Activation showed dependence on side-chain length and the chemical head-group. The data revealed a previously unrecognized lysophospholipid-sensing capability of TRPC5 that confers the property of a lipid ionotropic receptor.

Sensing of lysophospholipids by TRPC5 calcium channel.

In a screen of potential lipid regulators of transient receptor potential (TRP) channels, we identified sphingosine-1-phosphate (S1P) as an activator of TRPC5. We explored the relevance to vascular biology because S1P is a key cardiovascular signaling molecule. TRPC5 is expressed in smooth muscle cells of human vein along with TRPC1, which forms a complex with TRPC5. Importantly, S1P also activates the TRPC5-TRPC1 heteromultimeric channel. Because TRPC channels are linked to neuronal growth cone extension, we considered a related concept for smooth muscle. We find S1P stimulates smooth muscle cell motility, and that this is inhibited by E3-targeted anti-TRPC5 antibody. Ion permeation involving TRPC5 is crucial because S1P-evoked motility is also suppressed by the channel blocker 2-aminoethoxydiphenyl borate or a TRPC5 ion-pore mutant. S1P acts on TRPC5 via two mechanisms, one extracellular and one intracellular, consistent with its bipolar signaling functions. The extracellular effect appears to have a primary role in S1P-evoked cell motility. The data suggest S1P sensing by TRPC5 calcium channel is a mechanism contributing to vascular smooth muscle adaptation.

A sphingosine-1-phosphate-activated calcium channel controlling vascular smooth muscle cell motility.

Mammalian homologues of Drosophila melanogaster transient receptor potential (TRP) are a large family of multimeric cation channels that act, or putatively act, as sensors of one or more chemical factor. Major research objectives are the identification of endogenous activators and the determination of cellular and tissue functions of these channels. Here we show the activation of TRPC5 (canonical TRP 5) homomultimeric and TRPC5-TRPC1 heteromultimeric channels by extracellular reduced thioredoxin, which acts by breaking a disulphide bridge in the predicted extracellular loop adjacent to the ion-selectivity filter of TRPC5. Thioredoxin is an endogenous redox protein with established intracellular functions, but it is also secreted and its extracellular targets are largely unknown. Particularly high extracellular concentrations of thioredoxin are apparent in rheumatoid arthritis, an inflammatory joint disease that disables millions of people worldwide. We show that TRPC5 and TRPC1 are expressed in secretory fibroblast-like synoviocytes from patients with rheumatoid arthritis, that endogenous TRPC5-TRPC1 channels of the cells are activated by reduced thioredoxin, and that blockade of the channels enhances secretory activity and prevents the suppression of secretion by thioredoxin. The data indicate the presence of a previously unrecognized ion-channel activation mechanism that couples extracellular thioredoxin to cell function.

TRPC channel activation by extracellular thioredoxin.

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&#x673A;&#x5668;&#x4EBA;&#x591A;&#x4E95;&#x5E73;&#x9762;&#x819C;&#x7247;&#x94B3;&#x4E3A;&#x672C;&#x673A;&#x548C;&#x521D;&#x7EA7;&#x7684;&#x54FA;&#x4E73;&#x52A8;&#x7269;&#x7EC6;&#x80DE;&#x7684;&#x3002;

Robotic multiwell planar patch-clamp has become common in drug development and safety programs because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. It has not, however, been adopted significantly in other important areas of ion channel research, where conventional patch-clamp remains the favored method. Here, we show the wider potential of the multiwell approach with the ability for efficient intracellular solution exchange, describing protocols and success rates for recording from a range of native and primary mammalian cells derived from blood vessels, arthritic joints and the immune and central nervous systems. The protocol involves preparing a suspension of single cells to be dispensed robotically into 4-8 microfluidic chambers each containing a glass chip with a small aperture. Under automated control, giga-seals and whole-cell access are achieved followed by preprogrammed routines of voltage paradigms and fast extracellular or intracellular solution exchange. Recording from 48 chambers usually takes 1-6 h depending on the experimental design and yields 16-33 cell recordings.

Robotic multiwell planar patch-clamp for native and primary mammalian cells.

Robotique multipuit planar patch-clamp est devenu commun dans les programmes de d&eacute;veloppement et de la s&eacute;curit&eacute; des m&eacute;dicaments parce qu&#39;il permet efficace et syst&eacute;matique, essais de compos&eacute;s contre canaux ioniques pendant voltage clamp. Il n&#39;a pas, cependant, &eacute;t&eacute; adopt&eacute; sensiblement dans d&#39;autres domaines importants de la recherche de canal ionique, o&ugrave; patch-clamp de classique reste la m&eacute;thode favoris&eacute;e. Ici, nous montrons le plus large potentiel de l&#39;approche multipuit avec la capacit&eacute; d&#39;&eacute;change efficace solution intracellulaire, d&eacute;crivant des protocoles et des taux de r&eacute;ussite pour l&#39;enregistrement &agrave; partir d&#39;une plage de cellules de mammif&egrave;res indig&egrave;nes et primaires provenant des vaisseaux sanguins, articulations arthritiques et le syst&egrave;me immunitaire et le syst&egrave;me nerveux central. Le protocole consiste &agrave; pr&eacute;parer une suspension de cellules individuelles &agrave; pipeter robotis&eacute; en 4-8 microfluidique chambres, chacune contenant un verre &agrave; puce avec une petite ouverture. Sous contr&ocirc;le automatis&eacute;, giga-phoques et germes entiers acc&egrave;s sont atteints suivis des routines pr&eacute;programm&eacute;es des paradigmes de la tension et l&#39;&eacute;change de solution rapide extracellulaire ou intracellulaire. Enregistrement de 48 chambres habituellement prend de 1 &agrave; 6 h, selon le protocole exp&eacute;rimental et donne des enregistrements 16-33.

Robotique multipuit planar patch clamp pour les cellules mammif&egrave;res indig&egrave;nes et primaires.

Robot multiwell planare Patch-Clamp ist h&auml;ufig in Droge-Entwicklung und Sicherheit-Programme geworden, weil es erm&ouml;glicht effiziente und systematische Testen von Verbindungen gegen Ionenkan&auml;le w&auml;hrend Klemm-Spannung. Es wurde jedoch nicht, in anderen wichtigen Bereichen der Ionen-Kanal-Forschung, wo konventionelle Patch-Clamp die bevorzugte Methode bleibt deutlich angenommen. Hier zeigen wir die potenziellen multiwell Ansatz mit der F&auml;higkeit f&uuml;r effiziente intrazellul&auml;re L&ouml;sung Exchange, Beschreibung der Protokolle und Erfolgsraten f&uuml;r Aufnahmen aus einer Reihe von einheimischen und prim&auml;re S&auml;ugetier-Zellen, die Blutgef&auml;&szlig;e, arthritische Gelenke und das Immunsystem und zentralen Nervensystem abgeleitet. Das Protokoll umfasst das Vorbereiten einer Aussetzung der Einzelzellen auf robotically verzichtet werden in 4-8 mikrofluidische Kammern jeweils ein Glas chip mit einer kleinen Blende. Automatisierte Kontrolle werden Giga-Dichtungen und ganze Zellzugriff erreicht gefolgt von vorprogrammierten Routinen Spannung Paradigmen und schnell extrazellul&auml;re oder intrazellul&auml;ren L&ouml;sung Austausch. Aufnahme von 48 Chambers in der Regel dauert 1-6 h je nach der Versuchsanordnung und ergibt 16-33-Zelle-Aufnahmen.

Robot multiwell planare Patch-Clamp f&uuml;r native und prim&auml;re S&auml;ugetier-Zellen.

Robotica vetrini planari patch-clamp &egrave; diventato comune in programmi di sviluppo e sicurezza del farmaco perch&eacute; consente di efficiente e sistematico test di composti contro canali ionici durante la tensione-morsetto. Tuttavia, non &egrave; stato adottato in modo significativo in altre importanti aree di ricerca canale ionico, dove convenzionale patch-clamp rimane il metodo favorito. Qui, vi mostriamo il potenziale pi&ugrave; ampio dell&#39;approccio del multiwell con la capacit&agrave; di scambio efficiente soluzione intracellulare, che descrive i protocolli e i tassi di successo per la registrazione da una gamma di cellule di mammifero native e primarie derivata da vasi sanguigni, articolazioni artritiche e il sistema immunitario e sistema nervoso centrale. Il protocollo prevede di preparare una sospensione di cellule singole di essere dispensati robotizzato in 4-8 microfluidica camere ognuna contenente un vetro chip con una piccola apertura. Sotto controllo automatizzato, giga-guarnizioni e cellule intere accesso vengono raggiunti seguita da routine preprogrammate di paradigmi di tensione e di scambio di soluzione veloce extracellulare o intracellulare. Registrazione da 48 camere di solito prende 1-6 h a seconda del disegno sperimentale e produce registrazioni cella 16-33.

Robotica vetrini planari patch-clamp per cellule di mammifero native e primarie.

&#x52B9;&#x7387;&#x7684;&#x304B;&#x3064;&#x4F53;&#x7CFB;&#x7684;&#x306A;&#x5316;&#x5408;&#x7269;&#x306B;&#x5BFE;&#x3059;&#x308B;&#x30A4;&#x30AA;&#x30F3; &#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306E;&#x96FB;&#x5727;&#x30AF;&#x30E9;&#x30F3;&#x30D7;&#x4E2D;&#x306B;&#x30C6;&#x30B9;&#x30C8;&#x3067;&#x304D;&#x308B;&#x305F;&#x3081;&#x3001;&#x30ED;&#x30DC;&#x30C3;&#x30C8; multiwell &#x5E73;&#x9762;&#x30D1;&#x30C3;&#x30C1; &#x30FB; &#x30AF;&#x30E9;&#x30F3;&#x30D7;&#x306F;&#x85AC;&#x7269;&#x306E;&#x958B;&#x767A;&#x3068;&#x5B89;&#x5168;&#x6027;&#x306E;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30E0;&#x3067;&#x5171;&#x901A;&#x3068;&#x306A;&#x3063;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x305D;&#x308C;&#x306F;&#x3057;&#x304B;&#x3057;&#x3001;&#x3055;&#x308C;&#x3066;&#x3044;&#x306A;&#x3044;&#x3001;&#x5F93;&#x6765;&#x30D1;&#x30C3;&#x30C1; &#x30FB; &#x30AF;&#x30E9;&#x30F3;&#x30D7;&#x306E;&#x307E;&#x307E;&#x652F;&#x6301;&#x3055;&#x308C;&#x305F;&#x30E1;&#x30BD;&#x30C3;&#x30C9;&#x4ED6;&#x306E;&#x91CD;&#x8981;&#x306A;&#x5206;&#x91CE;&#x306E;&#x30A4;&#x30AA;&#x30F3; &#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306E;&#x7814;&#x7A76;&#x3092;&#x5927;&#x5E45;&#x306B;&#x63A1;&#x7528;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x6211;&#x3005; multiwell &#x306E;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x306E;&#x3088;&#x308A;&#x5E83;&#x3044;&#x53EF;&#x80FD;&#x6027;&#x52B9;&#x7387;&#x7684;&#x7D30;&#x80DE;&#x5185;&#x30BD;&#x30EA;&#x30E5;&#x30FC;&#x30B7;&#x30E7;&#x30F3; exchange &#x306E;&#x80FD;&#x529B;&#x3092;&#x6301;&#x3064;&#x30D7;&#x30ED;&#x30C8;&#x30B3;&#x30EB;&#x304A;&#x3088;&#x3073;&#x8840;&#x7BA1;&#x3001;&#x95A2;&#x7BC0;&#x708E;&#x306E;&#x95A2;&#x7BC0;&#x3068;&#x514D;&#x75AB;&#x304A;&#x3088;&#x3073;&#x4E2D;&#x67A2;&#x795E;&#x7D4C;&#x7CFB;&#x304B;&#x3089;&#x6D3E;&#x751F;&#x3057;&#x305F;&#x30CD;&#x30A4;&#x30C6;&#x30A3;&#x30D6;&#x3068;&#x30D7;&#x30E9;&#x30A4;&#x30DE;&#x30EA;&#x306E;&#x54FA;&#x4E73;&#x985E;&#x30BB;&#x30EB;&#x306E;&#x7BC4;&#x56F2;&#x304B;&#x3089;&#x306E;&#x9332;&#x97F3;&#x306E;&#x305F;&#x3081;&#x306E;&#x6210;&#x529F;&#x7387;&#x3092;&#x8A18;&#x8FF0;&#x3059;&#x308B;&#x8868;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x30D7;&#x30ED;&#x30C8;&#x30B3;&#x30EB;&#x306F;&#x3001;&#x5404;&#x30AC;&#x30E9;&#x30B9;&#x3092;&#x542B;&#x3080;&#x30C1;&#x30C3;&#x30D7; 4 8 &#x30DE;&#x30A4;&#x30AF;&#x30ED;&#x30D5;&#x30EB;&#x30A4;&#x30C7;&#x30A3;&#x30C3;&#x30AF; &#x30FB; &#x30C1;&#x30A7;&#x30F3;&#x30D0;&#x30FC;&#x306B;&#x5FAE;&#x5C0F;&#x958B;&#x53E3;&#x3092;&#x6301;&#x3064;&#x8ABF;&#x5264;&#x3059;&#x308B;&#x30ED;&#x30DC;&#x30C3;&#x30C8;&#x306B;&#x5358;&#x4E00;&#x7D30;&#x80DE;&#x306E;&#x61F8;&#x6FC1;&#x6DB2;&#x3092;&#x6E96;&#x5099;&#x3057;&#x307E;&#x3059;&#x3002;&#x81EA;&#x52D5;&#x5236;&#x5FA1;&#x306E;&#x4E0B;&#x3067;&#x3001;&#x30AE;&#x30AC; &#x30B7;&#x30FC;&#x30EB;&#x3068;&#x5168;&#x4F53;&#x30BB;&#x30EB;&#x3078;&#x306E;&#x30A2;&#x30AF;&#x30BB;&#x30B9;&#x3092;&#x96FB;&#x5727;&#x306E;&#x30D1;&#x30E9;&#x30C0;&#x30A4;&#x30E0;&#x3068;&#x901F;&#x304F;&#x7D30;&#x80DE;&#x307E;&#x305F;&#x306F;&#x7D30;&#x80DE;&#x5185;&#x30BD;&#x30EA;&#x30E5;&#x30FC;&#x30B7;&#x30E7;&#x30F3; exchange &#x306E;&#x3042;&#x3089;&#x304B;&#x3058;&#x3081;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30E0;&#x3055;&#x308C;&#x305F;&#x30EB;&#x30FC;&#x30C1;&#x30F3;&#x306B;&#x3088;&#x3063;&#x3066;&#x7D9A;&#x3044;&#x3066;&#x5B9F;&#x73FE;&#x3057;&#x307E;&#x3059;&#x3002;48 &#x5BA4;&#x901A;&#x5E38;&#x304B;&#x3089;&#x8A18;&#x9332;&#x5B9F;&#x9A13;&#x306E;&#x8A2D;&#x8A08;&#x306B;&#x5FDC;&#x3058;&#x3066; 1 6 h &#x3068;&#x308A; 16 33 &#x30BB;&#x30EB;&#x306E;&#x9332;&#x97F3;&#x304C;&#x5F97;&#x3089;&#x308C;&#x307E;&#x3059;&#x3002;

&#x30ED;&#x30DC;&#x30C3;&#x30C8; multiwell &#x5E73;&#x9762;&#x30D1;&#x30C3;&#x30C1;&#x30AF; &#x30CD;&#x30A4;&#x30C6;&#x30A3;&#x30D6;&#x3068;&#x30D7;&#x30E9;&#x30A4;&#x30DE;&#x30EA;&#x54FA;&#x4E73;&#x985E;&#x7D30;&#x80DE;&#x3002;

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&#xB85C;&#xBD07; multiwell &#xD3C9;&#xBA74; &#xD328;&#xCE58; &#xD074;&#xB7A8;&#xD504; &#xB124;&#xC774;&#xD2F0;&#xBE0C; &#xBC0F; &#xAE30;&#xBCF8; &#xD3EC;&#xC720;&#xB958; &#xC138;&#xD3EC;&#xC5D0; &#xB300; &#xD55C;.

Rob&oacute;tica pocillos planar patch-clamp se ha vuelto com&uacute;n en programas de desarrollo y seguridad de la droga porque permite eficiente y sistem&aacute;tica pruebas de compuestos contra canales i&oacute;nicos durante voltaje-abrazadera. Sin embargo, no ha sido adoptado significativamente en otras &aacute;reas importantes de investigaci&oacute;n del canal del ion, donde patch-clamp convencional sigue siendo el m&eacute;todo favorecido. A continuaci&oacute;n, os mostramos el mayor potencial del enfoque pocillos con la capacidad para el intercambio eficiente soluci&oacute;n intracelular, describir los protocolos y las tasas de &eacute;xito para la grabaci&oacute;n de una serie de c&eacute;lulas de mam&iacute;fero nativas y primarias, derivado de los vasos sangu&iacute;neos, articulaciones artr&iacute;ticas e inmune y sistema nervioso central. El protocolo consiste en preparar una suspensi&oacute;n de c&eacute;lulas a dispensar robot en 4-8 microflu&iacute;dicos c&aacute;maras cada una con un vidrio de la viruta con una peque&ntilde;a abertura. Bajo control automatizado, giga-sellos y acceso de celulares alcanzan seguido rutinas preprogramadas de paradigmas de tensi&oacute;n e intercambio de soluci&oacute;n r&aacute;pida extracelular o intracelular. Grabaci&oacute;n de 48 c&aacute;maras generalmente toma de 1-6 h seg&uacute;n el dise&ntilde;o experimental y produce grabaciones de c&eacute;lula 16-33.

Rob&oacute;tica pocillos planar patch-clamp para c&eacute;lulas de mam&iacute;fero nativas y primarias.

Transient receptor potential melastatin (TRPM)3 is a calcium-permeable ion channel activated by the neurosteroid pregnenolone sulfate and positively coupled to insulin secretion in beta cells. Although vascular TRPM3 mRNA has been reported, there is no knowledge of TRPM3 protein or its regulation and function in the cardiovascular system.

Pregnenolone sulphate- and cholesterol-regulated TRPM3 channels coupled to vascular smooth muscle secretion and contraction.

&#x4EE5;&#x786E;&#x5B9A;&#x9499;&#x6E17;&#x900F;&#x6E20;&#x9053;&#x662F;&#x5426;&#x6C27;&#x5316;&#x78F7;&#x8102;&#x7684;&#x76EE;&#x6807;&#xFF1A; 1-&#x68D5;&#x6988;-2-glutaroyl-&#x78F7;&#x8102;&#x9170;&#x80C6;&#x78B1; (PGPC) &#x548C; 1-&#x68D5;&#x6988;-2-oxovaleroyl-&#x78F7;&#x8102;&#x9170;&#x80C6;&#x78B1; (POVPC)&#x3002;

&#x9499;&#x900F;&#x77AC;&#x65F6;&#x53D7;&#x4F53;&#x6F5C;&#x5728;&#x89C4;&#x8303; 5 &#x542B;&#x6E20;&#x9053;&#x7531;&#x6C27;&#x5316;&#x78F7;&#x8102;&#x7684;&#x77ED;&#x671F;&#x523A;&#x6FC0;&#x3002;

To determine whether calcium-permeable channels are targets for the oxidized phospholipids: 1-palmitoyl-2-glutaroyl-phosphatidylcholine (PGPC) and 1-palmitoyl-2-oxovaleroyl-phosphatidylcholine (POVPC).

Short-term stimulation of calcium-permeable transient receptor potential canonical 5-containing channels by oxidized phospholipids.

Pour d&eacute;terminer si des canaux calcium-perm&eacute;ables sont des cibles pour les phospholipides oxyd&eacute;s: 1-palmitoyl-2-glutaroyl-phosphatidylcholine (PGPC) et 1-palmitoyl-2-oxovaleroyl-phosphatidylcholine (POVPC).

Stimulation &agrave; court terme des canaux contenant 5 canonique perm&eacute;able au calcium transient receptor potentiels, de phospholipides oxyd&eacute;s.

Um festzustellen, ob die Kalzium-durchl&auml;ssige Kan&auml;le Ziele f&uuml;r die oxidierte Phospholipide sind: 1-Palmitoyl-2-Glutaroyl-Phosphatidylcholin (PGPC) und 1-Palmitoyl-2-Oxovaleroyl-Phosphatidylcholin (POVPC).

Kurzfristige Stimulierung der Kalzium-durchl&auml;ssigen Transienten Rezeptor potenzielle kanonische 5-haltigen Channels von oxidierte Phospholipide.

Per determinare se i canali calcio-permeabile sono gli obiettivi per i fosfolipidi ossidati: 1-palmitoil-2-glutaroyl-fosfatidilcolina (PGPC) e 1-palmitoil-2-oxovaleroyl-fosfatidilcolina (POVPC).

Stimolazione a breve termine di calcio-permeabile transient receptor potenziali canonica contenente 5 canali da fosfolipidi ossidati.

&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x900F;&#x904E;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x9178;&#x5316;&#x30EA;&#x30F3;&#x8102;&#x8CEA;&#x306E;&#x5BFE;&#x8C61;&#x306B;&#x306A;&#x308B;&#x304B;&#x3069;&#x3046;&#x304B;&#x3092;&#x6C7A;&#x5B9A;&#x3057;&#x307E;&#x3059;: 1-&#x30D1;&#x30EB;&#x30DF;&#x30C8;&#x30A4;&#x30EB;-2-glutaroyl-&#x30DB;&#x30B9;&#x30D5;&#x30A1;&#x30C1;&#x30B8;&#x30EB;&#x30B3;&#x30EA;&#x30F3; (&#x30DE;&#x30BB;&#x30DE;&#x30C6;&#x30A3;&#x30AB;&#x30EB;) &#x304A;&#x3088;&#x3073; 1-&#x30D1;&#x30EB;&#x30DF;&#x30C8;&#x30A4;&#x30EB;-2-oxovaleroyl-&#x30DB;&#x30B9;&#x30D5;&#x30A1;&#x30C1;&#x30B8;&#x30EB;&#x30B3;&#x30EA;&#x30F3; (POVPC)&#x3002;

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&#xCE7C;&#xC298; &#xD22C;&#xACFC;&#xC131; &#xCC44;&#xB110; &#xC0B0;&#xD654; &#xC778;&#xC9C0;&#xC9C8;&#xC5D0; &#xB300; &#xD55C; &#xB300;&#xC0C1; &#xC778;&#xC9C0; &#xD655;&#xC778; &#xD558;&#xAE30; &#xC704;&#xD574;: 1-palmitoyl-2-glutaroyl-phosphatidylcholine (PGPC) &#xBC0F; 1-palmitoyl-2-oxovaleroyl-phosphatidylcholine (POVPC).

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Para determinar si los canales de calcio-permeables son objetivos para los fosfol&iacute;pidos oxidados: 1-palmitoil-2-glutaroyl-fosfatidilcolina (PGPC) y 1-palmitoil-2-oxovaleroyl-fosfatidilcolina (POVPC).

Est&iacute;mulo a corto plazo del receptor transitoria permeable al calcio potenciales can&oacute;nica que contienen 5 canales por fosfol&iacute;pidos oxidados.

Calcium-permeable channels are known to have roles in many mammalian cell types but the expression and contribution of such ion channels in synovial cells is mostly unknown. The objective of this study was to investigate the potential relevance of Transient Receptor Potential Melastatin 3 (TRPM3) channel to fibroblast-like synoviocytes (FLSs) of patients with rheumatoid arthritis.

TRPM3 channel stimulated by pregnenolone sulphate in synovial fibroblasts and negatively coupled to hyaluronan.

Transient receptor potential canonical 5 (TRPC5) forms cationic channels that are polymodal sensors of factors including oxidized phospholipids, hydrogen peroxide, and reduced thioredoxin. The aim of this study was to expand knowledge of the chemical-sensing capabilities of TRPC5 by investigating dietary antioxidants. Human TRPC5 channels were expressed in HEK 293 cells and studied by patch clamp and intracellular Ca(2+) recording. GFP- and HA-tagged channels were used to quantify plasma membrane localization. Gallic acid and vitamin C suppressed TRPC5 activity if it was evoked by exogenous hydrogen peroxide or lanthanide ions but not by lysophosphatidylcholine or carbachol. Catalase mimicked the effects, suggesting that lanthanide-evoked activity depended on endogenous hydrogen peroxide. Trans-resveratrol, by contrast, inhibited all modes of TRPC5, and its effect was additive with that of vitamin C, suggesting antioxidant-independent action. The IC(50) was ∼10 μM. Diethylstilbestrol, a related hydroxylated stilbene, inhibited TRPC5 with a similar IC(50), but its action contrasted sharply with that of resveratrol in outside-out membrane patches where diethylstilbestrol caused strong and reversible inhibition and resveratrol had no effect, suggesting indirect modulation by resveratrol. Resveratrol did not affect channel surface density, but its effect was calcium-sensitive, indicating an action via a calcium-dependent intermediate. The data suggest previously unrecognized chemical-sensing properties of TRPC5 through multiple mechanisms: (i) inhibition by scavengers of reactive oxygen species because a mode of TRPC5 activity depends on endogenous hydrogen peroxide; (ii) direct channel blockade by diethylstilbestrol; and (iii) indirect, antioxidant-independent inhibition by resveratrol.

TRPC5 channel sensitivities to antioxidants and hydroxylated stilbenes.

The aim of this study was to generate new insight into chemical regulation of transient receptor potential (TRP) channels with relevance to glucose homeostasis and the metabolic syndrome. Human TRP melastatin 2 (TRPM2), TRPM3, and TRP canonical 5 (TRPC5) were conditionally overexpressed in human embryonic kidney 293 cells and studied by using calcium-measurement and patch-clamp techniques. Rosiglitazone and other peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists were investigated. TRPM2 was unaffected by rosiglitazone at concentrations up to 10 μM but was inhibited completely at higher concentrations (IC(50), ∼22.5 μM). TRPM3 was more potently inhibited, with effects occurring in a biphasic concentration-dependent manner such that there was approximately 20% inhibition at low concentrations (0.1-1 μM) and full inhibition at higher concentrations (IC(50), 5-10 μM). PPAR-γ antagonism by 2-chloro-5-nitrobenzanilide (GW9662) did not prevent inhibition of TRPM3 by rosiglitazone. TRPC5 was strongly stimulated by rosiglitazone at concentrations of ≥10 μM (EC(50), ∼30 μM). Effects on TRPM3 and TRPC5 occurred rapidly and reversibly. Troglitazone and pioglitazone inhibited TRPM3 (IC(50), 12 μM) but lacked effect on TRPC5, suggesting no relevance of PPAR-γ or the thiazolidinedione moiety to rosiglitazone stimulation of TRPC5. A rosiglitazone-related but nonthiazolidinedione PPAR-γ agonist, N-(2-benzoylphenyl)-O-[2-(methyl-2-pyridinylamino)ethyl]-l-tyrosine (GW1929), was a weak stimulator of TRPM3 and TRPC5. The natural PPAR-γ agonist 15-deoxy prostaglandin J(2), had no effect on TRPM3 or TRPC5. The data suggest that rosiglitazone contains chemical moieties that rapidly, strongly, and differentially modulate TRP channels independently of PPAR-γ, potentially contributing to biological consequences of the agent and providing the basis for novel TRP channel pharmacology.

Rapid and contrasting effects of rosiglitazone on transient receptor potential TRPM3 and TRPC5 channels.

The Transient Receptor Potential Canonical 5 (TRPC5) protein forms calcium-permeable cationic channels that are stimulated by G protein-coupled receptor agonists. The signaling pathways of such agonist effects are poorly understood. Here we investigated the potential for involvement of lysophosphatidylcholine (LPC) and arachidonic acid generated by group 6 (GVI) phospholipase A2 (PLA2) enzymes, focusing on stimulation of TRPC5 by sphingosine-1-phosphate (S1P) which acts via a pertussis toxin-sensitive (Gi/o protein) pathway without Ca2+-release. Experiments were on HEK 293 cells containing conditional expression of human TRPC5. Channel activity was recorded using an intracellular calcium indicator or whole-cell patch-clamp and PLA2 activity was detected using 3H-arachidonic acid. S1P stimulated PLA2 and TRPC5 activities. Both effects were suppressed by the GVI PLA2 inhibitor bromoenol lactone. Knock-down of GVI PLA2 by RNA interference suppressed channel activity evoked by S1P whereas activity evoked by the direct channel stimulator LPC was unaffected. Arachidonic acid did not stimulate the channels. Prior exposure of channels to LPC but not arachidonic acid suppressed channel activity evoked by S1P but not gadolinium, a putative direct stimulator of the channels. The data suggest roles of LPC and GVI PLA2 in S1P-evoked TRPC5 activity.

GVI phospholipase A2 role in the stimulatory effect of sphingosine-1-phosphate on TRPC5 cationic channels.

Transient Receptor Potential Melastatin 3 (TRPM3) is a widely expressed calcium-permeable non-selective cation channel that is stimulated by high concentrations of nifedipine or by physiological steroids that include pregnenolone sulphate. Here we sought to identify steroids that inhibit TRPM3. Channel activity was studied using calcium-measurement and patch-clamp techniques. Progesterone (0.01-10μM) suppressed TRPM3 activity evoked by pregnenolone sulphate. Progesterone metabolites and 17β-oestradiol were also inhibitory but the effects were relatively small. Dihydrotestosterone was an inhibitor at concentrations higher than 1μM. Corticosteroids lacked effect. Overlay assays indicated that pregnenolone sulphate, progesterone and dihydrotestosterone bound to TRPM3. In contrast to dihydrotestosterone, progesterone inhibited nifedipine-evoked TRPM3 activity or activity in the absence of an exogenous activator, suggesting a pregnenolone sulphate-independent mechanism of action. Dihydrotestosterone, like a non-steroid look-alike compound, acted as a competitive antagonist at the pregnenolone sulphate binding site. Progesterone inhibited endogenous TRPM3 in vascular smooth muscle cells. Relevance of TRPM3 or the progesterone effect to ovarian cells, which have been suggested to express TRPM3, was not identified. The data further define a chemical framework for competition with pregnenolone sulphate at TRPM3 and expand knowledge of steroid interactions with TRPM3, suggesting direct steroid binding and pregnenolone sulphate-independent inhibition by progesterone.

Pregnenolone sulphate-independent inhibition of TRPM3 channels by progesterone.

The Sigma-1 receptor (Sig1R) impacts on calcium ion signalling and has a plethora of ligands. This study investigated Sig1R and its ligands in relation to endogenous calcium events of endothelial cells and transient receptor potential (TRP) channels.

Inhibition of endothelial cell Ca²⁺ entry and transient receptor potential channels by Sigma-1 receptor ligands.

There is demand for isoform-specific ion channel inhibitors as tools to investigate the biology of -endogenous ion channels and validate them as targets in drug discovery programs. There is also hope that such inhibitors may be new therapeutic agents or provide the foundation for such agents. However, in practice, it is commonly experienced that inhibitors lack sufficient specificity, fail to distinguish between members of a class of ion channel, or have other (non-ion channel) off-target effects. Due to their extraordinary specificity, antibodies offer a potentially attractive strategy for overcoming these problems. Inhibitory antibodies acting at the extracellular face of ion channels are particularly attractive because there is enhanced possibility for specificity and intracellular delivery methods are not required. Here we describe experience with such an antibody approach and methodology for generating agents based on anti-peptide polyclonal antibodies.

Generation of antibodies that are externally acting isoform-specific inhibitors of ion channels.

Biologics represent a fast-growing class of therapeutics in the pharmaceutical sector. Discovery of therapeutic antibodies and characterization of peptides can necessitate high expression of the target gene requiring the generation of clonal stably transfected cell lines. Traditional challenges of stable cell line transfection include gene silencing and cell-to-cell variability. Our inability to control these can present challenges in lead isolation. Recent progress in site-specific targeting of transgene to specific genomic loci has transformed the ability to generate stably transfected mammalian cell lines. In this article, we describe how the use of the Jump-In platform (Life Technologies, Carlsbad, CA) has been applied to drug discovery projects. It can easily and rapidly generate homogeneous high-expressing cell pools with a high degree of reproducibility. Their use in cell-based screening to identify specific binders, identify binding to relevant species variants, or detect functionally relevant therapeutic antibodies is central in driving drug discovery.

Use of the site-specific retargeting jump-in platform cell line to support biologic drug discovery.

Routine peptide structure-activity relationship screening requires the serial dilution of peptides to produce full concentration-response curves. Established tip-based protocols involve multiple tip changes and high exposure to plasticware. In the case of peptides, this becomes a challenge, since peptides can adsorb to plastic, resulting in an observed loss of potency. Various methods can be employed to prevent peptide loss during compound handling, such as the inclusion of bovine serum albumin or solvents in assay buffer and the siliconization of plasticware, yet protein binding remains unpredictable. The degree of variation by which peptides will adhere to plasticware can confuse results and cause inaccuracies in potency predictions. We evaluated acoustic noncontact methods for peptide serial dilution and compared it with traditional tip-based methods, on the effect on potency curves for glucagon-like peptide-1 and glucagon peptide analogues. The current study demonstrates the benefits of noncontact dispensing for high-density microplate assay preparation of peptides using nanoliter droplets across our entire drug discovery workflow, from in vitro high-throughput screening to drug exposure determinations from in vivo samples.

Acoustic Dispensing Preserves the Potency of Therapeutic Peptides throughout the Entire Drug Discovery Workflow.

The TRPC5 proteins assemble to create calcium-permeable, non-selective, cationic channels. We sought novel modulators of these channels through studies of natural products.

Natural and synthetic flavonoid modulation of TRPC5 channels.

Dual-agonist molecules combining glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) activity represent an exciting therapeutic strategy for diabetes treatment. Although challenging due to shared downstream signalling pathways, determining the relative activity of dual agonists at each receptor is essential when developing potential novel therapeutics. The challenge is exacerbated in physiologically relevant cell systems expressing both receptors. To this end, either GIP receptors (GIPR) or GLP-1 receptors (GLP-1R) were ablated via RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 endonucleases in the INS-1 pancreatic β-cell line. Multiple clonal cell lines harbouring gene disruptions for each receptor were isolated and assayed for receptor activity to identify functional knockouts (KOs). cAMP production in response to GIPR or GLP-1R activation was abolished and GIP- or GLP-1-induced potentiation of glucose-stimulated insulin secretion (GSIS) was attenuated in the cognate KO cell lines. The contributions of individual receptors derived from cAMP and GSIS assays were confirmed in vivo using GLP-1R KO mice in combination with a monoclonal antibody antagonist of GIPR. We have successfully applied CRISPR/Cas9-engineered cell lines to determining selectivity and relative potency contributions of dual-agonist molecules targeting receptors with overlapping native expression profiles and downstream signalling pathways. Specifically, we have characterised molecules as biased towards GIPR or GLP-1R, or with relatively balanced potency in a physiologically relevant β-cell system. This demonstrates the broad utility of CRISPR/Cas9 when applied to native expression systems for the development of drugs that target multiple receptors, particularly where the balance of receptor activity is critical.

Jacqueline Naylor

Cardiovascular & Metabolic Disease

MedImmune

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

Jacqueline Naylor

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Cardiovascular & Metabolic Disease

MedImmune

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

Cardiovascular & Metabolic Disease